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Sample records for reca-dependent recombinational dna

  1. The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA.

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    Rand, Lucinda; Hinds, Jason; Springer, Burkhard; Sander, Peter; Buxton, Roger S; Davis, Elaine O

    2003-11-01

    In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.

  2. The Walker A motif mutation recA4159 abolishes the SOS response and recombination in a recA730 mutant of Escherichia coli.

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    Šimatović, Ana; Mitrikeski, Petar T; Vlašić, Ignacija; Sopta, Mary; Brčić-Kostić, Krunoslav

    2016-01-01

    In bacteria, the RecA protein forms recombinogenic filaments required for the SOS response and DNA recombination. In order to form a recombinogenic filament, wild type RecA needs to bind ATP and to interact with mediator proteins. The RecA730 protein is a mutant version of RecA with superior catalytic abilities, allowing filament formation without the help of mediator proteins. The mechanism of RecA730 filament formation is not well understood, and the question remains as to whether the RecA730 protein requires ATP binding in order to become competent for filament formation. We examined two mutants, recA730,4159 (presumed to be defective for ATP binding) and recA730,2201 (defective for ATP hydrolysis), and show that they have different properties with respect to SOS induction, conjugational recombination and double-strand break repair. We show that ATP binding is essential for all RecA730 functions, while ATP hydrolysis is required only for double-strand break repair. Our results emphasize the similarity of the SOS response and conjugational recombination, neither of which requires ATP hydrolysis by RecA730. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. DNA compaction in the early part of the SOS response is dependent on RecN and RecA.

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    Odsbu, Ingvild; Skarstad, Kirsten

    2014-05-01

    The nucleoids of undamaged Escherichia coli cells have a characteristic shape and number, which is dependent on the growth medium. Upon induction of the SOS response by a low dose of UV irradiation an extensive reorganization of the nucleoids occurred. Two distinct phases were observed by fluorescence microscopy. First, the nucleoids were found to change shape and fuse into compact structures at midcell. The compaction of the nucleoids lasted for 10-20 min and was followed by a phase where the DNA was dispersed throughout the cells. This second phase lasted for ~1 h. The compaction was found to be dependent on the recombination proteins RecA, RecO and RecR as well as the SOS-inducible, SMC (structural maintenance of chromosomes)-like protein RecN. RecN protein is produced in high amounts during the first part of the SOS response. It is possible that the RecN-mediated 'compact DNA' stage at the beginning of the SOS response serves to stabilize damaged DNA prior to recombination and repair.

  4. Mycobacterium tuberculosis UvrD1 and UvrA proteins suppress DNA strand exchange promoted by cognate and noncognate RecA proteins.

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    Singh, Pawan; Patil, K Neelakanteshwar; Khanduja, Jasbeer Singh; Kumar, P Sanjay; Williams, Alan; Rossi, Franca; Rizzi, Menico; Davis, Elaine O; Muniyappa, K

    2010-06-15

    DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coli RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

  5. RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA

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    Coutelle Charles

    2006-03-01

    Full Text Available Abstract Background Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. Results We describe here the construction of the RecA-deficient minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure minicircle DNA from the obtained recA- producer strain HB101Cre. The minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric minicircle DNA forms, all containing the hybrid loxP66

  6. Recovery from ultraviolet light-induced inhibition of DNA synthesis requires umuDC gene products in recA718 mutant strains but not in recA+ strains of Escherichia coli

    International Nuclear Information System (INIS)

    Witkin, E.M.; Roegner-Maniscalco, V.; Sweasy, J.B.; McCall, J.O.

    1987-01-01

    Ultraviolet light (UV) inhibits DNA replication in Eschericia coli and induces the SOS response, a set of survival-enhancing phenotypes due to derepression of DNA damage-inducible genes, including recA and umuDC. Recovery of DNA synthesis after UV irradiation (induced replisome reactivation, or IRR) is an SOS function requiring RecA protein and postirradiation synthesis of additional protein(s), but this recovery does not require UmuDC protein. IRR occurs in strains carrying either recA718 (which does not reduce recombination, SOS inducibility, or UV mutagenesis) or umuC36 (which eliminates UV mutability), but not in recA718 umuC36 double mutants. In recA430 mutant strains, IRR does not occur whether or not functional UmuDC protein is present. IRR occurs in lexA-(Ind-) (SOS noninducible) strains if they carry an operator-constitutive recA allele and are allowed to synthesize proteins after irradiation. We conclude the following: (i) that UmuDC protein corrects or complements a defect in the ability of RecA718 protein (but not of RecA430 protein) to promote IRR and (ii) that in lexA(Ind-) mutant strains, IRR requires amplification of RecA+ protein (but not of any other LexA-repressed protein) plus post-UV synthesis of at least one other protein not controlled by LexA protein. We discuss the results in relation to the essential, but unidentified, roles of RecA and UmuDC proteins in UV mutagenesis

  7. DNA degradation in minicells of Escherichia coli K-12. Pt. 2. Effect of recA1 and recB21 mutations on DNA degradation in minicells and detection of exonuclease V activity

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    Khachatourians, G G [Saskatchewan Univ., Saskatoon (Canada). Dept. of Microbiology; Oak Ridge National Lab., Tenn. (USA). Biology Div.); Paterson, M C [Tennessee Univ., Oak Ridge (USA). Graduate School of Biomedical Sciences; Rijksuniversiteit Leiden (Netherlands). Lab. voor Stralengenetica); Sheehy, R J [Tennessee Univ., Oak Ridge (USA). Graduate School of Biomedical Sciences; Dorp, B Van [Rijksuniversiteit Leiden (Netherlands). Lab. voor Stralengenetica; Worthy, T E [Tennessee Univ., Knoxville (USA). Inst. of Radiation Biology

    1975-06-01

    The properties of minicell producing mutants of Escherichia coli deficient in genetic recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC-phenotypes are unaffected by min/sup +///sup -/ genotypes in whole cells. In contrast to minicells produced by rec/sup +/ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transferred DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permits unrestricted Hfr DNA breakdown in minicells produced by a recA1 strain. This results in an increase in TGA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R64-11 or lambdadv, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and (/sup 32/P)-labelled linear DNA from bacteriophage T/sub 7/ for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in rec/sup +/ and recA/sup -/ minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC/sup -/ minicells results in isolation of larger than average Hfr DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.

  8. RecA: Regulation and Mechanism of a Molecular Search Engine.

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    Bell, Jason C; Kowalczykowski, Stephen C

    2016-06-01

    Homologous recombination maintains genomic integrity by repairing broken chromosomes. The broken chromosome is partially resected to produce single-stranded DNA (ssDNA) that is used to search for homologous double-stranded DNA (dsDNA). This homology driven 'search and rescue' is catalyzed by a class of DNA strand exchange proteins that are defined in relation to Escherichia coli RecA, which forms a filament on ssDNA. Here, we review the regulation of RecA filament assembly and the mechanism by which RecA quickly and efficiently searches for and identifies a unique homologous sequence among a vast excess of heterologous DNA. Given that RecA is the prototypic DNA strand exchange protein, its behavior affords insight into the actions of eukaryotic RAD51 orthologs and their regulators, BRCA2 and other tumor suppressors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Roles of Bacillus subtilis DprA and SsbA in RecA-mediated genetic recombination.

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    Yadav, Tribhuwan; Carrasco, Begoña; Serrano, Ester; Alonso, Juan C

    2014-10-03

    Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP · Mg(2+)-bound form (RecA · ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA · ATP. This work demonstrates that RecA · ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA · dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis.

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    Pham, Phuong; Seitz, Erica M; Saveliev, Sergei; Shen, Xuan; Woodgate, Roger; Cox, Michael M; Goodman, Myron F

    2002-08-20

    SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates. Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis. One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass. A separate RecA mode is necessary for translesion synthesis. The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD'(2)C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode. Data are presented suggesting that the two RecA modes are "nonfilamentous". That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates. Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3' primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass. We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion. However, it is only a RecA molecule located at the 3' filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis.

  11. Genetic dependence of recombination in recD mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Lovett, S.T.; Luisi-DeLuca, C.; Kolodner, R.D.

    1988-01-01

    RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations

  12. Differences in mutagenic and recombinational DNA repair in enterobacteria

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    Sedgwick, S.G.; Goodwin, P.A.

    1985-01-01

    The incidence of recombinational DNA repair and inducible mutagenic DNA repair has been examined in Escherichia coli and 11 related species of enterobacteria. Recombinational repair was found to be a common feature of the DNA repair repertoire of at least 6 genera of enterobacteria. This conclusion is based on observations of (i) damage-induced synthesis of RecA-like proteins, (ii) nucleotide hybridization between E. coli recA sequences and some chromosomal DNAs, and (iii) recA-negative complementation by plasmids showing SOS-inducible expression of truncated E. coli recA genes. The mechanism of DNA damage-induced gene expression is therefore sufficiently conserved to allow non-E. coli regulatory elements to govern expression of these cloned truncated E. coli recA genes. In contrast, the process of mutagenic repair, which uses umuC+ umuD+ gene products in E. coli, appeared less widespread. Little ultraviolet light-induced mutagenesis to rifampicin resistance was detected outside the genus Escherichia, and even within the genus induced mutagenesis was detected in only 3 out of 6 species. Nucleotide hybridization showed that sequences like the E. coli umuCD+ gene are not found in these poorly mutable organisms. Evolutionary questions raised by the sporadic incidence of inducible mutagenic repair are discussed

  13. Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity.

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    Moreb, Eirik Adim; Hoover, Benjamin; Yaseen, Adam; Valyasevi, Nisakorn; Roecker, Zoe; Menacho-Melgar, Romel; Lynch, Michael D

    2017-12-15

    Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well as mutation rates through induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant negative form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are coexpressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E. coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10 000 guides covering the E. coli chromosome. The use of dominant negative RecA or homologues may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.

  14. Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis.

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    Korycka-Machala, Malgorzata; Brzostek, Anna; Rozalska, Sylwia; Rumijowska-Galewicz, Anna; Dziedzic, Renata; Bowater, Richard; Dziadek, Jaroslaw

    2006-05-01

    Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway.

  15. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

    International Nuclear Information System (INIS)

    Moreau, P.L.

    1988-01-01

    Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

  16. New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis

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    Dutreix, M.; Moreau, P.L.; Bailone, A.; Galibert, F.; Battista, J.R.; Walker, G.C.; Devoret, R.

    1989-05-01

    To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+.

  17. New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis

    International Nuclear Information System (INIS)

    Dutreix, M.; Moreau, P.L.; Bailone, A.; Galibert, F.; Battista, J.R.; Walker, G.C.; Devoret, R.

    1989-01-01

    To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+

  18. Relative roles of uvrA and recA genes in the recovery of Escherichia coli and phage lambda after ultraviolet irradiation

    International Nuclear Information System (INIS)

    Salaj-Smic, E.; Petranovic, D.; Petranovic, M.; Trgovcevic, Z.

    1980-01-01

    The action of the host-cell repair system on recovery from uv damage to bacterial and phage DNA was studied. lambda cI857 ind red lysogens were used. These lysogens, although noninducible by uv light, can be induced by raising the temperature from 30 to 42 0 C. Sensitivity of the phage in relation to its host was analyzed in various bacterial backgrounds. Relative sensitivity of the phage and its host is the same if the uv survival curve for colonies is 80 times steeper than for plaques. This same relative sensitivity is observed if the host cell does not possess any mechanism for DNA repair (uvrA recA background). In the uvrA recA + background, the plaque survival is not significantly increased above the level observed in the uvrA recA double mutant. recA-dependent recombinational postreplication repair does not operate on the phage DNA in the cytoplasm; relative sensitivity of the phage is therefore much higher than that of the host. If the lysogenic induction is delayed, a marked increase in the plaque count is seen so the phage shows the same relative sensitivity as the bacterial cell. Short-patch excision repair operates on both phage and bacterial DNA but less efficiently on phage DNA. In the wild-type (uvrA + recA + ) host, the highest survival of plaques and colonies is obtained. Relative sensitivity of the phage is nevertheless 50 times higher then that of the bacterial cell. This may mean the recA gene product is involved in copy-choice excision and/or long-patch excision and/or incision-promoted recombination repair of the phage DNA but it remains unable to mediate its recombinational postreplication repair

  19. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    International Nuclear Information System (INIS)

    Galvão, C.W.; Souza, E.M.; Etto, R.M.; Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G.; Schumacher, J.; Buck, M.; Steffens, M.B.R.

    2012-01-01

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs ) can interact with the H. seropedicae RecA protein (RecA Hs ) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs . RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions

  20. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

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    C.W. Galvão

    2012-12-01

    Full Text Available DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs can interact with the H. seropedicaeRecA protein (RecA Hs and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA, inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

  1. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae.

    Science.gov (United States)

    Galvão, C W; Souza, E M; Etto, R M; Pedrosa, F O; Chubatsu, L S; Yates, M G; Schumacher, J; Buck, M; Steffens, M B R

    2012-12-01

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

  2. Cre-dependent DNA recombination activates a STING-dependent innate immune response

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    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit

    2016-01-01

    Abstract Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  3. Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

    Energy Technology Data Exchange (ETDEWEB)

    Kokjohn, T.A.; Miller, R.V.

    1987-04-01

    We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants.

  4. Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene

    International Nuclear Information System (INIS)

    Kokjohn, T.A.; Miller, R.V.

    1987-01-01

    We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants

  5. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    International Nuclear Information System (INIS)

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro

  6. The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

    Science.gov (United States)

    Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

    2012-01-01

    Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

  7. Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

    DEFF Research Database (Denmark)

    Thresher, RJ; Christiansen, Gunna; Griffith, JD

    1988-01-01

    We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional Rec......M in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein...... assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein...

  8. DNA degradation, UV sensitivity and SOS-mediated mutagenesis in strains of Escherichia coli deficient in single-strand DNA binding protein: Effects of mutations and treatments that alter levels of exonuclease V or RecA protein

    International Nuclear Information System (INIS)

    Lieberman, H.B.; Witkin, E.M.

    1983-01-01

    Certain strains suppress the temperature-sensitivity caused by ssb-1, which encodes a mutant ssDNA binding protein (SSB). At 42 0 C, such strains are extremely UV-sensitive, degrade their DNA extensively after UV irradiation, and are defficient in UV mutability and UV induction of recA protein synthesis. We transduced recC22, which eliminates Exonuclease V activity, and recAo281, which causes operator-constitutive synthesis of recA protein, into such an ssb-1 strain. Both double mutants degraded their DNA extensively at 42 0 C after UV irradiation, and both were even more UV-sensitive than the ssb-1 single mutant. We conclude that one or more nucleases other than Exonuclease V degrades DNA in the ssb recC strain, and that recA protein, even if synthesized copiously, can function efficiently in recombinational DNA repair and in control of post-UV DNA degradation only if normal SSB is also present. Pretreatment with nalidixic acid at 30 0 C restored normal UV mutability at 42 0 C, but did not increase UV resistance, in an ssb-1 strain. Another ssb allele, ssb-113, which blocks SOS induction at 30 0 C, increases spontaneous mutability more than tenfold. The ssb-113 allele was transduced into the SOS-constitutive recA730 strain SC30. This double mutant expressed the same elevated spontaneous and UV-induced mutability at 30 0 C as the ssb + recA730 strain, and was three times more UV-resistant than its ssb-113 recA + parent. We conclude that ssb-1 at 42 0 C and ssb-113 at 30 0 C block UV-induced activation of recA protease, but that neither allele interferes with subsequent steps in SOS-mediated mutagenesis. (orig.)

  9. A homozygous recA mutant of Synechocystis PCC6803: construction strategy and characteristics eliciting a novel RecA independent UVC resistance in dark.

    Science.gov (United States)

    Minda, Renu; Ramchandani, Jyoti; Joshi, Vasudha P; Bhattacharjee, Swapan Kumar

    2005-12-01

    We report here the construction of a homozygous recA460::cam insertion mutant of Synechocystis sp. PCC 6803 that may be useful for plant molecular genetics by providing a plant like host free of interference from homologous recombination. The homozygous recA460::cam mutant is highly sensitive to UVC under both photoreactivating and non-photoreactivating conditions compared to the wild type (WT). The liquid culture of the mutant growing in approximately 800 lx accumulates nonviable cells to the tune of 86% as estimated by colony counts on plates incubated at the same temperature and light intensity. The generation time of recA mutant in standard light intensity (2,500 lx) increases to 50 h compared to 28 h in lower light intensity (approximately 800 lx) that was used for selection, thus explaining the earlier failures to obtain a homozygous recA mutant. The WT, in contrast, grows at faster rate (23 h generation time) in standard light intensity compared to that at approximately 800 lx (26 h). The Synechocystis RecA protein supports homologous recombination during conjugation in recA (-) mutant of Escherichia coli, but not the SOS response as measured by UV sensitivity. It is suggested that using this homozygous recA460::cam mutant, investigations can now be extended to dissect the network of DNA repair pathways involved in housekeeping activities that may be more active in cyanobacteria than in heterotrophs. Using this mutant for the first time we provide a genetic evidence of a mechanism independent of RecA that causes enhanced UVC resistance on light to dark transition.

  10. Genetic requirements for high constitutive SOS expression in recA730 mutants of Escherichia coli.

    Science.gov (United States)

    Vlašić, Ignacija; Šimatović, Ana; Brčić-Kostić, Krunoslav

    2011-09-01

    The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5'-3' exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5'-3' exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

  11. Prophage induction and differential RecA and UmuDAb transcriptome regulation in the DNA damage responses of Acinetobacter baumannii and Acinetobacter baylyi.

    Directory of Open Access Journals (Sweden)

    Janelle M Hare

    Full Text Available The SOS response to DNA damage that induces up to 10% of the prokaryotic genome requires RecA action to relieve LexA transcriptional repression. In Acinetobacter species, which lack LexA, the error-prone polymerase accessory UmuDAb is instead required for ddrR induction after DNA damage, suggesting it might be a LexA analog. RNA-Seq experiments defined the DNA damage transcriptome (mitomycin C-induced of wild type, recA and umuDAb mutant strains of both A. baylyi ADP1 and A. baumannii ATCC 17978. Of the typical SOS response genes, few were differentially regulated in these species; many were repressed or absent. A striking 38.4% of all ADP1 genes, and 11.4% of all 17978 genes, were repressed under these conditions. In A. baylyi ADP1, 66 genes (2.0% of the genome, including a CRISPR/Cas system, were DNA damage-induced, and belonged to four regulons defined by differential use of recA and umuDAb. In A. baumannii ATCC 17978, however, induction of 99% of the 152 mitomycin C-induced genes depended on recA, and only 28 of these genes required umuDAb for their induction. 90% of the induced A. baumannii genes were clustered in three prophage regions, and bacteriophage particles were observed after mitomycin C treatment. These prophages encoded esvI, esvK1, and esvK2, ethanol-stimulated virulence genes previously identified in a Caenorhabditis elegans model, as well as error-prone polymerase alleles. The induction of all 17978 error-prone polymerase alleles, whether prophage-encoded or not, was recA dependent, but only these DNA polymerase V-related genes were de-repressed in the umuDAb mutant in the absence of DNA damage. These results suggest that both species possess a robust and complex DNA damage response involving both recA-dependent and recA-independent regulons, and further demonstrates that although umuDAb has a specialized role in repressing error-prone polymerases, additional regulators likely participate in these species' transcriptional

  12. Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins.

    Science.gov (United States)

    Rangarajan, Savithri; Woodgate, Roger; Goodman, Myron F

    2002-02-01

    In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.

  13. Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein.

    Science.gov (United States)

    Sharadamma, N; Harshavardhana, Y; Singh, Pawan; Muniyappa, K

    2010-06-01

    A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination.

  14. Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis.

    OpenAIRE

    Korycka-Machala, M; Brzostek, A; Rozalska, S; Rumijowska-Galewicz, A; Dziedzic, R; Bowater, R; Dziadek, J

    2006-01-01

    Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the respon...

  15. Specificity in suppression of SOS expression by recA4162 and uvrD303.

    Science.gov (United States)

    Massoni, Shawn C; Sandler, Steven J

    2013-12-01

    Detection and repair of DNA damage is essential in all organisms and depends on the ability of proteins recognizing and processing specific DNA substrates. In E. coli, the RecA protein forms a filament on single-stranded DNA (ssDNA) produced by DNA damage and induces the SOS response. Previous work has shown that one type of recA mutation (e.g., recA4162 (I298V)) and one type of uvrD mutation (e.g., uvrD303 (D403A, D404A)) can differentially decrease SOS expression depending on the type of inducing treatments (UV damage versus RecA mutants that constitutively express SOS). Here it is tested using other SOS inducing conditions if there is a general feature of ssDNA generated during these treatments that allows recA4162 and uvrD303 to decrease SOS expression. The SOS inducing conditions tested include growing cells containing temperature-sensitive DNA replication mutations (dnaE486, dnaG2903, dnaN159, dnaZ2016 (at 37°C)), a del(polA)501 mutation and induction of Double-Strand Breaks (DSBs). uvrD303 could decrease SOS expression under all conditions, while recA4162 could decrease SOS expression under all conditions except in the polA strain or when DSBs occur. It is hypothesized that recA4162 suppresses SOS expression best when the ssDNA occurs at a gap and that uvrD303 is able to decrease SOS expression when the ssDNA is either at a gap or when it is generated at a DSB (but does so better at a gap). Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage

    International Nuclear Information System (INIS)

    Ennis, D.G.; Ossanna, N.; Mount, D.W.

    1989-01-01

    Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein

  17. Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium.

    Directory of Open Access Journals (Sweden)

    Solenne Ithurbide

    2015-10-01

    Full Text Available The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA+ as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA pathway, strongly reduces the frequency of RecA- (and RecO- independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA+ cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA+ bacteria exposed to ionizing radiation.

  18. Formation of base triplets by non-Watson-Crick bonds mediates homologous recognition in RecA recombination filaments.

    OpenAIRE

    Rao, B J; Radding, C M

    1994-01-01

    Whereas complementary strands of DNA recognize one another by forming Watson-Crick base pairs, the way in which RecA protein enables a single strand to recognize homology in duplex DNA has remained unknown. Recent experiments, however, have shown that a single plus strand in the RecA filament can recognize an identical plus strand via bonds that, by definition, are non-Watson-Crick. In experiments reported here, base substitutions had the same qualitative and quantitative effects on the pairi...

  19. The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

    Energy Technology Data Exchange (ETDEWEB)

    Galvão, C.W. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Souza, E.M. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Etto, R.M. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Schumacher, J.; Buck, M. [Department of Life Sciences, Imperial College London, London (United Kingdom); Steffens, M.B.R. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

    2012-10-15

    DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX{sub Hs}) can interact with the H. seropedicae RecA protein (RecA{sub Hs}) and that RecA{sub Hs} possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX{sub Hs} inhibited 90% of the RecA{sub Hs} DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA{sub Hs}. RecA{sub Hs} ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX{sub Hs} was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX{sub Hs} protein negatively modulates the RecA{sub Hs} activities by protein-protein interactions and also by DNA-protein interactions.

  20. Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

    Science.gov (United States)

    Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

    2016-01-01

    Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings

  1. Molecular Interaction and Cellular Location of RecA and CheW Proteins in Salmonella enterica during SOS Response and Their Implication in Swarming.

    Science.gov (United States)

    Irazoki, Oihane; Aranda, Jesús; Zimmermann, Timo; Campoy, Susana; Barbé, Jordi

    2016-01-01

    In addition to its role in DNA damage repair and recombination, the RecA protein, through its interaction with CheW, is involved in swarming motility, a form of flagella-dependent movement across surfaces. In order to better understand how SOS response modulates swarming, in this work the location of RecA and CheW proteins within the swarming cells has been studied by using super-resolution microscopy. Further, and after in silico docking studies, the specific RecA and CheW regions associated with the RecA-CheW interaction have also been confirmed by site-directed mutagenesis and immunoprecipitation techniques. Our results point out that the CheW distribution changes, from the cell poles to foci distributed in a helical pattern along the cell axis when SOS response is activated or RecA protein is overexpressed. In this situation, the CheW presents the same subcellular location as that of RecA, pointing out that the previously described RecA storage structures may be modulators of swarming motility. Data reported herein not only confirmed that the RecA-CheW pair is essential for swarming motility but it is directly involved in the CheW distribution change associated to SOS response activation. A model explaining not only the mechanism by which DNA damage modulates swarming but also how both the lack and the excess of RecA protein impair this motility is proposed.

  2. Molecular interaction and cellular location of RecA and CheW proteins in Salmonella enterica during SOS response and their implication in swarming

    Directory of Open Access Journals (Sweden)

    Oihane Irazoki

    2016-10-01

    Full Text Available In addition to its role in DNA damage repair and recombination, the RecA protein, through its interaction with CheW, is involved in swarming motility, a form of flagella-dependent movement across surfaces. In order to better understand how SOS response modulates swarming, in this work the location of RecA and CheW proteins within the swarming cells has been studied by using super-resolution microscopy. Further, and after in silico docking studies, the specific RecA and CheW regions associated with the RecA-CheW interaction have also been confirmed by site-directed mutagenesis and immunoprecipitation techniques. Our results point out that the CheW distribution changes, from the cell poles to foci distributed in a helical pattern along the cell axis when SOS response is activated or RecA protein is overexpressed. In this situation, the CheW presents the same subcellular location as that of RecA, pointing out that the previously described RecA storage structures may be modulators of swarming motility. Data reported herein not only confirmed that the RecA-CheW pair is essential for swarming motility but it is directly involved in the CheW distribution change associated to SOS response activation. A model explaining not only the mechanism by which DNA damage modulates swarming but also how both the lack and the excess of RecA protein impair this motility is proposed.

  3. Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament.

    Directory of Open Access Journals (Sweden)

    Wellington C Leite

    Full Text Available The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA. HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

  4. Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament.

    Science.gov (United States)

    Leite, Wellington C; Galvão, Carolina W; Saab, Sérgio C; Iulek, Jorge; Etto, Rafael M; Steffens, Maria B R; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L; Cox, Michael M

    2016-01-01

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

  5. A Structure-Function Study of RecA: The Structural Basis for ATP Specificity in the Strand Exchange Reaction

    Science.gov (United States)

    Gegner, Julie; Spruill, Natalie; Plesniak, Leigh A.

    1999-11-01

    The terms "structure" and "function" can assume a variety of meanings. In biochemistry, the "structure" of a protein can refer to its sequence of amino acids, the three-dimensional arrangement of atoms within a subunit, or the arrangement of subunits into a larger oligomeric or filamentous state. Likewise, the function of biological macromolecules can be examined at many levels. The function of a protein can be described by its role in an organism's survival or by a chemical reaction that it promotes. We have designed a three-part biochemical laboratory experiment that characterizes the structure and function of the Escherichia coli RecA protein. The first part examines the importance of RecA in the survival of bacteria that have been exposed to UV light. This is the broadest view of function of the enzyme. Second, the students use an in vitro assay of RecA whereby the protein promotes homologous recombination. Because RecA functions not catalytically, but rather stoichiometrically, in this recombination reaction, the oligomeric state of RecA in complex with DNA must also be discussed. Finally, through molecular modeling of X-ray crystallographic structures, students identify functionally important features of the ATP cofactor binding site of RecA.

  6. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

    Energy Technology Data Exchange (ETDEWEB)

    Leite, Wellington C.; Galvão, Carolina W.; Saab, Sérgio C.; Iulek, Jorge; Etto, Rafael M.; Steffens, Maria B.R.; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L.; Cox, Michael M. (UW); (UW-MED); (Ponta Grossa)

    2016-07-22

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. In conclusion, our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

  7. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

    Science.gov (United States)

    Galvão, Carolina W.; Saab, Sérgio C.; Iulek, Jorge; Etto, Rafael M.; Steffens, Maria B. R.; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L.; Cox, Michael M.

    2016-01-01

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament. PMID:27447485

  8. Complementation pattern of lexB and recA mutations in Escherichia coli K12; mapping of tif-1, lexB and recA mutations

    International Nuclear Information System (INIS)

    Morand, P.; Goze, A.; Devoret, R.

    1977-01-01

    Three lexB mutations, whose phenotypes have been previously characterized, are studied here in relation to a few recA mutations as to their complementation pattern and relative location. The restoration of resistance to UV-light and to X-rays in the hetero-allelic diploid bacteria was used as a test for dominance and complementation. The wild type allele was always dominant over the mutant allele. Only partial complementation was found between lexB and two rexA alleles. There was no complementation between the recA alleles. All the data taken together strongly suggest that the complementations found are intragenic: lexB and recA mutations are in one gene. Mapping of lexB, recA and tif-1 mutations in relation to srl-1 and cysC by phage P1 transduction shows that lexB and the tif-1 mutations form a cluster proximal to srl-1 whereas recA mutations are located at the other extremity of the gene. Variability with temperature of cotransduction frequencies as well as their extended range of values prevent a meaningful calculation of the length of the recA gene. Our hypothesis is that the recA protein has two functional regions called A and B respectively defined at the genetical level by recA and lexB mutations and that it is, in vivo, an oligomeric protein forming a complex with the lexA protein. This complex is postulated to be multifunctional: recombination and control of exonuclease V are effected by the A region while the B region and lexA protein effect induced DNA repair and lysogenic induction. (orig.) [de

  9. Bacillus halodurans RecA-DNA binding and RecAmediated ...

    African Journals Online (AJOL)

    Abstract. In Escherichia coli, RecA protein catalyzes DNA pairing and strand exchange activities essential for genetic recombination. This is critical for normal cellular function under conditions that lead to altered. DNA metabolism and DNA damage. The RecA proteins of E. coli and Bacillus halodurans both can bind to DNA ...

  10. Differential requirements of two recA mutants for constitutive SOS expression in Escherichia coli K-12.

    Directory of Open Access Journals (Sweden)

    Jarukit Edward Long

    Full Text Available Repairing DNA damage begins with its detection and is often followed by elicitation of a cellular response. In E. coli, RecA polymerizes on ssDNA produced after DNA damage and induces the SOS Response. The RecA-DNA filament is an allosteric effector of LexA auto-proteolysis. LexA is the repressor of the SOS Response. Not all RecA-DNA filaments, however, lead to an SOS Response. Certain recA mutants express the SOS Response (recA(C in the absence of external DNA damage in log phase cells.Genetic analysis of two recA(C mutants was used to determine the mechanism of constitutive SOS (SOS(C expression in a population of log phase cells using fluorescence of single cells carrying an SOS reporter system (sulAp-gfp. SOS(C expression in recA4142 mutants was dependent on its initial level of transcription, recBCD, recFOR, recX, dinI, xthA and the type of medium in which the cells were grown. SOS(C expression in recA730 mutants was affected by none of the mutations or conditions tested above.It is concluded that not all recA(C alleles cause SOS(C expression by the same mechanism. It is hypothesized that RecA4142 is loaded on to a double-strand end of DNA and that the RecA filament is stabilized by the presence of DinI and destabilized by RecX. RecFOR regulate the activity of RecX to destabilize the RecA filament. RecA730 causes SOS(C expression by binding to ssDNA in a mechanism yet to be determined.

  11. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    DEFF Research Database (Denmark)

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan

    2013-01-01

    V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) ar......V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA......-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA...... when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA...

  12. Unveiling novel RecO distant orthologues involved in homologous recombination.

    Directory of Open Access Journals (Sweden)

    Stéphanie Marsin

    2008-08-01

    Full Text Available The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.

  13. Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination

    International Nuclear Information System (INIS)

    Madiraju, M.V.; Templin, A.; Clark, A.J.

    1988-01-01

    A mutation partially suppressing the UV sensitivity caused by recF143 in a uvrA6 background was located at codon 37 of recA where GTG (valine) became ATG (methionine). This mutation, originally named srf-803, was renamed recA803. Little if any suppression of the recF143 defect in UV induction of a lexA regulon promoter was detected. This led to the hypothesis that a defect in recombination repair of UV damage was suppressed by recA803. The mutant RecA protein (RecA803) was purified and compared with wild-type protein (RecA+) as a catalyst of formation of joint molecules. Under suboptimal conditions, RecA803 produces both a higher rate of formation and a higher yield of joint molecules. The suboptimal conditions tested included addition of single-stranded DNA binding protein to single-stranded DNA prior to addition of RecA. We hypothesize that the ability of RecA803 to overcome interference by single-stranded DNA binding protein is the property that allows recA803 to suppress partially the deficiency in repair caused by recF mutations in the uvrA6 background. Implications of this hypothesis for the function of RecF protein in recombination are discussed

  14. SOS response in bacteria: Inhibitory activity of lichen secondary metabolites against Escherichia coli RecA protein.

    Science.gov (United States)

    Bellio, Pierangelo; Di Pietro, Letizia; Mancini, Alisia; Piovano, Marisa; Nicoletti, Marcello; Brisdelli, Fabrizia; Tondi, Donatella; Cendron, Laura; Franceschini, Nicola; Amicosante, Gianfranco; Perilli, Mariagrazia; Celenza, Giuseppe

    2017-06-15

    RecA is a bacterial multifunctional protein essential to genetic recombination, error-prone replicative bypass of DNA damages and regulation of SOS response. The activation of bacterial SOS response is directly related to the development of intrinsic and/or acquired resistance to antimicrobials. Although recent studies directed towards RecA inactivation via ATP binding inhibition described a variety of micromolar affinity ligands, inhibitors of the DNA binding site are still unknown. Twenty-seven secondary metabolites classified as anthraquinones, depsides, depsidones, dibenzofurans, diphenyl-butenolides, paraconic acids, pseudo-depsidones, triterpenes and xanthones, were investigated for their ability to inhibit RecA from Escherichia coli. They were isolated in various Chilean regions from 14 families and 19 genera of lichens. The ATP hydrolytic activity of RecA was quantified detecting the generation of free phosphate in solution. The percentage of inhibition was calculated fixing at 100µM the concentration of the compounds. Deeper investigations were reserved to those compounds showing an inhibition higher than 80%. To clarify the mechanism of inhibition, the semi-log plot of the percentage of inhibition vs. ATP and vs. ssDNA, was evaluated. Only nine compounds showed a percentage of RecA inhibition higher than 80% (divaricatic, perlatolic, alpha-collatolic, lobaric, lichesterinic, protolichesterinic, epiphorellic acids, sphaerophorin and tumidulin). The half-inhibitory concentrations (IC 50 ) calculated for these compounds were ranging from 14.2µM for protolichesterinic acid to 42.6µM for sphaerophorin. Investigations on the mechanism of inhibition showed that all compounds behaved as uncompetitive inhibitors for ATP binding site, with the exception of epiphorellic acid which clearly acted as non-competitive inhibitor of the ATP site. Further investigations demonstrated that epiphorellic acid competitively binds the ssDNA binding site. Kinetic data were

  15. Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange

    DEFF Research Database (Denmark)

    Register, JC; Christiansen, Gunna; Griffith, J

    1987-01-01

    examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally...

  16. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1

    Energy Technology Data Exchange (ETDEWEB)

    Gruenig, Marielle C.; Lu, Duo; Won, Sang Joon; Dulberger, Charles L.; Manlick, Angela J.; Keck, James L.; Cox, Michael M. (UW)

    2012-03-16

    The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme; that is, a RecA-dependent nuclease. Ref binds to ss- and dsDNA but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double-strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 {angstrom} resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded {beta}-hairpin that is sandwiched between several {alpha}-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.

  17. SOS-like induction in Bacillus subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains

    International Nuclear Information System (INIS)

    Lovett, C.M. Jr.; Love, P.E.; Yasbin, R.E.; Roberts, J.W.

    1988-01-01

    We quantitated the induction of the Bacillus subtilis Rec protein (the analog of Escherichia coli RecA protein) and the B. subtilis din-22 operon (representative of a set of DNA damage-inducible operons in B. subtilis) following DNA damage in Rec+ and DNA repair-deficient strains. After exposure to mitomycin C or UV irradiation, each of four distinct rec (recA1, recB2, recE4, and recM13) mutations reduced to the same extent the rates of both Rec protein induction (determined by densitometric scanning of immunoblot transfers) and din-22 operon induction (determined by assaying beta-galactosidase activity in din-22::Tn917-lacZ fusion strains). The induction deficiencies in recA1 and recE4 strains were partially complemented by the E. coli RecA protein, which was expressed on a plasmid in B. subtilis; the E. coli RecA protein had no effect on either induction event in Rec+, recB2, or recM13 strains. These results suggest that (i) the expression of both the B. subtilis Rec protein and the din-22 operon share a common regulatory component, (ii) the recA1 and recE4 mutations affect the regulation and/or activity of the B. subtilis Rec protein, and (iii) an SOS regulatory system like the E. coli system is highly conserved in B. subtilis. We also showed that the basal level of B. subtilis Rec protein is about 4,500 molecules per cell and that maximum induction by DNA damage causes an approximately fivefold increase in the rate of Rec protein accumulation

  18. Molecular cloning of the recA analog from the marine fish pathogen Vibrio anguillarum 775

    International Nuclear Information System (INIS)

    Singer, J.T.

    1989-01-01

    The recA analog from Vibrio anguillarum 775 was isolated by complementation of recA mutations in Escherichia coli, and its protein product was identified. The recA analog promoted recombination between two partially deleted lactose operons, stimulated both spontaneous and mitomycin C-induced phage production in RecA- lambda lysogens, and restored near wild-type levels of resistance to UV radiation and methyl methanesulfonate

  19. Mutations at the cysteine codons of the recA gene of Escherichia coli

    International Nuclear Information System (INIS)

    Weisemann, J.M.; Weinstock, G.M.

    1988-01-01

    Each of the three cysteine residues in the Escherichia coli RecA protein was replaced with a number of other amino acids. To do this, each cysteine codon was first converted to a chain-terminating amber codon by oligonucleotide-directed mutagenesis. These amber mutants were then either assayed for function in different suppressor strains or reverted by a second round of mutagenesis with oligonucleotides that had random sequences at the amber codon. Thirty-three different amino acid substitutions were obtained. Mutants were tested for three functions of RecA: survival following UV irradiation, homologous recombination, and induction of the SOS response. It was found that although none of the cysteines is essential for activity, mutations at each of these positions can affect one or more of the activities of RecA, depending on the particular amino acid substitution. In addition, the cysteine at position 116 appears to be involved in the RecA-promoted cleavage of the LexA protein

  20. Recombinational repair: workshop summary

    International Nuclear Information System (INIS)

    Howard-Flanders, P.

    1983-01-01

    Recombinational repair may or may not be synonymous with postreplication repair. Considerable progress has been made in the study of the relevant enzymes, particularly those from bacteria. In this workshop we focus on the recombination enzyme RecA protein. What structural changes take place in the protein and in DNA during repair. How does homologous pairing take place. How is ATP hydrolysis coupled to the stand exchange reaction and the formation of heteroduplx DNA. Turning to another enzyme needed for certain kinds of bacterial recombination, we will ask whether the purified recB protein and recC protein complement each other and are sufficient for exonuclease V activity. In higher cells, we would like to know whether sister exchanges, which occur in bacteria after uv irradiation, are also seen in animal cells

  1. Molecular cloning and characterization of the recA gene of Pseudomonas aeruginosa PAO

    Energy Technology Data Exchange (ETDEWEB)

    Kokjohn, T.A.; Miller, R.V.

    1985-08-01

    The recA gene of Pseudomonas aeruginosa PAO has been isolated and introduced into Escherichia coli K-12. Resistance to killing by UV irradiation was restored in several RecA-E. coli K-12 hosts by the P. aeruginosa gene, as was resistance to methyl methanesulfonate. Recombination proficiency was also restored, as measured by HfrH-mediated conjugation and by the ability to propagate Fec-phage lambda derivatives. The cloned P. aeruginosa recA gene restored both spontaneous and mitomycin C-stimulated induction of lambda prophage in lysogens of a recA strain of E. coli K-12.

  2. RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

    Directory of Open Access Journals (Sweden)

    Masaki Odahara

    2015-03-01

    Full Text Available Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

  3. Single Molecule Study of DNA Organization and Recombination

    Science.gov (United States)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  4. Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

    Science.gov (United States)

    Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

    2015-09-01

    Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence

  5. Comparative evolution of the recA gene of surface and deep subsurface microorganisms (an evolutionary clock of intermediate rate). Final report

    Energy Technology Data Exchange (ETDEWEB)

    Miller, R.V.

    1998-04-01

    Because of the ability of the recA protein product to maintain both DNA integrity and increase genetic diversity, this gene may be essential to the survival of microorganisms following the damaging effects of numerous environmental stresses such as exposure to solar UV radiation, exposure to gamma radiation, starvation, and changing environments. While the various activities and amino-acid sequence of recA have been highly conserved among the eubacteria and archaea, little is known as to whether a strict structure-function relationship has been conserved. In other words, are the same regions of this highly plastic, functionally heterogeneous protein involved in the same catalytic capacities throughout the bacterial kingdom? While it is reasonable to assume that this type of conservation has also occurred, we felt it necessary to test the assumption by demonstrating that mutations in different genera of bacteria which eliminate similar functions (i.e., lead to similar phenotypes) are caused by changes in the amino-acid sequence in the same regions of their recA proteins. Therefore, we located the changes in nucleotide sequence in two recA mutants of P. aeruginosa which displayed mutant phenotypes in recombination and UV resistance. Our assumption was that if structure-function relationships held, these mutations would be found in areas already identified as essential for the function of the E. coli recA protein.

  6. RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms.

    Science.gov (United States)

    Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael; Korolev, Sergey

    2014-10-17

    Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Effects of radiations on DNA and repair of the damage. Progress report, March 1, 1975--March 31, 1976

    International Nuclear Information System (INIS)

    Hutchinson, F.

    1976-01-01

    It was established that repair of radioinduced double-strand breaks in the DNA of E. coli AB2497 takes place. This repair can be eliminated by growing the cells in poor media so there is only 1+ genome/cell. There is no measurable repair in AB2487 recA - (otherwise isogenic with AB2497) or NH4803 recA - recB - cells. These results strongly suggest that DNA double-strand break repair occurs by a process involving recombination of the broken pieces with a homologous double hexix

  8. Expression of Mycobacterium tuberculosis Ku and Ligase D in Escherichia coli results in RecA and RecB-independent DNA end-joining at regions of microhomology.

    Science.gov (United States)

    Malyarchuk, Svitlana; Wright, Douglas; Castore, Reneau; Klepper, Emily; Weiss, Bernard; Doherty, Aidan J; Harrison, Lynn

    2007-10-01

    Unlike Escherichia coli, Mycobacterium tuberculosis (Mt) expresses a Ku-like protein and an ATP-dependent DNA ligase that can perform non-homologous end-joining (NHEJ). We have expressed the Mt-Ku and Mt-Ligase D in E. coli using an arabinose-inducible promoter and expression vectors that integrate into specific sites in the E. coli chromosome. E. coli strains have been generated that express the Mt-Ku and Mt-Ligase D on a genetic background that is wild-type for repair, or deficient in either the RecA or RecB protein. Transformation of these strains with linearized plasmid DNA containing a 2bp overhang has demonstrated that expression of both the Mt-Ku and Mt-Ligase D is required for DNA end-joining and that loss of RecA does not prevent this double-strand break repair. Analysis of the re-joined plasmid has shown that repair is predominantly inaccurate and results in the deletion of sequences. Loss of RecB did not prevent the formation of large deletions, but did increase the amount of end-joining. Sequencing the junctions has revealed that the majority of the ligations occurred at regions of microhomology (1-4bps), eliminating one copy of the homologous sequence at the junction. The Mt-Ku and Mt-Ligase D can therefore function in E. coli to re-circularize linear plasmid.

  9. A multicopy phr-plasmid increases the ultraviolet resistance of a recA strain of Escherichia coli

    International Nuclear Information System (INIS)

    Yamamoto, K.; Satake, M.; Shinagawa, H.

    1984-01-01

    It has been previously reported that the ultraviolet sensitivity of recA strains of Escherichia coli in the dark is suppressed by a plasmid pKY1 which carries the phr gene, suggesting that this is due to a novel effect of photoreactivating enzyme (PRE) of E. coli in the dark. In this work, it is observed that an increase of UV-resistance by pKY1 in the dark is not apparent in strains with a mutation in either uvrA, uvrB, uvrC, lexA, recBC or recF. The sensitivity of recA lexA and recA recBC multiple mutants to UV is suppressed by the plasmid but that of recA uvrA, recA uvrB and recA uvrC is not. Host-cell reactivation of UV-irradiated lambda phage is slightly more efficient in the recA/pKY1 strain compared with the parental recA strain. On the other hand, the recA and recA/pKY1 strains do not differ significantly in the following properties: Hfr recombination, induction of lambda by UV, and mutagenesis. It is suggested that dark repair of PRE is correlated with its capacity of excision repair. (Auth.)

  10. Tuberculosis vaccine strain Mycobacterium bovis BCG Russia is a natural recA mutant

    Directory of Open Access Journals (Sweden)

    Böttger Erik C

    2008-07-01

    Full Text Available Abstract Background The current tuberculosis vaccine is a live vaccine derived from Mycobacterium bovis and attenuated by serial in vitro passaging. All vaccine substrains in use stem from one source, strain Bacille Calmette-Guérin. However, they differ in regions of genomic deletions, antigen expression levels, immunogenicity, and protective efficacy. Results As a RecA phenotype increases genetic stability and may contribute restricting the ongoing evolution of the various BCG substrains while maintaining their protective efficacy, we aimed to inactivate recA by allelic replacement in BCG vaccine strains representing different phylogenetic lineages (Pasteur, Frappier, Denmark, Russia. Homologous gene replacement was achieved successfully in three out of four strains. However, only illegitimate recombination was observed in BCG substrain Russia. Sequence analyses of recA revealed that a single nucleotide insertion in the 5' part of recA led to a translational frameshift with an early stop codon making BCG Russia a natural recA mutant. At the protein level BCG Russia failed to express RecA. Conclusion According to phylogenetic analyses BCG Russia is an ancient vaccine strain most closely related to the parental M. bovis. We hypothesize that recA inactivation in BCG Russia occurred early and is in part responsible for its high degree of genomic stability, resulting in a substrain that has less genetic alterations than other vaccine substrains with respect to M. bovis AF2122/97 wild-type.

  11. Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

    Science.gov (United States)

    Loutre, Romuald; Heckel, Anne-Marie; Jeandard, Damien; Tarassov, Ivan; Entelis, Nina

    2018-01-01

    Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion

  12. Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication.

    Science.gov (United States)

    Steigemann, Birthe; Schulz, Annina; Werten, Sebastiaan

    2013-11-15

    The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors. © 2013.

  13. Functions and structures of eukaryotic recombination proteins

    International Nuclear Information System (INIS)

    Ogawa, Tomoko

    1994-01-01

    We have found that Rad51 and RecA Proteins form strikingly similar structures together with dsDNA and ATP. Their right handed helical nucleoprotein filaments extend the B-form DNA double helixes to 1.5 times in length and wind the helix. The similarity and uniqueness of their structures must reflect functional homologies between these proteins. Therefore, it is highly probable that similar recombination proteins are present in various organisms of different evolutional states. We have succeeded to clone RAD51 genes from human, mouse, chicken and fission yeast genes, and found that the homologues are widely distributed in eukaryotes. The HsRad51 and MmRad51 or ChRad51 proteins consist of 339 amino acids differing only by 4 or 12 amino acids, respectively, and highly homologous to both yeast proteins, but less so to Dmcl. All of these proteins are homologous to the region from residues 33 to 240 of RecA which was named ''homologous core. The homologous core is likely to be responsible for functions common for all of them, such as the formation of helical nucleoprotein filament that is considered to be involved in homologous pairing in the recombination reaction. The mouse gene is transcribed at a high level in thymus, spleen, testis, and ovary, at lower level in brain and at a further lower level in some other tissues. It is transcribed efficiently in recombination active tissues. A clear functional difference of Rad51 homologues from RecA was suggested by the failure of heterologous genes to complement the deficiency of Scrad51 mutants. This failure seems to reflect the absence of a compatible partner, such as ScRad52 protein in the case of ScRad51 protein, between different species. Thus, these discoveries play a role of the starting point to understand the fundamental gene targeting in mammalian cells and in gene therapy. (J.P.N.)

  14. Genetic relatedness and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe.

    Science.gov (United States)

    Kuzmanović, N; Biondi, E; Bertaccini, A; Obradović, A

    2015-09-01

    To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis. © 2015 The Society for Applied Microbiology.

  15. Site-specific DNA Inversion by Serine Recombinases

    Science.gov (United States)

    2015-01-01

    Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides. Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then discussed in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system, the assembly of the active recombination complex (invertasome) containing the Fis/enhancer, and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is emphasized. PMID:25844275

  16. Regulated expression of the dinR and recA genes during competence development and SOS induction in Bacillus subtilis

    NARCIS (Netherlands)

    Haijema, BJ; vanSinderen, D; Winterling, K; Kooistra, J; Venema, G; Hamoen, LW

    1996-01-01

    It has been hypothesized that the dinR gene product of Bacillus subtilis acts as a repressor of the SOS regulon by binding to DNA sequences located upstream of SOS genes, including dinR and recA. Following activation as a result of DNA damage, RecA is believed to catalyse DinR-autocleavage, thus

  17. Regulation of Recombination between gtfB/gtfC Genes in Streptococcus mutans by Recombinase A

    Directory of Open Access Journals (Sweden)

    Satoko Inagaki

    2013-01-01

    Full Text Available Streptococcus mutans produces 3 types of glucosyltransferases (GTFs, whose cooperative action is essential for cellular adhesion. The recombinase A (RecA protein is required for homologous recombination. In our previous study, we isolated several strains with a smooth colony morphology and low GTF activity, characteristics speculated to be derived from the GTF fusions. The purpose of the present study was to investigate the mechanism of those fusions. S. mutans strain MT8148 was grown in the presence of recombinant RecA (rRecA protein, after which smooth colonies were isolated. The biological functions and sequences of the gtfB and gtfC genes of this as well as other clinical strains were determined. The sucrose-dependent adherence rates of those strains were reduced as compared to that of MT8148. Determination of the sequences of the gtfB and gtfC genes showed that an approximately 3500 bp region was deleted from the area between them. Furthermore, expression of the recA gene was elevated in those strains as compared to MT8148. These results suggest that RecA has an important role in fusions of gtfB and gtfC genes, leading to alteration of colony morphology and reduction in sucrose-dependent adhesion.

  18. In vitro recombination of bacteriophage T7 DNA damaged by uv radiation

    International Nuclear Information System (INIS)

    Masker, W.E.; Kuemmerle, N.B.

    1980-01-01

    A system capable of in vitro packaging of exogenous bacteriophage T7 DNA has been used to monitor the biological activity of DNA replicated in vitro. This system has been used to follow the effects of uv radiation on in vitro replication and recombination. During the in vitro replication process, a considerable exchange of genetic information occurs between T7 DNA molecules present in the reaction mixture. This in vitro recombination is reflected in the genotype of the T7 phage produced after in vitro encapsulation; depending on the genetic markers selected, recombinants can comprise nearly 20% of the total phage production. When uv-irradiated DNA is incubated in this system, the amount of in vitro synthesis is reduced and the total amount of viable phage produced after in vitro packaging is diminished. In vitro recombination rates are also lower when the participating DNA molecules have been exposed to uv. However, biochemical and genetic measurements confirmed that there is little or no transfer of pyrimidine dimers from irradiated DNA into undamaged molecules

  19. Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

    International Nuclear Information System (INIS)

    Lin, Y.H.; Keil, R.L.

    1991-01-01

    HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

  20. Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Lynn C. Thomason

    2016-09-01

    Full Text Available Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion.

  1. Autogenous regulation and kinetics of induction of Pseudomonas aeruginosa recA transcription as analyzed with operon fusions

    International Nuclear Information System (INIS)

    Horn, J.M.; Ohman, D.E.

    1988-01-01

    A promoterless chloramphenicol acetyltransferase gene (cat) was used to construct recA-cat operon fusions to quantitatively examine the transcriptional regulation of the Pseudomonas aeruginosa recA gene in P. aeruginosa PAO. Wild-type P. aeruginosa containing the recA8-cat fusion was treated with methyl methanesulfonate (MMS) and showed immediate induction of chloramphenicol acetyltransferase (CAT) specific activity, whereas a recA::Tn501 mutant of P. aeruginosa containing recA8-cat showed no induction with MMS. This indicated that a functional copy of recA was required for derepression of recA transcription and that P. aeruginosa recA protein was a positive regulatory factor promoting its own expression. Compared with that in the wild type, the uninduced level of CAT in recA8-cat-containing cells was reduced by approximately one-half in the recA::Tn501 mutant, indicating that recA+-dependent spontaneous induction contributes to the uninduced levels of recA expression in P. aeruginosa. MMS (0.012%) caused recA-directed CAT synthesis to increase almost immediately, with maximum CAT activity, fourfold higher than uninduced levels, attained at 60 min postinduction. The kinetics of recA8-cat fusion activity were shown to be directly related to the MMS doses used. Another fusion called recAa1-cat, where cat was located between the two transcriptional terminators of the P. aeruginosa recA gene, also showed dose-dependent induction by MMS, but the CAT activity from recAa1-cat was only one-half of that obtained with recA8-cat under the same conditions. Treatment of recA+ P. aeruginosa containing recA8-cat with UV irradiation produced an immediate effect on recA8-cat transcription and showed little UV dose dependency at doses of 5 J/m2 or greater

  2. Role of allosteric switch residue histidine 195 in maintaining active-site asymmetry in presynaptic filaments of bacteriophage T4 UvsX recombinase.

    Science.gov (United States)

    Farb, Joshua N; Morrical, Scott W

    2009-01-16

    Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

  3. Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

    Directory of Open Access Journals (Sweden)

    Nicole A. Najor

    2016-12-01

    Full Text Available In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control.

  4. Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA

    International Nuclear Information System (INIS)

    Paramio, J.M.; Bauluz, C.; Vidania, R. de

    1986-01-01

    Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs

  5. FBH1 helicase disrupts RAD51 filaments in vitro and modulates homologous recombination in mammalian cells

    DEFF Research Database (Denmark)

    Simandlova, Jitka; Zagelbaum, Jennifer; Payne, Miranda J

    2013-01-01

    Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD......51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences...... filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under...

  6. Single-Molecule Tethered Particle Motion: Stepwise Analyses of Site-Specific DNA Recombination

    Directory of Open Access Journals (Sweden)

    Hsiu-Fang Fan

    2018-05-01

    Full Text Available Tethered particle motion/microscopy (TPM is a biophysical tool used to analyze changes in the effective length of a polymer, tethered at one end, under changing conditions. The tether length is measured indirectly by recording the Brownian motion amplitude of a bead attached to the other end. In the biological realm, DNA, whose interactions with proteins are often accompanied by apparent or real changes in length, has almost exclusively been the subject of TPM studies. TPM has been employed to study DNA bending, looping and wrapping, DNA compaction, high-order DNA–protein assembly, and protein translocation along DNA. Our TPM analyses have focused on tyrosine and serine site-specific recombinases. Their pre-chemical interactions with DNA cause reversible changes in DNA length, detectable by TPM. The chemical steps of recombination, depending on the substrate and the type of recombinase, may result in a permanent length change. Single molecule TPM time traces provide thermodynamic and kinetic information on each step of the recombination pathway. They reveal how mechanistically related recombinases may differ in their early commitment to recombination, reversibility of individual steps, and in the rate-limiting step of the reaction. They shed light on the pre-chemical roles of catalytic residues, and on the mechanisms by which accessory proteins regulate recombination directionality.

  7. Escherichia coli ArgR mutants defective in cer/Xer recombination, but not in DNA binding.

    Science.gov (United States)

    Sénéchal, Hélène; Delesques, Jérémy; Szatmari, George

    2010-04-01

    The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls the expression of the arginine biosynthetic genes and is required as an accessory factor for Xer site-specific recombination at cer and related recombination sites in plasmids. We used the technique of pentapeptide scanning mutagenesis to isolate a series of ArgR mutants that were considerably reduced in cer recombination, but were still able to repress an argA::lacZ fusion. DNA sequence analysis showed that all of the mutants mapped to the same nucleotide, resulting in a five amino acid insertion between residues 149 and 150 of ArgR, corresponding to the end of the alpha6 helix. A truncated ArgR containing a stop codon at residue 150 displayed the same phenotype as the protein with the five amino acid insertion, and both mutants displayed sequence-specific DNA-binding activity that was L-arginine dependent. These results show that the C-terminus of ArgR is more important in cer/Xer site-specific recombination than in DNA binding.

  8. Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA

    International Nuclear Information System (INIS)

    Smider, V.; Rathmell, W.K.; Chu, G.; Lieber, M.R.

    1994-01-01

    Three genetic complementation groups of rodent cells are defective for both repair of x-ray-induced double-strand breaks and V(D)J recombination. Cells from one group lack a DNA end-binding activity that is biochemically and antigenically similar to the Ku autoantigen. Transfection of complementary DNA (cDNA) that encoded the 86-kilodalton subunit of Ku rescued these mutant cells for DNA end-binding activity, x-ray resistance, and V(D)J recombination activity. These results establish a role for Ku in DNA repair and recombination. Furthermore, as a component of a DNA-dependent protein kinase, Ku may initiate a signaling pathway induced by DNA damage

  9. Genetic control of near-UV (300-400 nm) sensitivity independent of the recA gene in strains of Escherichia coli K12

    International Nuclear Information System (INIS)

    Tuveson, R.W.; Jonas, R.B.

    1979-01-01

    Stationary cells of isogenic pairs of Escherichia coli K12 strains presumably differing only in the recA function, were inactivated with near-UV (300-400 nm) radiation. Based on near-UV inactivation kinetics, the strains can be divided into two discrete categories in which near-UV sensitivity does not necessarily correlate with far-UV sensitivity conferred by two different recA alleles. Lack of overlap between near-UV and far-UV (recA) sensitivity can be explained by assuming that a different chromosomal gene (nur) controls near-UV sensitivity. Support for this hypothesis came from a mating experiment in which four selected recombinants, isogenic with respect to auxotrophic markers, were identified exhibiting all four possible combinations of far-UV (recA1 vs recA + ) and near-UV sensitivity (nur vs nur + ). Transduction with phase P1 showed that introduction of the recA1 allele into a recA + recipient did not affect the near-UV sensitivity of the recipient. Additional matings together with transduction experiments suggested that the nur gene is located at a position on the E. coli linkage map clearly separable from recA (minute 58). (author)

  10. Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

    Science.gov (United States)

    Thomason, Lynn C; Costantino, Nina; Court, Donald L

    2016-09-13

    Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single

  11. DNA Sequence-Mediated, Evolutionarily Rapid Redistribution of Meiotic Recombination Hotspots

    Science.gov (United States)

    Wahls, Wayne P.; Davidson, Mari K.

    2011-01-01

    Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor. PMID:22084420

  12. RPA homologs and ssDNA processing during meiotic recombination.

    Science.gov (United States)

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

  13. Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry.

    Science.gov (United States)

    Zhao, Weixing; Vaithiyalingam, Sivaraja; San Filippo, Joseph; Maranon, David G; Jimenez-Sainz, Judit; Fontenay, Gerald V; Kwon, Youngho; Leung, Stanley G; Lu, Lucy; Jensen, Ryan B; Chazin, Walter J; Wiese, Claudia; Sung, Patrick

    2015-07-16

    The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI).

    Science.gov (United States)

    Fukuda, Tomoyuki; Ohya, Yoshikazu

    2006-02-01

    During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.

  15. Induction of the SOS response in ultraviolet-irradiated Escherichia coli analyzed by dynamics of LexA, RecA and SulA proteins

    International Nuclear Information System (INIS)

    Aksenov, S.V.

    1999-01-01

    The SOS response in Escherichia coli is induced after DNA-damaging treatments including ultraviolet light. Regulation of the SOS response is accomplished through specific interaction of the two SOS regulator proteins, LexA and RecA. In ultraviolet light treated cells nucleotide excision repair is the major system that removes the induced lesions from the DNA. Here, induction of the SOS response in Escherichia coli with normal and impaired excision repair function is studied by simulation of intracellular levels of regulatory LexA and RecA proteins, and SulA protein. SulA protein is responsible for SOS-inducible cell division inhibition. Results of the simulations show that nucleotide excision repair influences time-courses of LexA , RecA and SulA induction by modulating the dynamics of RecA protein distribution between its normal and SOS-activated forms

  16. Bacterial natural transformation by highly fragmented and damaged DNA

    DEFF Research Database (Denmark)

    Overballe-Petersen, Søren; Harms, Klaus; Orlando, Ludovic Antoine Alexandre

    2013-01-01

    for microbes, but not as potential substrate for bacterial evolution. Here, we show that fragmented DNA molecules (≥20 bp) that additionally may contain abasic sites, cross-links, or miscoding lesions are acquired by the environmental bacterium Acinetobacter baylyi through natural transformation. With uptake......DNA molecules are continuously released through decomposition of organic matter and are ubiquitous in most environments. Such DNA becomes fragmented and damaged (often DNA is recognized as nutrient source...... of DNA from a 43,000-y-old woolly mammoth bone, we further demonstrate that such natural transformation events include ancient DNA molecules. We find that the DNA recombination is RecA recombinase independent and is directly linked to DNA replication. We show that the adjacent nucleotide variations...

  17. Relationship among the repair mechanisms and the genetic recombination

    International Nuclear Information System (INIS)

    Alcantara D, D.

    1987-12-01

    In accordance with the previous reports of the Project BZ87 of the Department of Radiobiology, a dependent stimulation of the system exists in E.coli SOS, of the recombination of the bacteriophage Lambda whose genetic material has not been damaged. This stimulation is not due to the increase of the cellular concentration of the protein RecA and the mechanism but probable for which we find that it is carried out, it is through a cooperation among the product of the gene rec N of E. coli and the system Net of recombination of Lambda. The gene recN belongs to the group of genes SOS and its expression is induced when damaging the bacterial DNA where it intervenes in the repair of breaks of the double helix of the molecule (Picksley et, 1984). If the repair of breaks of this type is a factor that limits the speed with which it happens the recombination among viral chromosomes, then the biggest readiness in the protein RecN, due to the induction of the functions SOS, would facilitate the repair of such ruptures. In this new project it is to enlarge the knowledge about this phenomenon, it was, on one hand of corroborating in a way but he/she specifies the relationship between the recombinogenic response of Lambda and the System SOS of E. coli and for the other one to determine the effect that has the inhibition of the duplication of the DNA on the stimulation of the viral recombination. Everything it with the idea of making it but evident and to be able to use it as a system of genotoxic agents detection in E. coli. (Author)

  18. Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri.

    Science.gov (United States)

    Van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S; Britton, Robert A

    2012-01-01

    Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution.

  19. Molecular genetic and biochemical analyses of a DNA repair gene from Serratia marcescens

    International Nuclear Information System (INIS)

    Murphy, K.E.

    1989-01-01

    In Escherichia coli, the SOS response and two 3-methyladenine DNA glycosylases (TagI and TagII) are required for repair of DNA damaged by alkylating agents such as methyl methanesulfonate (MMS). Mutations of the recA gene eliminate the SOS response. TagI and TagII are encoded by the tag and alkA genes, respectively. A gene (rpr) encoding 3-methyladenine DNA glycosylase activity was isolated from the Gram-negative bacterium Serratia marcescens. The gene, localized to a 1.5-kilobase pair SmaI-HindIII restriction fragment, was cloned into plasmid pUC18. The clone complemented E. coli tag alkA and recA mutations for MMS resistance. The rpr gene did not, however, complement recA mutations for resistance to ultraviolet light or the ability to perform homologous recombination reactions, nor did it complement E. coli ada or alkB mutations. Two proteins of molecular weights 42,000 and 16,000 were produced from the rpr locus. Analysis of deletion and insertion mutants of rpr suggested that the 42kD molecule is the active protein. The 16kD protein may either be a breakdown product of the 42kD species or may be encoded by another gene overlapping the reading frame of the rpr gene. Biochemical assays showed that the rpr gene product (Rpr) possesses 3-methyladenine DNA glycosylase activity

  20. Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA.

    Science.gov (United States)

    Mori, Tetsuya; Saveliev, Sergei V; Xu, Yao; Stafford, Walter F; Cox, Michael M; Inman, Ross B; Johnson, Carl H

    2002-12-24

    KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecADnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecADnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.

  1. Somatic DNA recombination yielding circular DNA and deletion of a genomic region in embryonic brain

    International Nuclear Information System (INIS)

    Maeda, Toyoki; Chijiiwa, Yoshiharu; Tsuji, Hideo; Sakoda, Saburo; Tani, Kenzaburo; Suzuki, Tomokazu

    2004-01-01

    In this study, a mouse genomic region is identified that undergoes DNA rearrangement and yields circular DNA in brain during embryogenesis. External region-directed inverse polymerase chain reaction on circular DNA extracted from late embryonic brain tissue repeatedly detected DNA of this region containing recombination joints. Wide-range genomic PCR and digestion-circularization PCR analysis showed this region underwent recombination accompanied with deletion of intervening sequences, including the circularized regions. This region was mapped by fluorescence in situ hybridization to C1 on mouse chromosome 16, where no gene and no physiological DNA rearrangement had been identified. DNA sequence in the region has segmental homology to an orthologous region on human chromosome 3q.13. These observations demonstrated somatic DNA recombination yielding genomic deletions in brain during embryogenesis

  2. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    International Nuclear Information System (INIS)

    Golubnitchaya-Labudova, O.; Hoefer, M.; Portele, A.; Vacata, V.; Rink, H.; Lubec, G.

    1997-01-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer the question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the inserts of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9. (author)

  3. Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Hoang, Margaret L.; Tan, Frederick J.; Lai, David C.; Celniker, Sue E.; Hoskins, Roger A.; Dunham, Maitreya J.; Zheng, Yixian; Koshland, Douglas

    2010-08-27

    Genome rearrangements often result from non-allelic homologous recombination (NAHR) between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs) induce NAHR-dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR) occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR-dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer.

  4. Competitive repair by naturally dispersed repetitive DNA during non-allelic homologous recombination.

    Directory of Open Access Journals (Sweden)

    Margaret L Hoang

    2010-12-01

    Full Text Available Genome rearrangements often result from non-allelic homologous recombination (NAHR between repetitive DNA elements dispersed throughout the genome. Here we systematically analyze NAHR between Ty retrotransposons using a genome-wide approach that exploits unique features of Saccharomyces cerevisiae purebred and Saccharomyces cerevisiae/Saccharomyces bayanus hybrid diploids. We find that DNA double-strand breaks (DSBs induce NAHR-dependent rearrangements using Ty elements located 12 to 48 kilobases distal to the break site. This break-distal recombination (BDR occurs frequently, even when allelic recombination can repair the break using the homolog. Robust BDR-dependent NAHR demonstrates that sequences very distal to DSBs can effectively compete with proximal sequences for repair of the break. In addition, our analysis of NAHR partner choice between Ty repeats shows that intrachromosomal Ty partners are preferred despite the abundance of potential interchromosomal Ty partners that share higher sequence identity. This competitive advantage of intrachromosomal Tys results from the relative efficiencies of different NAHR repair pathways. Finally, NAHR generates deleterious rearrangements more frequently when DSBs occur outside rather than within a Ty repeat. These findings yield insights into mechanisms of repeat-mediated genome rearrangements associated with evolution and cancer.

  5. The transcription fidelity factor GreA impedes DNA break repair.

    Science.gov (United States)

    Sivaramakrishnan, Priya; Sepúlveda, Leonardo A; Halliday, Jennifer A; Liu, Jingjing; Núñez, María Angélica Bravo; Golding, Ido; Rosenberg, Susan M; Herman, Christophe

    2017-10-12

    Homologous recombination repairs DNA double-strand breaks and must function even on actively transcribed DNA. Because break repair prevents chromosome loss, the completion of repair is expected to outweigh the transcription of broken templates. However, the interplay between DNA break repair and transcription processivity is unclear. Here we show that the transcription factor GreA inhibits break repair in Escherichia coli. GreA restarts backtracked RNA polymerase and hence promotes transcription fidelity. We report that removal of GreA results in markedly enhanced break repair via the classic RecBCD-RecA pathway. Using a deep-sequencing method to measure chromosomal exonucleolytic degradation, we demonstrate that the absence of GreA limits RecBCD-mediated resection. Our findings suggest that increased RNA polymerase backtracking promotes break repair by instigating RecA loading by RecBCD, without the influence of canonical Chi signals. The idea that backtracked RNA polymerase can stimulate recombination presents a DNA transaction conundrum: a transcription fidelity factor that compromises genomic integrity.

  6. Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

    Science.gov (United States)

    Weller, Sandra K.; Sawitzke, James A.

    2015-01-01

    The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies. PMID:25002096

  7. Recombination-deficient mutants of Bacillus subtilis

    International Nuclear Information System (INIS)

    Sadaie, Y.; Kada, T.

    1976-01-01

    Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (uv), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation, SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr + capacity for uv-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 tranduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The uv impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene product whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strains NIG43 and NIG45 were not inducible, indicating involvement of rec-43 + or rec-45 + gene product in the development of SPO2 prophage to a vegetative form. The uv-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains

  8. Thio and hydrogen peroxide modofication of recA induction in UV-irradiated wild-type and catalase-deficient Escherichia coli K12

    International Nuclear Information System (INIS)

    Claycamp, H.G.; Kam-Kuen Ho; DeRose, C.; Iowa Univ., Iowa City, IA

    1990-01-01

    Induction of recA in Escherichia coli, monitored as β-D-galactosidase activity in recA-lacZ fusion strains, was shown to be elevated and prolonged by dithiothreitol (DTT) treatment after far-UV radiation. Pretreatment of UV-irradiated coltures using DTT led to a shortened recA response and little increase of β-Gal yield. Similar studies were performed using a catalase-deficient recA-lacZ strain in which the major feature was elevated levels of recA-lacZ induction. Catalase activity in UV-irradiated wild-type cells was reduced by DTT treatment to levels as low as in a katE mutant strain, leading to similar recA-lacZ induction patterns between the strains. Neither DTT nor H 2 O 2 treatment of cells could induce significant recA transcription in the absence of UV-radiation, implying that both agents modify recA activity primarily by interfering with repair of recA-inducing DNA lesions. The results confirm previous studies suggesting that modification of DNA repair is probably a significant portion of thiol radiation protection. (author). 36 refs.; 7 figs.; 1 tab

  9. Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

    Science.gov (United States)

    Hoolahan, Angelique H; Blok, Vivian C; Gibson, Tracey; Dowton, Mark

    2012-03-01

    Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.

  10. Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene.

    Science.gov (United States)

    Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Mu-Chiou; Wang, Li-Tin; Huang, Lina; Lee, Fwu-Ling

    2012-10-01

    To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing. Copyright © 2012 Society of Chemical Industry.

  11. Protection of DNA damage by radiation exposure

    International Nuclear Information System (INIS)

    Lee, Jeong Ho; Kim, In Gyu; Lee, Kang Suk; Kim, Kug Chan; Oh, Tae Jung

    1998-12-01

    The SOS response of Escherichia coli is positively regulated by RecA. To examine the effects of polyamines on The SOS response of E. Coli, we investigated the expression of recA gene in polyamine-deficient mutant and wild type carrying recA'::lacZ fusion gene. As a result, recA expression by mitomycin C is higher in wild type than that of polyamine-deficient mutant, but recA expression by UV radiation is higher in wild type than of mutant. We also found that exogenous polyamines restored the recA expression in the polyamine-deficient mutant to the wild type level. These results proposed that polyamines play an important role in mechanism of intracellular DNA protection by DNA damaging agents

  12. Protection of DNA damage by radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Ho; Kim, In Gyu; Lee, Kang Suk; Kim, Kug Chan; Oh, Tae Jung

    1998-12-01

    The SOS response of Escherichia coli is positively regulated by RecA. To examine the effects of polyamines on The SOS response of E. Coli, we investigated the expression of recA gene in polyamine-deficient mutant and wild type carrying recA'::lacZ fusion gene. As a result, recA expression by mitomycin C is higher in wild type than that of polyamine-deficient mutant, but recA expression by UV radiation is higher in wild type than of mutant. We also found that exogenous polyamines restored the recA expression in the polyamine-deficient mutant to the wild type level. These results proposed that polyamines play an important role in mechanism of intracellular DNA protection by DNA damaging agents.

  13. Protection of DNA damage by radiation exposure

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeong Ho; Kim, In Gyu; Lee, Kang Suk; Kim, Kug Chan; Oh, Tae Jung

    1998-12-01

    The SOS response of Escherichia coli is positively regulated by RecA. To examine the effects of polyamines on The SOS response of E. Coli, we investigated the expression of recA gene in polyamine-deficient mutant and wild type carrying recA'::lacZ fusion gene. As a result, recA expression by mitomycin C is higher in wild type than that of polyamine-deficient mutant, but recA expression by UV radiation is higher in wild type than of mutant. We also found that exogenous polyamines restored the recA expression in the polyamine-deficient mutant to the wild type level. These results proposed that polyamines play an important role in mechanism of intracellular DNA protection by DNA damaging agents.

  14. DNA: Polymer and molecular code

    Science.gov (United States)

    Shivashankar, G. V.

    1999-10-01

    The thesis work focusses upon two aspects of DNA, the polymer and the molecular code. Our approach was to bring single molecule micromanipulation methods to the study of DNA. It included a home built optical microscope combined with an atomic force microscope and an optical tweezer. This combined approach led to a novel method to graft a single DNA molecule onto a force cantilever using the optical tweezer and local heating. With this method, a force versus extension assay of double stranded DNA was realized. The resolution was about 10 picoN. To improve on this force measurement resolution, a simple light backscattering technique was developed and used to probe the DNA polymer flexibility and its fluctuations. It combined the optical tweezer to trap a DNA tethered bead and the laser backscattering to detect the beads Brownian fluctuations. With this technique the resolution was about 0.1 picoN with a millisecond access time, and the whole entropic part of the DNA force-extension was measured. With this experimental strategy, we measured the polymerization of the protein RecA on an isolated double stranded DNA. We observed the progressive decoration of RecA on the l DNA molecule, which results in the extension of l , due to unwinding of the double helix. The dynamics of polymerization, the resulting change in the DNA entropic elasticity and the role of ATP hydrolysis were the main parts of the study. A simple model for RecA assembly on DNA was proposed. This work presents a first step in the study of genetic recombination. Recently we have started a study of equilibrium binding which utilizes fluorescence polarization methods to probe the polymerization of RecA on single stranded DNA. In addition to the study of material properties of DNA and DNA-RecA, we have developed experiments for which the code of the DNA is central. We studied one aspect of DNA as a molecular code, using different techniques. In particular the programmatic use of template specificity makes

  15. Recent advances in yeast molecular biology: recombinant DNA

    International Nuclear Information System (INIS)

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis

  16. Self-regulation of recombinant DNA technology in Japan in the 1970s.

    Science.gov (United States)

    Nagai, Hiroyuki; Nukaga, Yoshio; Saeki, Koji; Akabayashi, Akira

    2009-07-01

    Recombinant DNA technology was developed in the United States in the early 1970s. Leading scientists held an international Asilomar Conference in 1975 to examine the self regulation of recombinant DNA technology, followed by the U.S. National Institutes of Health drafting the Recombinant DNA Research Guidelines in 1976. The result of this conference significantly affected many nations, including Japan. However, there have been few historical studies on the self-regulation of recombinant technologies conducted by scientists and government officials in Japan. The purpose of this paper is to analyze how the Science Council of Japan, the Ministry of Education, Science adn Culture, and the Science and Technology Agency developed self-regulation policies for recombinant DNA technology in Japan in the 1970s. Groups of molecular biologist and geneticists played a key role in establishing guidelines in cooperation with government officials. Our findings suggest that self-regulation policies on recombinant DNA technology have influenced safety management for the life sciences and establishment of institutions for review in Japan.

  17. Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Jennifer R Gruhn

    Full Text Available Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.

  18. Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

    Science.gov (United States)

    2013-01-01

    SUMMARY Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells. PMID:24006472

  19. Construction of recombinant DNA clone for bovine viral diarrhea virus

    International Nuclear Information System (INIS)

    Yeo, S.G.; Cho, H.J.; Masri, S.A.

    1992-01-01

    Molecular cloning was carried out on the Danish strain of bovine viral diarrhea virus (BVDV) to construct strategy for the diagnostic tools and effective vaccine of BVD afterwards. A recombinant DNA clone (No. 29) was established successfully from cDNA for viral RNA tailed with adenine homopolymer at 3 -end. 32 P-labeled DNA probes of 300~1, 800bp fragments, originating from the clone 29, directed specific DNA-RNA hybridization results with BVDV RNA. Recombinant DNA of the clone 29 was about 5,200bp representing 41.6% of the full length of Danish strain's RNA, and restriction sites were recognized for EooR I, Sst I, Hind III and Pst I restriction enzymes in the DNA fragment

  20. Transcription and recombination: when RNA meets DNA.

    Science.gov (United States)

    Aguilera, Andrés; Gaillard, Hélène

    2014-08-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  1. Characterization of the Pseudomonas aeruginosa recA gene: the Les- phenotype

    International Nuclear Information System (INIS)

    Kokjohn, T.A.; Miller, R.V.

    1988-01-01

    The Les- phenotype (lysogeny establishment deficient) is a pleiotropic effect of the lesB908 mutation of Pseudomonas aeruginosa PAO. lesB908-containing strains are also (i) deficient in general recombination, (ii) sensitive to UV irradiation, and (iii) deficient in UV-stimulated induction of prophages. The P. aeruginosa recA-containing plasmid pKML3001 complemented each of these pleiotropic characteristics of the lesB908 mutation, supporting the hypothesis that lesB908 is an allele of the P. aeruginosa recA gene. The phenotypic effects of the lesB908 mutation may be best explained by the hypothesis that the lesB908 gene product is altered in such a way that it has lost synaptase activity but possesses intrinsic protease activity in the absence of DNA damage. The Les- phenotype is a result of the rapid destruction of newly synthesized phage repressor, resulting in lytic growth of the infecting virus. This hypothesis is consistent with the observations that increasing the number of copies of the phage repressor gene by increasing the multiplicity of infection (i.e., average number of phage genomes per cell) or by introducing the cloned phage repressor gene into a lesB908 mutant will also suppress the Les- phenotype in a phage-specific fashion

  2. Recent advances in DNA repair and recombination.

    Science.gov (United States)

    Iwanejko, L A; Jones, N J

    1998-09-11

    The subjects of the talks at this 1-day DNA Repair Network meeting, held at City University, London on December 15, 1997, encompassed a range of topics and reflected some of the current areas of research in the United Kingdom. Topics included DNA double-strand break repair, V(D)J recombination, DNA ligases, the RecQ family of helicases and Bloom's syndrome, UVB and immunosuppression, the repair of oxidative damage and mismatch repair mechanisms.

  3. DNA repair and its relation to recombination-deficient and other mutations in Bacillus subtilis

    International Nuclear Information System (INIS)

    Ganesan, A.T.

    1975-01-01

    DNA repair processes operating in Bacillus subtilis are similar to other transformable bacterial systems. Radiation-sensitive, recombination-deficient mutants are blocked in distinct steps leading to recombination. DNA polymerase I is essential for the repair of x-ray-induced damage to DNA but not for recombination

  4. The Mitochondrial DNA (mtDNA)-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination

    KAUST Repository

    Blomme, Jonas

    2017-04-19

    In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana. Gainand loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.

  5. Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

    Science.gov (United States)

    Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

    2014-01-01

    HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

  6. Role of RecA protein in untargeted UV mutagenesis of bacteriophage lambda: evidence for the requirement for the dinB gene

    International Nuclear Information System (INIS)

    Brotcorne-Lannoye, A.; Maenhaut-Michel, G.

    1986-01-01

    Untargeted UV mutagenesis of bacteriophage lambda--i.e., the increased recovery of lambda mutants when unirradiated lambda infects UV-irradiated Escherichia coli--is thought to be mediated by a transient decrease in DNA replication fidelity, generating mutations in the newly synthesized strands. Using the bacteriophage lambda cI857----lambda c mutation system, we provide evidence that the RecA protein, shown previously to be required for this mutagenic pathway, is no longer needed when the LexA protein is inactivated by mutation. We suggest that the error-prone DNA replication responsible for UV-induced untargeted mutagenesis is turned on by the presence of replication-blocking lesions in the host cell DNA and that the RecA protein is required only to derepress the relevant din gene(s). This is in contrast to mutagenesis of irradiated bacteria or irradiated phage lambda, in which activated RecA protein has a second role in mutagenesis in addition to the cleavage of the LexA protein. Among the tested din genes, the dinB gene product (in addition to the uvrA and uvrB gene products) was found to be required for untargeted mutagenesis of bacteriophage lambda. To our knowledge, a phenotype associated with the dinB gene has not been reported previously

  7. Effects of radiations on DNA and repair of the damage. Progress report, May 1, 1974--June 30, 1977

    International Nuclear Information System (INIS)

    Hutchinson, F.

    1977-01-01

    Repair of DNA double-strand breaks produced by gamma rays takes place in E. coli. Such repair requires recA function and the presence of another DNA molecule of the same base sequence, so it may involve a recombination-like event. Ultraviolet light acting on DNA containing bromouracil produces doublestrand breaks by single photochemical events, and a simple model can explain this, as well as other results. Bromouracil mutagenesis of either E. coli or lambda phage does not involve the recA or red functions. Bromouracil mutagenesis is greatly increased in E. coli mutants such as uvrE, mutL, mutR and mutS, which are defective in mismatch repair. This, and other results, suggest that bromouracil mutagenesis occurs when cell enzymes fail to remove mismatched bases. Ultraviolet mutagenesis of lambda phage may be a useful model for the study of mutagenesis in cells, because the effects of lesions in the gene mutated (i.e. in the phage) and changes in enzyme systems (by treating the host cells) can be examined separately. Quantitative data support this approach

  8. Does RecA have a role in Borrelia recurrentis?

    OpenAIRE

    Cutler, S.J.; Rinky, I.J.; Bonilla, E.M.

    2011-01-01

    Genomic sequencing of two relapsing fever spirochaetes showed truncation of recA in Borrelia recurrentis, but not in Borrelia duttonii. RecA has an important role among bacteria; we investigated whether this characteristic was representative of B. recurrentis, or an artefact following in vitro cultivation. We sequenced recA directly from samples of patient with louse-borne relapsing fever (B. recurrentis) or tick-borne relapsing fever (B. duttonii). We confirmed the premature stop codon in se...

  9. Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats

    Directory of Open Access Journals (Sweden)

    Daniël O. Warmerdam

    2016-03-01

    Full Text Available rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5 as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability.

  10. Mycobacterium smegmatis PafBC is involved in regulation of DNA damage response.

    Science.gov (United States)

    Fudrini Olivencia, Begonia; Müller, Andreas U; Roschitzki, Bernd; Burger, Sibylle; Weber-Ban, Eilika; Imkamp, Frank

    2017-10-25

    Two genes, pafB and pafC, are organized in an operon with the Pup-ligase gene pafA, which is part of the Pup-proteasome system (PPS) present in mycobacteria and other actinobacteria. The PPS is crucial for Mycobacterium tuberculosis resistance towards reactive nitrogen intermediates (RNI). However, pafB and pafC apparently play only a minor role in RNI resistance. To characterize their function, we generated a pafBC deletion in Mycobacterium smegmatis (Msm). Proteome analysis of the mutant strain revealed decreased cellular levels of various proteins involved in DNA damage repair, including recombinase A (RecA). In agreement with this finding, Msm ΔpafBC displayed increased sensitivity to DNA damaging agents. In mycobacteria two pathways regulate DNA repair genes: the LexA/RecA-dependent SOS response and a predominant pathway that controls gene expression via a LexA/RecA-independent promoter, termed P1. PafB and PafC feature winged helix-turn-helix DNA binding motifs and we demonstrate that together they form a stable heterodimer in vitro, implying a function as a heterodimeric transcriptional regulator. Indeed, P1-driven transcription of recA was decreased in Msm ΔpafBC under standard conditions and induction of recA expression upon DNA damage was strongly impaired. Taken together, our data indicate an important regulatory function of PafBC in the mycobacterial DNA damage response.

  11. Mycobacterium tuberculosis DNA repair in response to subinhibitory concentrations of ciprofloxacin.

    Science.gov (United States)

    O'Sullivan, D M; Hinds, J; Butcher, P D; Gillespie, S H; McHugh, T D

    2008-12-01

    To investigate how the SOS response, an error-prone DNA repair pathway, is expressed following subinhibitory quinolone treatment of Mycobacterium tuberculosis. Genome-wide expression profiling followed by quantitative RT (qRT)-PCR was used to study the effect of ciprofloxacin on M. tuberculosis gene expression. Microarray analysis showed that 16/110 genes involved in DNA protection, repair and recombination were up-regulated. There appeared to be a lack of downstream genes involved in the SOS response. qRT-PCR detected an induction of lexA and recA after 4 h and of dnaE2 after 24 h of subinhibitory treatment. The pattern of gene expression observed following subinhibitory quinolone treatment differed from that induced after other DNA-damaging agents (e.g. mitomycin C). The expression of the DnaE2 polymerase response was significantly delayed following subinhibitory quinolone exposure.

  12. Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

    International Nuclear Information System (INIS)

    Burkovics, Peter; Sebesta, Marek; Kolesar, Peter; Sisakova, Alexandra; Marini, Victoria; Plault, Nicolas; Szukacsov, Valeria; Pinter, Lajos; Haracska, Lajos; Robert, Thomas; Kolesar, Peter; Gangloff, Serge; Krejci, Lumir

    2013-01-01

    Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. (authors)

  13. Characterization of a bacteriophage T4 mutant lacking DNA-dependent ATPase

    International Nuclear Information System (INIS)

    Behme, M.T.; Ebisuzaki, K.

    1975-01-01

    A DNA-dependent ATPase has previously been purified from bacteriophage T4-infected Escherichia coli. A mutant phage strain lacking this enzyme has been isolated and characterized. Although the mutant strain produced no detectable DNA-dependent ATPase, growth properties were not affected. Burst sizes were similar for the mutant phage and T4D in polAl, recB, recC, uvrA, uvrB, uvrC, and various DNA-negative E. coli. UV sensitivity and genetic recombination were normal in a variety of E. coli hosts. Mapping data indicate that the genetic locus controlling the mutant occurs near gene 56. The nonessential nature of this gene is discussed

  14. Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

    Directory of Open Access Journals (Sweden)

    Ken Motohashi

    2017-03-01

    Full Text Available Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA− strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid

  15. ATP-dependent chromatin remodeling in the DNA-damage response

    Directory of Open Access Journals (Sweden)

    Lans Hannes

    2012-01-01

    Full Text Available Abstract The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.

  16. Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

    Science.gov (United States)

    Warmerdam, Daniël O; van den Berg, Jeroen; Medema, René H

    2016-03-22

    rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  17. A role for recombination junctions in the segregation of mitochondrial DNA in yeast.

    Science.gov (United States)

    Lockshon, D; Zweifel, S G; Freeman-Cook, L L; Lorimer, H E; Brewer, B J; Fangman, W L

    1995-06-16

    In S. cerevisiae, mitochondrial DNA (mtDNA) molecules, in spite of their high copy number, segregate as if there were a small number of heritable units. The rapid segregation of mitochondrial genomes can be analyzed using mtDNA deletion variants. These small, amplified genomes segregate preferentially from mixed zygotes relative to wild-type mtDNA. This segregation advantage is abolished by mutations in a gene, MGT1, that encodes a recombination junction-resolving enzyme. We show here that resolvase deficiency causes a larger proportion of molecules to be linked together by recombination junctions, resulting in the aggregation of mtDNA into a small number of cytological structures. This change in mtDNA structure can account for the increased mitotic loss of mtDNA and the altered pattern of mtDNA segregation from zygotes. We propose that the level of unresolved recombination junctions influences the number of heritable units of mtDNA.

  18. Role of the RecF pathway of recombination in the metabolism of uv-irradiated DNA in Escherichia coli K-12

    International Nuclear Information System (INIS)

    Rothman, R.H.

    1976-01-01

    The RecF pathway of genetic recombination in Escherichia coli is potentially capable of supporting wild type levels of recombination, but in wild type cells it plays a relatively minor role in this process. RecF and recL single mutants were found to be ultraviolet-sensitive but recombination proficient. These observations led to the hypothesis that the main function of the RecF pathway lies in the metabolism of uv-damaged DNA. The role of reF and recL in pathways of recovery from uv-irradiation has been examined. Both recF - and recL - inhibited post-replication joining of DNA fragments synthesized on uv-damaged DNA templates (post-replication repair). The addition of a uvrB5 mutation to the single mutants did not affect the cell's ability to complete post-replication repair in the case of recL, but did completely prevent completion of joining in the case of recF. It was hypothesized that recF is an endonuclease weakly indirectly suppressible by the presence of functional correndo II. It is suggested that recF is necessary to cleave the crossed strand intermediate at the end of repair. RecL, in addition to its involvement in post-replication repair, was also found to be involved in excision repair. A uvrB recB recC recF multiple mutant was as sensitive as a uvrB recA strain, suggesting that it is devoid of any repair abilities. RecB - was shown to have an inhibitory effect of post-replication repair. The uvrB recF mutant, however, was totally devoid of post-replication repair even though recB + contributed to the recovery of the strain. Thus the role of recB in post-replication repair is unclear. Lastly, the effects of recF and recL on uv-inducible repair was studied. W-reactivation of uv-irradiated lambda was used as an assay for inducible repair. The conclusions from these experiments were unclear. They seemed to imply that W-reactivation is effected by the combined action of excision repair and post-replication repair

  19. DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair.

    Science.gov (United States)

    Serrano, M A; Li, Z; Dangeti, M; Musich, P R; Patrick, S; Roginskaya, M; Cartwright, B; Zou, Y

    2013-05-09

    Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

  20. MEIOB targets single-strand DNA and is necessary for meiotic recombination.

    Directory of Open Access Journals (Sweden)

    Benoit Souquet

    Full Text Available Meiotic recombination is a mandatory process for sexual reproduction. We identified a protein specifically implicated in meiotic homologous recombination that we named: meiosis specific with OB domain (MEIOB. This protein is conserved among metazoan species and contains single-strand DNA binding sites similar to those of RPA1. Our studies in vitro revealed that both recombinant and endogenous MEIOB can be retained on single-strand DNA. Those in vivo demonstrated the specific expression of Meiob in early meiotic germ cells and the co-localization of MEIOB protein with RPA on chromosome axes. MEIOB localization in Dmc1 (-/- spermatocytes indicated that it accumulates on resected DNA. Homologous Meiob deletion in mice caused infertility in both sexes, due to a meiotic arrest at a zygotene/pachytene-like stage. DNA double strand break repair and homologous chromosome synapsis were impaired in Meiob (-/- meiocytes. Interestingly MEIOB appeared to be dispensable for the initial loading of recombinases but was required to maintain a proper number of RAD51 and DMC1 foci beyond the zygotene stage. In light of these findings, we propose that RPA and this new single-strand DNA binding protein MEIOB, are essential to ensure the proper stabilization of recombinases which is required for successful homology search and meiotic recombination.

  1. Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

    Science.gov (United States)

    Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

    2015-01-01

    Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  2. Expression of the recA gene of Pseudomonas aeruginosa PAO is inducible by DNA-damaging agents

    International Nuclear Information System (INIS)

    Miller, R.V.; Kokjohn, T.A.

    1988-01-01

    Western (immunoblot) analysis using Escherichia coli anti-RecA antiserum revealed that expression of the RecA protein of Pseudomonas aeruginosa PAO is induced upon exposure of the bacterium to UV irradiation or norfloxacin, a quinolone related to nalidixic acid

  3. ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

    DEFF Research Database (Denmark)

    Zhao, A.; Gray, F. C; MacNeill, S. A.

    2006-01-01

    DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile....... volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that Lig...

  4. DNA fragmentation and cytotoxicity by recombinant human tumor necrosis factor in L929 fibroblast cells

    International Nuclear Information System (INIS)

    Kosaka, T.; Kuwabara, M.; Koide, F.

    1992-01-01

    Induction of cell DNA fragmentation by treatment of recombinant human Tumor Necrosis Factor alpha (rhTNF alpha) was examined by using mouse L929 cells derived from mouse fibroblast cells. The amount of DNA fragments derived from rhTNF alpha-treated cells, detected by alkaline elution technique, was smaller than that derived from X-irradiated cells. The rhTNF alpha caused the DNA fragmentation depending on its incubation time and concentration. The DNA damage caused by rhTNF alpha treatment correlated with its cytotoxicity. This result suggested that the DNA fragmentation is one of causes of cell death. The treatment with proteinase K of DNA obtained from rhTNF alpha-treated cells did not increase the amount of DNA fragmentation, which indicates that rhTNF alpha causes DNA-fragmentation but not DNA-protein cross-linking

  5. Xrcc1-dependent and Ku-dependent DNA double-strand break repair kinetics in Arabidopsis plants.

    Science.gov (United States)

    Charbonnel, Cyril; Gallego, Maria E; White, Charles I

    2010-10-01

    Double-strand breakage (DSB) of DNA involves loss of information on the two strands of the DNA fibre and thus cannot be repaired by simple copying of the complementary strand which is possible with single-strand DNA damage. Homologous recombination (HR) can precisely repair DSB using another copy of the genome as template and non-homologous recombination (NHR) permits repair of DSB with little or no dependence on DNA sequence homology. In addition to the well-characterised Ku-dependent non-homologous end-joining (NHEJ) pathway, much recent attention has been focused on Ku-independent NHR. The complex interrelationships and regulation of NHR pathways remain poorly understood, even more so in the case of plants, and we present here an analysis of Ku-dependent and Ku-independent repair of DSB in Arabidopsis thaliana. We have characterised an Arabidopsis xrcc1 mutant and developed quantitative analysis of the kinetics of appearance and loss of γ-H2AX foci as a tool to measure DSB repair in dividing root tip cells of γ-irradiated plants in vivo. This approach has permitted determination of DSB repair kinetics in planta following a short pulse of γ-irradiation, establishing the existence of a Ku-independent, Xrcc1-dependent DSB repair pathway. Furthermore, our data show a role for Ku80 during the first minutes post-irradiation and that Xrcc1 also plays such a role, but only in the absence of Ku. The importance of Xrcc1 is, however, clearly visible at later times in the presence of Ku, showing that alternative end-joining plays an important role in DSB repair even in the presence of active NHEJ. © 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.

  6. Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

    Energy Technology Data Exchange (ETDEWEB)

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

  7. DNA-dependent protein kinase (DAN-PK), a key enzyme in the re-ligation of DNA double-strand breaks

    International Nuclear Information System (INIS)

    Hennequin, C.; Averbeck, D.

    1999-01-01

    Repair pathways of DNA are now defined and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEH) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEj implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridization in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catalytic subunit (DNA-PK cs ) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immuno-deficient (scid) mice are deficient in DNA-PK cs : this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylate Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival. (authors)

  8. Relationship among the repair mechanisms and the genetic recombination; Relacion entre los mecanismos de reparacion y la recombinacion genetica

    Energy Technology Data Exchange (ETDEWEB)

    Alcantara D, D [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    1987-12-15

    In accordance with the previous reports of the Project BZ87 of the Department of Radiobiology, a dependent stimulation of the system exists in E.coli SOS, of the recombination of the bacteriophage Lambda whose genetic material has not been damaged. This stimulation is not due to the increase of the cellular concentration of the protein RecA and the mechanism but probable for which we find that it is carried out, it is through a cooperation among the product of the gene rec N of E. coli and the system Net of recombination of Lambda. The gene recN belongs to the group of genes SOS and its expression is induced when damaging the bacterial DNA where it intervenes in the repair of breaks of the double helix of the molecule (Picksley et, 1984). If the repair of breaks of this type is a factor that limits the speed with which it happens the recombination among viral chromosomes, then the biggest readiness in the protein RecN, due to the induction of the functions SOS, would facilitate the repair of such ruptures. In this new project it is to enlarge the knowledge about this phenomenon, it was, on one hand of corroborating in a way but he/she specifies the relationship between the recombinogenic response of Lambda and the System SOS of E. coli and for the other one to determine the effect that has the inhibition of the duplication of the DNA on the stimulation of the viral recombination. Everything it with the idea of making it but evident and to be able to use it as a system of genotoxic agents detection in E. coli. (Author)

  9. Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA; Implicacion de los genes uvrA de E. coli K12 en la reparacion de monoaductos y entrecruzamien tos inducidos en DNA plasmidico por 8-metoxipso raleno mas luz ultravioleta A

    Energy Technology Data Exchange (ETDEWEB)

    Paramio, J M; Bauluz, C; Vidania, R de

    1986-07-01

    Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs.

  10. Genomic analysis of murine DNA-dependent protein kinase

    International Nuclear Information System (INIS)

    Fujimori, A.; Abe, M.

    2003-01-01

    Full text: The gene of catalytic subunit of DNA dependent protein kinase is responsible gene for SCID mice. The molecules play a critical role in non-homologous end joining including the V(D)J recombination. Contribution of the molecules to the difference of radiosensitivity and the susceptibility to cancer has been suggested. Here we show the entire nucleotide sequence of approximately 193 kbp and 84 kbp genomic regions encoding the entire DNA-PKcs gene in the mouse and chicken respectively. Retroposon was found in the intron 51 of mouse genomic DNA-PKcs gene but in human and chicken. Comparative analysis of these two species strongly suggested that only two genes, DNA-PKcs and MCM4, exist in the region of both species. Several conserved sequences and cis elements, however, were predicted. Recently, the orthologous region for the human DNA-PKcs locus was completed. The results of further comparative study will be discussed

  11. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    Science.gov (United States)

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

  12. Factors limiting SOS expression in log-phase cells of Escherichia coli.

    Science.gov (United States)

    Massoni, Shawn C; Leeson, Michael C; Long, Jarukit Edward; Gemme, Kristin; Mui, Alice; Sandler, Steven J

    2012-10-01

    In Escherichia coli, RecA-single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25%) log-phase cells have RecA filaments, few (about 1%) are induced for SOS. It is hypothesized that RecA's ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2- to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91%) nearly equals the percentage with at least one RecA-GFP focus (83%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell.

  13. The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

    DEFF Research Database (Denmark)

    Burkovics, Peter; Dome, Lili; Juhasz, Szilvia

    2016-01-01

    to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during...... recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits...

  14. Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats

    OpenAIRE

    Warmerdam, Daniël O.; van den Berg, Jeroen; Medema, René H.

    2016-01-01

    rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of b...

  15. Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation.

    Science.gov (United States)

    Lerat, Sylvain; Gulden, Robert H; Hart, Miranda M; Powell, Jeff R; England, Laura S; Pauls, K Peter; Swanton, Clarence J; Klironomos, John N; Trevors, Jack T

    2007-12-12

    The presence of the recombinant cp4 epsps gene from Roundup Ready (RR) corn and RR soybean was quantified using real-time PCR in soil samples from a field experiment growing RR and conventional corn and soybean in rotation. RR corn and RR soybean cp4 epsps persisted in soil for up to 1 year after seeding. The concentration of recombinant DNA in soil peaked in July and August in RR corn and RR soybean plots, respectively. A small fraction of soil samples from plots seeded with conventional crops contained recombinant DNA, suggesting transgene dispersal by means of natural process or agricultural practices. This research will aid in the understanding of the persistence of recombinant DNA in agricultural cropping systems.

  16. AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

    Science.gov (United States)

    Kobayashi, Maki; Aida, Masatoshi; Nagaoka, Hitoshi; Begum, Nasim A; Kitawaki, Yoko; Nakata, Mikiyo; Stanlie, Andre; Doi, Tomomitsu; Kato, Lucia; Okazaki, Il-mi; Shinkura, Reiko; Muramatsu, Masamichi; Kinoshita, Kazuo; Honjo, Tasuku

    2009-12-29

    To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

  17. Structure of the hDmc1-ssDNA filament reveals the principles of its architecture.

    Directory of Open Access Journals (Sweden)

    Andrei L Okorokov

    Full Text Available In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1, a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active and compressed (inactive. However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds DNA nucleoprotein filaments, the extended (active state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers

  18. Does RecA have a role in Borrelia recurrentis?

    Science.gov (United States)

    Cutler, S J; Rinky, I J; Bonilla, E M

    2011-02-01

    Genomic sequencing of two relapsing fever spirochaetes showed truncation of recA in Borrelia recurrentis, but not in Borrelia duttonii. RecA has an important role among bacteria; we investigated whether this characteristic was representative of B. recurrentis, or an artefact following in vitro cultivation. We sequenced recA directly from samples of patient with louse-borne relapsing fever (B. recurrentis) or tick-borne relapsing fever (B. duttonii). We confirmed the premature stop codon in seven louse-borne relapsing fever samples, and its absence from three tick-borne relapsing fever samples. Furthermore, specific signature polymorphisms were found that could differentiate between these highly similar spirochaetes. © 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases.

  19. Recombinational DNA repair and human disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Larry H.; Schild, David

    2002-11-30

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

  20. Recombinational DNA repair and human disease

    International Nuclear Information System (INIS)

    Thompson, Larry H.; Schild, David

    2002-01-01

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities

  1. Rogue athletes and recombinant DNA technology: challenges for doping control.

    Science.gov (United States)

    Azzazy, Hassan M E; Mansour, Mai M H

    2007-10-01

    The quest for athletic excellence holds no limit for some athletes, and the advances in recombinant DNA technology have handed these athletes the ultimate doping weapons: recombinant proteins and gene doping. Some detection methods are now available for several recombinant proteins that are commercially available as pharmaceuticals and being abused by dopers. However, researchers are struggling to come up with efficient detection methods in preparation for the imminent threat of gene doping, expected in the 2008 Olympics. This Forum article presents the main detection strategies for recombinant proteins and the forthcoming detection strategies for gene doping as well as the prime analytical challenges facing them.

  2. Recombinant DNA. Rifkin's regulatory revivalism runs riot.

    Science.gov (United States)

    David, P

    Jeremy Rifkin, activist opponent of genetic engineering, has adopted tactics of litigation, persuasion, and confrontation in his campaign to halt genetic experimentation. The Recombinant DNA Advisory Committee of the National Institutes of Health has often been the target of his criticism, most recently for its failure to prepare an environmental risk assessment for some DNA tests it approved. Rifkin has won support for his position from religious organizations in the United States, and in June 1983 persuaded an ecumenical group of religious leaders to ask Congress to ban genetic experiments that would affect the human germ line.

  3. Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

    International Nuclear Information System (INIS)

    Iwanaga, Kentaro; Sueoka, Naoko; Sato, Akemi; Hayashi, Shinichiro; Sueoka, Eisaburo

    2005-01-01

    Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression

  4. Genetic recombination in escherichia coli and its relationship with DNA replication

    International Nuclear Information System (INIS)

    Siddiqui, O.

    1974-01-01

    Relationship of DNA replication with genetic recombination in Escherichia Coli was investigated by mating Hfr donors labelled with H 3 -thymine, C 13 and N 15 to C 13 N 15 labelled recipients. The DNA extracted from the zygotes was analysed on CsCl density gradients. The results show that all of the biparentally labelled DNA arises from the single strand insertions of the donor DNA. (M.G.B.)

  5. Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.

    Science.gov (United States)

    Wu, Min; Li, Jin; Yue, Lei; Bai, Lu; Li, Yaming; Chen, Jieliang; Zhang, Xiaonan; Yuan, Zhenghong

    2018-04-01

    Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  6. [Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

    Science.gov (United States)

    Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

    2016-01-01

    Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

  7. Jeremy Rifkin challenges recombinant DNA research: A rhetoric of heresy

    Energy Technology Data Exchange (ETDEWEB)

    Futrell, W.M.

    1992-01-01

    One significant issue to come before the public in recent years is recombinant DNA research or genetic engineering and its applications. An important spokesman on this issue is Jeremy Rifkin. Rifkin is of rhetorical interest because of his strategies to sustain the dialogue and define the parameters in which it occurs. This dissertation analyzes a broad range of Rifkin's rhetorical artifacts and those of scientists engaged in recombinant DNA research. They are examined against criteria developed to identify and understand heresy. The five areas of analysis are: the nearness/remoteness phenomenon, the social construction of heresy, the social consequences of heresy, the doctrinal consequences of heresy, and the heresy-hunt ritual. The first two criteria focus on the rhetorical strategies of the heretic. The last three concentrate on the rhetorical strategies of the defenders of the institutional orthodoxy. This dissertation examines the rhetorical strategies of a heretical challenge to the scientific establishment and the consequences of that challenge. This dissertation also analyzes the rhetorical strategies employed by the defenders of the scientific orthodoxy. Although an understanding of the rhetorical strategies employed on both sides of this conflict is important, the implications for the role of rhetoric in highly controversial issues such as recombinant DNA are even more critical.

  8. RecA-mediated cleavage reaction of Lambda repressor and DNA ...

    African Journals Online (AJOL)

    PRECIOUS

    2010-01-11

    Jan 11, 2010 ... hydrolyze ATP at all, but fulfills RecA functions such as cleavage of Lambda repressor and strand .... DNA binding properties of RecA and may result in an in- .... AMP-PNP there is no cleavage of Lambda repressor (Figure.

  9. Recombinational DNA repair is regulated by compartmentalization of DNA lesions at the nuclear pore complex

    DEFF Research Database (Denmark)

    Géli, Vincent; Lisby, Michael

    2015-01-01

    and colleagues shows that also physiological threats to genome integrity such as DNA secondary structure-forming triplet repeat sequences relocalize to the NPC during DNA replication. Mutants that fail to reposition the triplet repeat locus to the NPC cause repeat instability. Here, we review the types of DNA...... lesions that relocalize to the NPC, the putative mechanisms of relocalization, and the types of recombinational repair that are stimulated by the NPC, and present a model for NPC-facilitated repair....

  10. Selection of LNA-containing DNA aptamers against recombinant human CD73

    DEFF Research Database (Denmark)

    Elle, Ida C; Karlsen, Kasper K; Terp, Mikkel G

    2015-01-01

    tested by surface plasmon resonance. Truncated variants of these aptamers and variants where the LNA nucleotides were substituted for the DNA equivalent also exhibited affinity for the recombinant CD73 in the low nanomolar range. In enzyme inhibition assays with recombinant CD73 the aptamer sequences......LNA-containing DNA aptamers against CD73 (human ecto-5'-nucleotidase), a protein frequently overexpressed in solid tumours, were isolated by SELEX. A pre-defined stem-loop library, containing LNA in the forward primer region, was enriched with CD73 binding sequences through six rounds of SELEX...... with recombinant his-tagged CD73 immobilised on anti-his plates. Enriched pools isolated from rounds one, three and six were subjected to next-generation sequencing and analysed for enrichment using custom bioinformatics software. The software identified aptamer sequences via the primers and then performed several...

  11. Modes of DNA repair and replication

    International Nuclear Information System (INIS)

    Hanawalt, P.; Kondo, S.

    1979-01-01

    Modes of DNA repair and replication require close coordination as well as some overlap of enzyme functions. Some classes of recovery deficient mutants may have defects in replication rather than repair modes. Lesions such as the pyrimidine dimers produced by ultraviolet light irradiation are the blocks to normal DNA replication in vivo and in vitro. The DNA synthesis by the DNA polymerase 1 of E. coli is blocked at one nucleotide away from the dimerized pyrimidines in template strands. Thus, some DNA polymerases seem to be unable to incorporate nucleotides opposite to the non-pairing lesions in template DNA strands. The lesions in template DNA strands may block the sequential addition of nucleotides in the synthesis of daughter strands. Normal replication utilizes a constitutive ''error-free'' mode that copies DNA templates with high fidelity, but which may be totally blocked at a lesion that obscures the appropriate base pairing specificity. It might be expected that modified replication system exhibits generally high error frequency. The error rate of DNA polymerases may be controlled by the degree of phosphorylation of the enzyme. Inducible SOS system is controlled by recA genes that also control the pathways for recombination. It is possible that SOS system involves some process other than the modification of a blocked replication apparatus to permit error-prone transdimer synthesis. (Yamashita, S.)

  12. LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms.

    Science.gov (United States)

    Yang, Peng; Wu, Min; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

    2014-02-17

    As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Recently, an algorithm called "LDsplit" has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of

  13. Choreography of recombination proteins during the DNA damage response

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2009-01-01

    Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such...... research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells....

  14. Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik

    2013-01-01

    Background Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described....... This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. Results A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable...

  15. RecA: a universal drug target in pathogenic bacteria.

    Science.gov (United States)

    Pavlopoulou, Athanasia

    2018-01-01

    The spread of bacterial infectious diseases due to the development of resistance to antibiotic drugs in pathogenic bacteria is an emerging global concern. Therefore, the efficacious management and prevention of bacterial infections are major public health challenges. RecA is a pleiotropic recombinase protein that has been demonstrated to be implicated strongly in the bacterial drug resistance, survival and pathogenicity. In this minireview, RecA's role in the development of antibiotic resistance and its potential as an antimicrobial drug target are discussed.

  16. Mechanisms of sister chromatid recombination

    International Nuclear Information System (INIS)

    Nakai, Sayaka; Machida, Isamu; Tsuji, Satsuki

    1985-01-01

    Studies using T948 as a model system have been carried out aimed at elucidating the mechanism of sister chromatid recombination (SCR). Characterization of U.V. light- and x-ray-induced SCR, the relationiship between SCR induction and DNA repair using rad mutations, and the relationship between SCR induction and the time of cell division using cdc mutations are presented. It has been supposed that SCR is induced at the phase of S-G 2 following DNA replication, that postreplication break of DNA strands is strongly involved in the induction of SCR, and that induction type of SCR, i.e., conversion type or recombination type, is dependent upon the type of molecular damage of DNA. (Namekawa, K.)

  17. Direct Single-Molecule Observation of Mode and Geometry of RecA-Mediated Homology Search.

    Science.gov (United States)

    Lee, Andrew J; Endo, Masayuki; Hobbs, Jamie K; Wälti, Christoph

    2018-01-23

    Genomic integrity, when compromised by accrued DNA lesions, is maintained through efficient repair via homologous recombination. For this process the ubiquitous recombinase A (RecA), and its homologues such as the human Rad51, are of central importance, able to align and exchange homologous sequences within single-stranded and double-stranded DNA in order to swap out defective regions. Here, we directly observe the widely debated mechanism of RecA homology searching at a single-molecule level using high-speed atomic force microscopy (HS-AFM) in combination with tailored DNA origami frames to present the reaction targets in a way suitable for AFM-imaging. We show that RecA nucleoprotein filaments move along DNA substrates via short-distance facilitated diffusions, or slides, interspersed with longer-distance random moves, or hops. Importantly, from the specific interaction geometry, we find that the double-stranded substrate DNA resides in the secondary DNA binding-site within the RecA nucleoprotein filament helical groove during the homology search. This work demonstrates that tailored DNA origami, in conjunction with HS-AFM, can be employed to reveal directly conformational and geometrical information on dynamic protein-DNA interactions which was previously inaccessible at an individual single-molecule level.

  18. Specific modifications of histone tails, but not DNA methylation, mirror the temporal variation of mammalian recombination hotspots.

    Science.gov (United States)

    Zeng, Jia; Yi, Soojin V

    2014-10-16

    Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called "recombination hotspot paradox") remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy "bivalent" chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for

  19. Proteomics of post-irradiation recovery in D. radiodurans

    International Nuclear Information System (INIS)

    Basu, Bhakti; Apte, Shree Kumar

    2012-01-01

    An extremophile Deinococcus radiodurans is bestowed with an extraordinary DNA repair ability that renders it virtually resistant to all known forms of DNA damage caused by ionizing radiations (10 kGy of gamma rays), UV (1 kJ/m 2 ) or weeks of desiccation etc. The genome of D. radiodurans encodes a unique combination of DNA repair pathways such as prokaryotic type RecFOR mediated homologous recombination (HR) and nucleotide/base excision repair along with eukaryotic type strand annealing (SA) and non-homologous end joining (NHEJ), but is devoid of universal prokaryotic DNA repair pathways such as RecBCD mediated HR, photo-reactivation and SOS response. Collective evidence obtained so far from multiple approaches, have indicated (i) that all genes essential for DNA repair are not necessarily induced following radiation stress (ii) early RecA independent DNA assembly occurs, and (iii) absolute necessity of RecA dependent HR for final genome restitution. The 6 kGy gamma irradiation inducible proteome dynamics were mapped during the post-irradiation growth arrest phase by 2D protein electrophoresis coupled with mass spectrometry. Radiation inducible expression of at least 33 proteins was evident in the first 1h of post irradiation recovery

  20. Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity

    International Nuclear Information System (INIS)

    Cai Xuwei; Fu Xiaolong; Yang Jian; Song Houyan

    2005-01-01

    Objective: To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods: Based on Adeno- X TM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then sub-cloned into the Adeno-X TM genome, which was transformed into E. coli to get recombinant Adeno-X TM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated , and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results: Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0 x 10 11 TCID 50 /ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions: With the aid of Adeno-X TM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose-dependent

  1. VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner.

    Science.gov (United States)

    Fukuda, Tomoyuki; Nogami, Satoru; Ohya, Yoshikazu

    2003-07-01

    Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus. We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5delta clb6delta mutation reduces VDE-mediated DSBs. The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism.

  2. Over a Decade of recA and tly Gene Sequence Typing of the Skin Bacterium Propionibacterium acnes: What Have We Learnt?

    Directory of Open Access Journals (Sweden)

    Andrew McDowell

    2017-12-01

    Full Text Available The Gram-positive, anaerobic bacterium Propionibacterium acnes forms part of the normal microbiota on human skin and mucosal surfaces. While normally associated with skin health, P. acnes is also an opportunistic pathogen linked with a range of human infections and clinical conditions. Over the last decade, our knowledge of the intraspecies phylogenetics and taxonomy of this bacterium has increased tremendously due to the introduction of DNA typing schemes based on single and multiple gene loci, as well as whole genomes. Furthermore, this work has led to the identification of specific lineages associated with skin health and human disease. In this review we will look back at the introduction of DNA sequence typing of P. acnes based on recA and tly loci, and then describe how these methods provided a basic understanding of the population genetic structure of the bacterium, and even helped characterize the grapevine-associated lineage of P. acnes, known as P. acnes type Zappe, which appears to have undergone a host switch from humans-to-plants. Particular limitations of recA and tly sequence typing will also be presented, as well as a detailed discussion of more recent, higher resolution, DNA-based methods to type P. acnes and investigate its evolutionary history in greater detail.

  3. Characterization of new radiation-sensitive mutant, Escherichia coli K-12 radC102

    International Nuclear Information System (INIS)

    Felzenszwalb, I.; Sargentini, N.J.; Smith, K.C.

    1984-01-01

    A new radiation-sensitive mutant, radC, has been isolated. The radC gene is located at 81.0 min on the Escherichia coli K-12 linkage map. The radC mutation sensitized cells to uv radiation, but unlike most DNA repair mutations, sensitization to X rays was observed only for rich medium-grown cells. For cells grown in rich medium, the radC mutant was normal for γ radiation mutagenesis, but showed less uv-radiation mutagenesis than the wild-type strain; it showed normal amount of X- and uv-radiation-induced DNA degradation, and it wasapprox. =60% deficient in recombination ability. The radC strain was normal for host cell reactivation of γ and uv-irradiated bacteriophage the radC mutation did not sensitize a recA strain, but did sensitize a radA and a polA strain to X and uv radiation and a uvrA strain to uv radiation. Therefore, it is suggested that the radC gene product plays a role in the growth medium-dependent, recA gene-dependent repair of DNA single-strand breaks after X irradiation, and in postreplication repair after uv irradiation

  4. Successful development of recombinant DNA-derived pharmaceuticals.

    Science.gov (United States)

    Werner, R G; Pommer, C H

    1990-11-01

    Successful development of recombinant DNA-derived pharmaceuticals, a new class of therapeutic agents, is determined by a variety of factors affecting the selection and positioning of the compound under development. For an efficient development it is of utmost importance that the mechanism of action of the compound selected be understood on a molecular level. The compound's potential therapeutical profile and a strong patent position are key positioning considerations, as well as vital elements in shortening the development phase and protecting innovation. Installation of an interdisciplinary project management team, along with a clear definition of team members' responsibilities, is required to avoid delays and improve communication during development. Selection of the organism to be used in production must take into consideration both the structure of the protein and the quality and safety of the final product. New technologies require a considerable investment in new manufacturing facilities and equipment. Often, the decision for such an investment must be made early and with a high degree of uncertainty. Desired product yield, expected dosage, and estimated market potential are the most important considerations in this decision. Following public disclosure of the plan to develop recombinant DNA-derived products, approval of the production plant and expansion or adaptation to the new process and technology may be delayed. For this reason, they should be considered as a critical step in the overall development phase. Recruitment of qualified staff is a time-consuming and critical element of the production process. Its impact on the product timeline should not be underestimated, especially if such technologies are new to the company. The entire production process must be validated in respect to identity, purity, and safety of the product to guarantee constant product quality, as well as for safety aspects in the environment. Adequate in-process and final product

  5. Recombinant methods for screening human DNA excision repair proficiency

    International Nuclear Information System (INIS)

    Athas, W.F.

    1988-01-01

    A method for measuring DNA excision repair in response to ultraviolet radiation (UV)-induced DNA damage has been developed, validated, and field-tested in cultured human lymphocytes. The methodology is amenable to population-based screening and should facilitate future epidemiologic studies seeking to investigate associations between excision repair proficiency and cancer susceptibility. The impetus for such endeavors derives from the belief that the high incidence of skin cancer in the genetic disorder xeroderma pigmentosum (XP) primarily is a result of the reduced capacity of patients cells to repair UV-induced DNA damage. For assay, UV-irradiated non-replicating recombinant plasmid DNA harboring a chloramphenicol acetyltransferase (CAT) indicator gene is introduced into lymphocytes using DEAE-dextran short-term transfection conditions. Exposure to UV induces transcriptionally-inactivating DNA photoproducts in the plasmid DNA which inactivate CAT gene expression. Excision repair of the damaged CAT gene is monitored indirectly as a function of reactivated CAT enzyme activity following a 40 hour repair/expression incubation period

  6. Molecular requirements for radiation-activated recombination

    International Nuclear Information System (INIS)

    Stevens, Craig W.; Zeng Ming; Stamato, Thomas; Cerniglia, George

    1997-01-01

    Purpose/Objective: The major stumbling block to successful gene therapy today is poor gene transfer. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. We further hypothesized that known DNA-damage-repair proteins might also be important in radiation-activated recombination. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human (A549 and 39F) and rodent (NIH/3T3) cell lines. Continuous low dose rate and multiple radiation fractions were also tested. Nuclear extracts were made and the effect of irradiation on inter-plasmid recombination/ligation determined. Multiple DNA damage-repair deficient cell lines were tested for radiation-activated recombination. Results: A significant radiation dose-dependent improvement in stable plasmid transfection (by as much as 1300 fold) is demonstrated in neoplastic and primary cells. An improvement in transient plasmid transfection is also seen, with as much as 85% of cells transiently expressing b-galactosidase (20-50 fold improvement). Stable transfection is only improved for linearized or nicked plasmids. Cells have improved gene transfer for at least 96 hours after irradiation. Both fractionated and continuous low dose rate irradiation are effective at improving stable gene transfer in mammalian cells, thus making relatively high radiation dose delivery clinically feasible. Inter-plasmid recombination is radiation dose dependent in nuclear extract assays, and the type of overhang (3', 5' or blunt end) significantly affects recombination efficiency and the type of product. The most common end-joining activity involves filling-in of the overhang followed by blunt end ligation. Adenovirus is a linear, double stranded DNA virus. We demonstrate that adenoviral infection efficiency is increased by irradiation. The duration of transgene expression is lengthened because the virus integrates with high efficiency (∼10

  7. DNA degradation and reduced recombination following UV irradiation during meiosis in yeast (Saccharomyces cerevisiae)

    International Nuclear Information System (INIS)

    Salts, Y.; Pinon, R.; Simchen, G.

    1976-01-01

    Irradiation of meiotic yeast cells with moderate doses of ultraviolet irradiation (1,600 erg/mm 2 ) leads to the arrest of premeiotic DNA synthesis, massive (5-40%) DNA degradation, and a 40-50% loss of cell viability. In contrast, such doses of UV irradiation had a minor effect on viability (15-20% loss) of logarithmically growing cells, and no comparable DNA degradation was observed in irradiated synchronized vegetative cells. Meiotic recombination is also affected by UV irradiation. When administered at a stage comparable to meiotic prophase, low doses of irradiation result in a reduction in recombination frequency without significantly affecting cell viability. (orig.) [de

  8. RPA accumulation during class switch recombination represents 5'-3' DNA-end resection during the S-G2/M phase of the cell cycle.

    Science.gov (United States)

    Yamane, Arito; Robbiani, Davide F; Resch, Wolfgang; Bothmer, Anne; Nakahashi, Hirotaka; Oliveira, Thiago; Rommel, Philipp C; Brown, Eric J; Nussenzweig, Andre; Nussenzweig, Michel C; Casellas, Rafael

    2013-01-31

    Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Ensuring an exit strategy: RTEL1 restricts rogue recombination.

    Science.gov (United States)

    Villeneuve, Anne M

    2008-10-17

    Success of homologous recombination-based DNA repair depends not only on recombinases, which promote invasion of the homologous DNA duplex that serves as a template for repair, but also on antirecombinases, which dismantle recombination intermediates to allow completion of repair. In this issue, Barber et al. (2008) identify a previously elusive antirecombinase activity important for maintaining genome stability in animals.

  10. Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

    Directory of Open Access Journals (Sweden)

    Luisina De Tullio

    2017-10-01

    Full Text Available Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second in the 3′→5′ direction along ssDNA saturated with replication protein A (RPA. We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.

  11. Activated RecA protein may induce expression of a gene that is not controlled by the LexA repressor and whose function is required for mutagenesis and repair of UV-irradiated bacteriophage lambda

    International Nuclear Information System (INIS)

    Calsou, P.; Villaverde, A.; Defais, M.

    1987-01-01

    The activated form of the RecA protein (RecA) is known to be involved in the reactivation and mutagenesis of UV-irradiated bacteriophage lambda and in the expression of the SOS response in Escherichia coli K-12. The expression of the SOS response requires cleavage of the LexA repressor by RecA and the subsequent expression of LexA-controlled genes. The evidence presented here suggests that RecA induces the expression of a gene(s) that is not under LexA control and that is also necessary for maximal repair and mutagenesis of damaged phage. This conclusion is based on the chloramphenicol sensitivity of RecA -dependent repair and mutagenesis of damaged bacteriophage lambda in lexA(Def) hosts

  12. An att site-based recombination reporter system for genome engineering and synthetic DNA assembly.

    Science.gov (United States)

    Bland, Michael J; Ducos-Galand, Magaly; Val, Marie-Eve; Mazel, Didier

    2017-07-14

    Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla). The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested. The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

  13. Failure to induce a DNA repair gene, RAD54, in Saccharomyces cerevisiae does not affect DNA repair or recombination phenotypes

    International Nuclear Information System (INIS)

    Cole, G.M.; Mortimer, R.K.

    1989-01-01

    The Saccharomyces cerevisiae RAD54 gene is transcriptionally regulated by a broad spectrum of DNA-damaging agents. Induction of RAD54 by DNA-damaging agents is under positive control. Sequences responsible for DNA damage induction (the DRS element) lie within a 29-base-pair region from -99 to -70 from the most proximal transcription start site. This inducible promoter element is functionally separable from a poly(dA-dT) region immediately downstream which is required for constitutive expression. Deletions which eliminate induction of RAD54 transcription by DNA damage but do not affect constitutive expression have no effect on growth or survival of noninducible strains relative to wild-type strains in the presence of DNA-damaging agents. The DRS element is also not required for homothallic mating type switching, transcriptional induction of RAD54 during meiosis, meiotic recombination, or spontaneous or X-ray-induced mitotic recombination. We find no phenotype for a lack of induction of RAD54 message via the damage-inducible DRS, which raises significant questions about the physiology of DNA damage induction in S. cerevisiae

  14. Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats

    NARCIS (Netherlands)

    Warmerdam, Daniel O.; van den Berg, Jeroen; Medema, Rene H.

    2016-01-01

    rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded

  15. Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1.

    Science.gov (United States)

    Pryce, David W; Ramayah, Soshila; Jaendling, Alessa; McFarlane, Ramsay J

    2009-03-24

    DNA replication stress has been implicated in the etiology of genetic diseases, including cancers. It has been proposed that genomic sites that inhibit or slow DNA replication fork progression possess recombination hotspot activity and can form potential fragile sites. Here we used the fission yeast, Schizosaccharomyces pombe, to demonstrate that hotspot activity is not a universal feature of replication fork barriers (RFBs), and we propose that most sites within the genome that form RFBs do not have recombination hotspot activity under nonstressed conditions. We further demonstrate that Swi1, the TIMELESS homologue, differentially controls the recombination potential of RFBs, switching between being a suppressor and an activator of recombination in a site-specific fashion.

  16. Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

    Science.gov (United States)

    Tu, Qiang; Yin, Jia; Fu, Jun; Herrmann, Jennifer; Li, Yuezhong; Yin, Yulong; Stewart, A Francis; Müller, Rolf; Zhang, Youming

    2016-04-20

    Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.

  17. Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation.

    Science.gov (United States)

    De Tullio, Luisina; Kaniecki, Kyle; Kwon, Youngho; Crickard, J Brooks; Sung, Patrick; Greene, Eric C

    2017-10-17

    Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3'→5' direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Formation of (DNA)2-LNA triplet with recombinant base recognition: A quantum mechanical study

    Science.gov (United States)

    Mall, Vijaya Shri; Tiwari, Rakesh Kumar

    2018-05-01

    The formation of DNA triple helix offers the verity of new possibilities in molecular biology. However its applications are limited to purine and pyrimidine rich sequences recognized by forming Hoogsteen/Reverse Hoogsteen triplets in major groove sites of DNA duplex. To overcome this drawback modification in bases backbone and glucose of nucleotide unit of DNA have been proposed so that the third strand base recognized by both the bases of DNA duplex by forming Recombinant type(R-type) of bonding in mixed sequences. Here we performed Quanrum Mechanical (Hartree-Fock and DFT) methodology on natural DNA and Locked Nucleic Acids(LNA) triplets using 6-31G and some other new advance basis sets. Study suggests energetically stable conformation has been observed for recombinant triplets in order of G-C*G > A-T*A > G-C*C > T-A*T for both type of triplets. Interestingly LNA leads to more stable conformation in all set of triplets, clearly suggests an important biological tool to overcome above mentioned drawbacks.

  19. Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways.

    Science.gov (United States)

    Gupta, Richa; Barkan, Daniel; Redelman-Sidi, Gil; Shuman, Stewart; Glickman, Michael S

    2011-01-01

    Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in Escherichia coli and constitute the major pathway of error-free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via non-homologous end-joining (NHEJ) catalysed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways. © 2010 Blackwell Publishing Ltd.

  20. Postreplicational formation and repair of DNA double-strand breaks in UV-irradiated Escherichia coli uvrB cells

    International Nuclear Information System (INIS)

    Wang, Tzuchien V.; Smith, K.C.

    1986-01-01

    The number of DNA double-strand breaks formed in UV-irradiated uvrB recF recB cells correlates with the number of unrepaired DNA daughter-strand gaps, and is dependent on DNA synthesis after UV-irradiation. These results are consistent with the model that the DNA double-strand breaks that are produced in UV-irradiated excision-deficient cells occur as the result of breaks in the parental DNA opposite unrepaired DNA daughter-strand gaps. By employing a temperature-sensitive recA200 mutation, we have devised an improved assay for studying the formation and repair of these DNA double-strand breaks. Possible mechanisms for the postreplication repair of DNA double-strand breaks are discussed. (Auth.)

  1. Heteroduplex DNA position defines the roles of the Sgs1, Srs2, and Mph1 helicases in promoting distinct recombination outcomes.

    Directory of Open Access Journals (Sweden)

    Katrina Mitchel

    Full Text Available The contributions of the Sgs1, Mph1, and Srs2 DNA helicases during mitotic double-strand break (DSB repair in yeast were investigated using a gap-repair assay. A diverged chromosomal substrate was used as a repair template for the gapped plasmid, allowing mismatch-containing heteroduplex DNA (hDNA formed during recombination to be monitored. Overall DSB repair efficiencies and the proportions of crossovers (COs versus noncrossovers (NCOs were determined in wild-type and helicase-defective strains, allowing the efficiency of CO and NCO production in each background to be calculated. In addition, the products of individual NCO events were sequenced to determine the location of hDNA. Because hDNA position is expected to differ depending on whether a NCO is produced by synthesis-dependent-strand-annealing (SDSA or through a Holliday junction (HJ-containing intermediate, its position allows the underlying molecular mechanism to be inferred. Results demonstrate that each helicase reduces the proportion of CO recombinants, but that each does so in a fundamentally different way. Mph1 does not affect the overall efficiency of gap repair, and its loss alters the CO-NCO by promoting SDSA at the expense of HJ-containing intermediates. By contrast, Sgs1 and Srs2 are each required for efficient gap repair, strongly promoting NCO formation and having little effect on CO efficiency. hDNA analyses suggest that all three helicases promote SDSA, and that Sgs1 and Srs2 additionally dismantle HJ-containing intermediates. The hDNA data are consistent with the proposed role of Sgs1 in the dissolution of double HJs, and we propose that Srs2 dismantles nicked HJs.

  2. Structural studies on Mycobacterium tuberculosis RecA

    Indian Academy of Sciences (India)

    Structures of crystals of Mycobacterium tuberculosis RecA, grown and analysed under different conditions, provide insights into hitherto underappreciated details of molecular structure and plasticity. In particular, they yield information on the invariant and variable features of the geometry of the P-loop, whose binding to ATP ...

  3. Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate.

    Science.gov (United States)

    Kaiser, Gitte S; Germann, Susanne M; Westergaard, Tine; Lisby, Michael

    2011-08-01

    Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted. 2011 Elsevier B.V. All rights reserved.

  4. SAMHD1 Promotes DNA End Resection to Facilitate DNA Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Waaqo Daddacha

    2017-08-01

    Full Text Available DNA double-strand break (DSB repair by homologous recombination (HR is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity.

  5. Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells.

    Science.gov (United States)

    Murakawa, Yasuhiro; Sonoda, Eiichiro; Barber, Louise J; Zeng, Weihua; Yokomori, Kyoko; Kimura, Hiroshi; Niimi, Atsuko; Lehmann, Alan; Zhao, Guang Yu; Hochegger, Helfrid; Boulton, Simon J; Takeda, Shunichi

    2007-09-15

    Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.

  6. Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

    Directory of Open Access Journals (Sweden)

    Seto Anita G

    2000-11-01

    Full Text Available Abstract Background NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. Results We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. Conclusions We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

  7. FBH1 influences DNA replication fork stability and homologous recombination through ubiquitylation of RAD51

    DEFF Research Database (Denmark)

    Chu, Wai Kit; Payne, Miranda J; Beli, Petra

    2015-01-01

    Unscheduled homologous recombination (HR) can lead to genomic instability, which greatly increases the threat of neoplastic transformation in humans. The F-box DNA helicase 1 (FBH1) is a 3'-5' DNA helicase with a putative function as a negative regulator of HR. It is the only known DNA helicase t...

  8. Structural studies on Mycobacterium tuberculosis RecA: Molecular ...

    Indian Academy of Sciences (India)

    2015-01-11

    Jan 11, 2015 ... The molecular geometry of RecA and the location of the nucleotide binding site ...... the residue in all the glycerol complexes clusters together along with the two ..... an X-ray and molecular dynamics investigation on banana.

  9. Mixed infection of Sida jamaicensis in Jamaica reveals the presence of three recombinant begomovirus DNA A components.

    Science.gov (United States)

    Stewart, Cheryl; Kon, Tatsuya; Rojas, Maria; Graham, André; Martin, Darren; Gilbertson, Robert; Roye, Marcia

    2014-09-01

    Begomoviruses impose serious constraints on agriculture throughout the temperate, tropical and subtropical regions. Previously, we characterised a sida golden yellow vein virus isolate, SiGYVV-[JM:Lig2:08] (HQ009519-20) from a symptomatic Sida jamaicensis plant. With the aim of establishing whether it was hosting a mixed infection that could facilitate recombination, PCR-RFLP was done on DNA extracted from this plant, and the results suggested the presence of two additional genetically distinct DNA-A molecules. Sequence analysis of these two DNA-A molecules (relying on BLAST searches and the CLUSTAL V algorithm within the DNASTAR MegAlign module) revealed that they belonged to novel species, and we have tentatively named these viruses sida golden mosaic Braco virus-[Jamaica:Liguanea:2008] and sida golden mosaic Liguanea virus-[Jamaica:1:2008]. Using RDP4 (recombination detection program), we determined that all three viruses were recombinant, with bases ~10 to ~440 of both SiGMLigV-[JM:Lig:08] and SiGYVV-[JM:Lig2:08] having been derived from a relative of SiGMBV-[JM:Lig:08] (P<2.070×10(-7) for all seven of the recombination detection methods). SiGMBV-[JM:Lig:08] was itself a product of recombination, deriving bases ~490-1195 from a virus that was ~92% similar to malvastrum yellow mosaic Helshire virus. Phylogenetically, these DNA-A components are most closely related to those of malvaceous weed-infecting begomoviruses from Jamaica, Cuba, Florida and Mexico. The SiGMBV DNA-A was able to elicit symptomatic infection in N. benthamiana.

  10. Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

    Science.gov (United States)

    Throop, Andrea L.; LaBaer, Joshua

    2015-01-01

    The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

  11. Cell survival, UV-reactivation and induction of prophage lambda in Escherichia coli K12 overproducing RecA protein

    International Nuclear Information System (INIS)

    Quillardet, P.; Moreau, P.L.; Devoret, R.; Ginsburg, H.; Mount, D.W.

    1982-01-01

    The effect of the cellular level of RecA protein on the ability of E. coli K12 bacteria to (I) survive UV-irradiation (II) promote UV-reactivation of UV-damaged phage lambda (III) induce prophage lambda was determined in bacterial mutants with discrete increasing levels of RecA protein. The various levels of RecA protein were obtained by combining lexA and recA alleles. Except for the double mutant lexA3 recAo98, whose repair ability was 25% less than that observed in wild type bacteria, bacterial survival was proportional to the level of ReCA protein measured after 90 min of incubation. In lexA3 recAo98 bacteria, RecA protein, at a constitutive high basal level, failed to compensate totally for the lack of LexA repressor cleavage; UV-reactivation of UV-damaged phage lambda was not restored; yet, prophage lambda was induced with 35% efficiency. Efficient UV-induction of prophage lambda is linked to the induction of lexA-controlled host processes that repair the UV-damaged prophage. (orig.)

  12. Blocking the RecA activity and SOS-response in bacteria with a short α-helical peptide.

    Science.gov (United States)

    Yakimov, Alexander; Pobegalov, Georgii; Bakhlanova, Irina; Khodorkovskii, Mikhail; Petukhov, Michael; Baitin, Dmitry

    2017-09-19

    The RecX protein, a very active natural RecA protein inhibitor, can completely disassemble RecA filaments at nanomolar concentrations that are two to three orders of magnitude lower than that of RecA protein. Based on the structure of RecX protein complex with the presynaptic RecA filament, we designed a short first in class α-helical peptide that both inhibits RecA protein activities in vitro and blocks the bacterial SOS-response in vivo. The peptide was designed using SEQOPT, a novel method for global sequence optimization of protein α-helices. SEQOPT produces artificial peptide sequences containing only 20 natural amino acids with the maximum possible conformational stability at a given pH, ionic strength, temperature, peptide solubility. It also accounts for restrictions due to known amino acid residues involved in stabilization of protein complexes under consideration. The results indicate that a few key intermolecular interactions inside the RecA protein presynaptic complex are enough to reproduce the main features of the RecX protein mechanism of action. Since the SOS-response provides a major mechanism of bacterial adaptation to antibiotics, these results open new ways for the development of antibiotic co-therapy that would not cause bacterial resistance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Are High School Students Ready for Recombinant DNA?: The UOP Experience.

    Science.gov (United States)

    Minch, Michael J.

    1989-01-01

    Discusses a three-week summer college honors course for talented high school juniors with three exams, lab six days a week, a research paper, field trips, and student panel discussions. Presents an overview of the course. Describes the lab which uses "E. coli" for DNA recombination. (MVL)

  14. Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA.

    Science.gov (United States)

    Choi, Kyuha; Yelina, Nataliya E; Serra, Heïdi; Henderson, Ian R

    2017-01-01

    During meiosis, homologous chromosomes undergo recombination, which can result in formation of reciprocal crossover molecules. Crossover frequency is highly variable across the genome, typically occurring in narrow hotspots, which has a significant effect on patterns of genetic diversity. Here we describe methods to measure crossover frequency in plants at the hotspot scale (bp-kb), using allele-specific PCR amplification from genomic DNA extracted from the pollen of F 1 heterozygous plants. We describe (1) titration methods that allow amplification, quantification and sequencing of single crossover molecules, (2) quantitative PCR methods to more rapidly measure crossover frequency, and (3) application of high-throughput sequencing for study of crossover distributions within hotspots. We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions.

  15. Effect of mutagens, chemotherapeutic agents and defects in DNA repair genes on recombination in F' partial diploid Escherichia coli

    International Nuclear Information System (INIS)

    Norin, A.J.; Goldschmidt, E.P.

    1979-01-01

    The ability of mutagenic agents, nonmutagenic substances and defects in DNA repair to alter the genotype of F' partial diploid (F30) Escherichia coli was determined. The frequency of auxotrophic mutants and histidine requiring (His - ) haploid colonies was increased by mutagen treatment but Hfr colonies were not detected in F30 E. coli even with specific selection techniques. Genotype changes due to nonreciprocal recombination were determined by measuring the frequency of His - homogenotes, eg. F' hisC780, hisI + /hisC780, hisI + , arising from a His + heterogenote, F' hisC780 hisI + /hisC + , his1903. At least 75% of the recombinants were homozygous for histidine alleles which were present on the F' plasmid (exogenote) of the parental hetergenote rather than for histidine alleles on the chromosome. Mutagens, chemotherapeutic agents which block DNA synthesis and a defective DNA polymerase I gene, polA1, were found to increase the frequency of nonreciprocal recombination. A defect in the ability to excise thymine dimers, uvrC34, did not increase spontaneous nonreciprocal recombination. However, UV irradiation but not methyl methanesulfonate (MMS) induced greater recombination in this excision-repair defective mutant than in DNA-repair-proficient strains. (Auth.)

  16. The "Frankenplasmid" Lab: An Investigative Exercise for Teaching Recombinant DNA Methods

    Science.gov (United States)

    Dean, Derek M.; Wilder, Jason A.

    2011-01-01

    We describe an investigative laboratory module designed to give college undergraduates strong practical and theoretical experience with recombinant DNA methods within 3 weeks. After deducing restriction enzyme maps for two different plasmids, students ligate the plasmids together in the same reaction, transform "E. coli" with this mixture of…

  17. The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.

    Science.gov (United States)

    Gammon, Don B; Evans, David H

    2009-05-01

    Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

  18. Role of teh Rad52 Amino-terminal DNA Binding Activity in DNA Strand Capture in Homologous Recombination

    DEFF Research Database (Denmark)

    Shi, Idina; Hallwyl, Swee Chuang Lim; Seong, Changhyun

    2009-01-01

    Saccharomyces cerevisiae Rad52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified rad52-R70A mutant protein, with a compromised amino-ter...

  19. Induction of UV-resistant DNA replication in Escherichia coli: Induced stable DNA replication as an SOS function

    International Nuclear Information System (INIS)

    Kogoma, T.; Torrey, T.A.; Connaughton, M.J.

    1979-01-01

    The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA + lexA + -dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication. The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with nalidixic acid was severely suppressed in tif-1 lex A mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-5l mutation, an intragenic suppressor of lexA3. Induced stable DNA replication was found to be considerably more resistant to UV irradiation than normal replication both in a uvr A6 strain and a uvr + strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed. (orig.)

  20. Regulatory Control of the Resolution of DNA Recombination Intermediates during Meiosis and Mitosis

    OpenAIRE

    Matos, Joao; Blanco, Miguel G.; Maslen, Sarah; Skehel, J. Mark; West, Stephen C.

    2011-01-01

    The efficient and timely resolution of DNA recombination intermediates is essential for bipolar chromosome segregation. Here, we show that the specialized chromosome segregation patterns of meiosis and mitosis, which require the coordination of recombination with cell-cycle progression, are achieved by regulating the timing of activation of two crossover-promoting endonucleases. In yeast meiosis, Mus81-Mms4 and Yen1 are controlled by phosphorylation events that lead to their sequential activa...

  1. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    Science.gov (United States)

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  2. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    DEFF Research Database (Denmark)

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas Salhøj

    2014-01-01

    falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome...... of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens....

  3. Expression of human DNA polymerase β in Escherichia coli and characterization of the recombinant enzyme

    International Nuclear Information System (INIS)

    Abbotts, J.; SenGupta, D.N.; Zmudzka, B.; Widen, S.G.; Notario, V.; Wilson, S.H.

    1988-01-01

    The coding region of a human β-polymerase cDNA, predicting a 335 amino acid protein, was subcloned in the Escherichia coli expression plasmid pRC23. After induction of transformed cells, the crude soluble extract was found to contain a new protein immunoreactive with β-polymerase antibody and corresponding in size to the protein deduced from the cDNA. This protein was purified in a yield of 1-2 mg/50 g of cells. The recombinant protein had about the same DNA polymerase specific activity as β-polymerase purified from mammalian tissues, and template-primer specificity and immunological properties of the recombinant polymerase were similar to those of natural β-polymerases. The purified enzyme was free of nuclease activity. The authors studied detailed catalytic properties of the recombinant β-polymerase using defined template-primer systems. The results indicate that this β-polymerase is essentially identical with natural β-polymerases. The recombinant enzyme is distributive in mode of synthesis and is capable of detecting changes in the integrity of the single-stranded template, such as methylated bases and a double-stranded region. The enzyme recognizes a template region four to seven bases downstream of the primer 3' end and utilizes alternative primers if this downstream template region is double stranded. The enzyme is unable to synthesize past methylated bases N 3 -methyl-dT or O 6 -methyl-dG

  4. Functional Analysis of Breast Cancer Susceptibility Gene BRCA2

    National Research Council Canada - National Science Library

    Wang, Yingcai

    1999-01-01

    ...- specific RecA homologue, but not with XRCC2, Rad51D or the replication Protein (RPA). The specific interaction of BRCA2 and hsDMCl suggests that BRCA2 may be involved in DNA recombination and repair both in germ and somatic cells...

  5. Comparative proteomics reveals key proteins recruited at the nucleoid of Deinococcus after irradiation-induced DNA damage

    International Nuclear Information System (INIS)

    Bouthier de la Tour, Claire; Passot, Fanny Marie; Toueille, Magali; Servant, Pascale; Sommer, Suzanne; Mirabella, Boris; Blanchard, Laurence; Groot, Arjan de; Guerin, Philippe; Armengaud, Jean

    2013-01-01

    The nucleoids of radiation-resistant Deinococcus species show a high degree of compaction maintained after ionizing irradiation. We identified proteins recruited after irradiation in nucleoids of Deinococcus radiodurans and Deinococcus deserti by means of comparative proteomics. Proteins in nucleoid-enriched fractions from unirradiated and irradiated Deinococcus were identified and semi quantified by shotgun proteomics. The ssDNA-binding protein SSB, DNA gyrase subunits GyrA and GyrB, DNA topoisomerase I, RecA recombinase, UvrA excinuclease, RecQ helicase, DdrA, DdrB, and DdrD proteins were found in significantly higher amounts in irradiated nucleoids of both Deinococcus species. We observed, by immunofluorescence microscopy, the subcellular localization of these proteins in D. radiodurans, showing for the first time the recruitment of the DdrD protein into the D. radiodurans nucleoid. We specifically followed the kinetics of recruitment of RecA, DdrA, and DdrD to the nucleoid after irradiation. Remarkably, RecA proteins formed irregular filament-like structures 1 h after irradiation, before being redistributed throughout the cells by 3 h post-irradiation. Comparable dynamics of DdrD localization were observed, suggesting a possible functional interaction between RecA and DdrD. Several proteins involved in nucleotide synthesis were also seen in higher quantities in the nucleoids of irradiated cells, indicative of the existence of a mechanism for orchestrating the presence of proteins involved in DNA metabolism in nucleoids in response to massive DNA damage. All MS data have been deposited in the ProteomeXchange with identifier PXD00196. (authors)

  6. Direct ATP photolabeling of Escherichia coli recA proteins: identification of regions required for ATP binding

    International Nuclear Information System (INIS)

    Banks, G.R.; Sedgwick, S.G.

    1986-01-01

    When the Escherichia coli RecA protein is UV irradiated in the presence of [alpha- 32 P]ATP, a labeled protein--ATP adduct is formed. All the experimental evidence indicates that, in forming such an adduct, the ATP becomes specifically immobilized in the catalytically relevant ATP binding site. The adduct can also be identified after irradiation of E. coli cell lysates in a similar manner. This direct ATP photolabeling of RecA proteins has been used to identify regions of the polypeptide chain involved in the binding of ATP. The photolabeling of a RecA protein that lacks wild-type carboxy-terminal amino acids is not detectable. A RecA protein in which the amino-terminal sequence NH2-Ala-Ile-Asp-Glu-Asn- is replaced by NH2-Thr-Met-Ile-Thr-Asn-Ser-Ser-Ser- is only about 5% as efficiently photolabeled as the wild-type protein. Both of these RecA protein constructions, however, contain all the elements previously implicated, directly or indirectly, in the binding of ATP. ATP-photolabeled RecA protein has also been chemically cleaved at specific amino acids in order to identify regions of the polypeptide chain to which the nucleotide becomes covalently photolinked. The evidence is consistent with a region comprising amino acids 116-170. Thus, this work and that of others suggest that several disparate regions of the unfolded polypeptide chain may combine to form the ATP binding site upon protein folding or may influence binding through long-range effects

  7. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs

    Directory of Open Access Journals (Sweden)

    Gong Shiaochin

    2009-03-01

    Full Text Available Abstract Background Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. Results We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1 subclone genes of interest into BAC linking vectors, (2 insert desired reporter genes into respective genes and (3 link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. Conclusion The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  8. A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

    Science.gov (United States)

    Maye, Peter; Stover, Mary Louise; Liu, Yaling; Rowe, David W; Gong, Shiaochin; Lichtler, Alexander C

    2009-03-13

    Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

  9. Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    OpenAIRE

    Davidson, John F.; Fox, Richard; Harris, Dawn D.; Lyons-Abbott, Sally; Loeb, Lawrence A.

    2003-01-01

    Insertion of the T3 DNA polymerase thioredoxin binding domain (TBD) into the distantly related thermostable Taq DNA polymerase at an analogous position in the thumb domain, converts the Taq DNA polymerase from a low processive to a highly processive enzyme. Processivity is dependent on the presence of thioredoxin. The enhancement in processivity is 20–50-fold when compared with the wild-type Taq DNA polymerase or to the recombinant polymerase in the absence of thioredoxin. The recombinant Taq...

  10. Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

    Science.gov (United States)

    Zelensky, Alex N.; Sanchez, Humberto; Ristic, Dejan; Vidic, Iztok; van Rossum-Fikkert, Sari E.; Essers, Jeroen; Wyman, Claire; Kanaar, Roland

    2013-01-01

    Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair. PMID:23666627

  11. Effects of DNA double-strand and single-strand breaks on intrachromosomal recombination events in cell-cycle-arrested yeast cells

    International Nuclear Information System (INIS)

    Galli, A.; Schiestl, R.H.

    1998-01-01

    Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both γ-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas γ-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBsbut not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage. (author)

  12. Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3′ to 5′ translocase and helicase activities

    OpenAIRE

    Yakovleva, Lyudmila; Shuman, Stewart

    2012-01-01

    Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and...

  13. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

    Directory of Open Access Journals (Sweden)

    Olivier J Becherel

    2013-04-01

    Full Text Available Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2, plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI. Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops, and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

  14. Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State

    Directory of Open Access Journals (Sweden)

    Federico González

    2013-03-01

    Full Text Available Induced pluripotent stem cells (iPSCs hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs. Additional mechanistic studies uncover a direct role of the homologous recombination (HR pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

  15. Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

    Science.gov (United States)

    Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

    2015-07-01

    The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. [HPV DNA vaccines expressing recombinant CRT/HPV6bE7 fusion protein inhibit tumor growth and angiogenic activity].

    Science.gov (United States)

    Xu, Yan; Cheng, Hao; Zhao, Ke-Jia; Zhu, Ke-Jian; Zhang, Xing

    2007-11-01

    This paper was to study the angiogenic inhibitory effect and the potential antitumor effect of the constructed recombinant DNA vaccine CRT/HPV6bE7 in vivo. The C57BL/6 mice were vaccinated respectively with recombinant CRT/HPV6bE7 DNA plamids. The inhibitory effects on angiogenesis of generated vaccines in vivo were evaluated by a bFGF-induced angiogenesis assay using the Matrigel kit. To investigate the potential antitumor effect, the mean tumor weights, sizes and tumor appearing times were measured in C57BL/6 mice treated with HPV6bE7-expressing B16 cells. The results indicated that the recombinants CRT180/HPV6bE7 and CRT180 showed strong anti-angiogenic effects in bFGF-induced angiogenesis in vivo. Moreover, CRT180/HPV6bE7 and CRT180 DNA vaccines could significantly inhibit the tumor growth in tumor challenge experiment, and CRT180/HPV6bE7 was superior to other vaccines in delaying tumor formation time, limiting tumor size and weight in tumor protection experiment. In conclusion, recombinant CRT180/HPV6bE7 DNA could elicit a most efficient anti-angiogenic effect and inhibit tumor growth in mice inoculated with DNA vaccines. The antiangiogenic activity of CRT were suggested residing in a domain between CRT 120-180 aa.

  17. A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily.

    Science.gov (United States)

    Sang, Pau Biak; Srinath, Thiruneelakantan; Patil, Aravind Goud; Woo, Eui-Jeon; Varshney, Umesh

    2015-09-30

    Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. A PHF8 homolog in C. elegans promotes DNA repair via homologous recombination.

    Directory of Open Access Journals (Sweden)

    Changrim Lee

    Full Text Available PHF8 is a JmjC domain-containing histone demethylase, defects in which are associated with X-linked mental retardation. In this study, we examined the roles of two PHF8 homologs, JMJD-1.1 and JMJD-1.2, in the model organism C. elegans in response to DNA damage. A deletion mutation in either of the genes led to hypersensitivity to interstrand DNA crosslinks (ICLs, while only mutation of jmjd-1.1 resulted in hypersensitivity to double-strand DNA breaks (DSBs. In response to ICLs, JMJD-1.1 did not affect the focus formation of FCD-2, a homolog of FANCD2, a key protein in the Fanconi anemia pathway. However, the dynamic behavior of RPA-1 and RAD-51 was affected by the mutation: the accumulations of both proteins at ICLs appeared normal, but their subsequent disappearance was retarded, suggesting that later steps of homologous recombination were defective. Similar changes in the dynamic behavior of RPA-1 and RAD-51 were seen in response to DSBs, supporting a role of JMJD-1.1 in homologous recombination. Such a role was also supported by our finding that the hypersensitivity of jmjd-1.1 worms to ICLs was rescued by knockdown of lig-4, a homolog of Ligase 4 active in nonhomologous end-joining. The hypersensitivity of jmjd-1.1 worms to ICLs was increased by rad-54 knockdown, suggesting that JMJD-1.1 acts in parallel with RAD-54 in modulating chromatin structure. Indeed, the level of histone H3 Lys9 tri-methylation, a marker of heterochromatin, was higher in jmjd-1.1 cells than in wild-type cells. We conclude that the histone demethylase JMJD-1.1 influences homologous recombination either by relaxing heterochromatin structure or by indirectly regulating the expression of multiple genes affecting DNA repair.

  19. RPA mediates recombination repair during replication stress and is displaced from DNA by checkpoint signalling in human cells

    DEFF Research Database (Denmark)

    Sleeth, Kate M; Sørensen, Claus Storgaard; Issaeva, Natalia

    2007-01-01

    The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited...... the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation...... and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role...

  20. Generating and repairing genetically programmed DNA breaks during immunoglobulin class switch recombination

    Science.gov (United States)

    Nicolas, Laura; Cols, Montserrat; Choi, Jee Eun; Chaudhuri, Jayanta; Vuong, Bao

    2018-01-01

    Adaptive immune responses require the generation of a diverse repertoire of immunoglobulins (Igs) that can recognize and neutralize a seemingly infinite number of antigens. V(D)J recombination creates the primary Ig repertoire, which subsequently is modified by somatic hypermutation (SHM) and class switch recombination (CSR). SHM promotes Ig affinity maturation whereas CSR alters the effector function of the Ig. Both SHM and CSR require activation-induced cytidine deaminase (AID) to produce dU:dG mismatches in the Ig locus that are transformed into untemplated mutations in variable coding segments during SHM or DNA double-strand breaks (DSBs) in switch regions during CSR. Within the Ig locus, DNA repair pathways are diverted from their canonical role in maintaining genomic integrity to permit AID-directed mutation and deletion of gene coding segments. Recently identified proteins, genes, and regulatory networks have provided new insights into the temporally and spatially coordinated molecular interactions that control the formation and repair of DSBs within the Ig locus. Unravelling the genetic program that allows B cells to selectively alter the Ig coding regions while protecting non-Ig genes from DNA damage advances our understanding of the molecular processes that maintain genomic integrity as well as humoral immunity. PMID:29744038

  1. Top3 processes recombination intermediates and modulates checkpoint activity after DNA damage

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Hickson, Ian D

    2006-01-01

    Mutation of TOP3 in Saccharomyces cerevisiae causes poor growth, hyperrecombination, and a failure to fully activate DNA damage checkpoints in S phase. Here, we report that overexpression of a dominant-negative allele of TOP3, TOP3(Y356F), which lacks the catalytic (decatenation) activity of Top3......, the catalytic activity of Top3 is not required for DNA damage checkpoint activation, but it is required for normal S-phase progression after DNA damage. We also present evidence that the checkpoint-mediated cell cycle delay and persistence of X-shaped DNA molecules resulting from overexpression of TOP3(Y356F......) are downstream of Rad51 function. We propose that Top3 functions in S phase to both process homologous recombination intermediates and modulate checkpoint activity....

  2. Methotrexate induces DNA damage and inhibits homologous recombination repair in choriocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Xie L

    2016-11-01

    Full Text Available Lisha Xie,1,* Tiancen Zhao,1,2,* Jing Cai,1 You Su,1 Zehua Wang,1 Weihong Dong1 1Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Department of Obstetrics and Gynecology, Central Hospital of Wuhan, Wuhan, China *These authors contributed equally to this work Objective: The objective of this study was to investigate the mechanism of sensitivity to methotrexate (MTX in human choriocarcinoma cells regarding DNA damage response. Methods: Two choriocarcinoma cancer cell lines, JAR and JEG-3, were utilized in this study. An MTX-sensitive osteosarcoma cell line MG63, an MTX-resistant epithelial ovarian cancer cell line A2780 and an MTX-resistant cervical adenocarcinoma cell line Hela served as controls. Cell viability assay was carried out to assess MTX sensitivity of cell lines. MTX-induced DNA damage was evaluated by comet assay. Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of BRCA1, BRCA2, RAD51 and RAD52. The protein levels of γH2AX, RAD 51 and p53 were analyzed by Western blot. Results: Remarkable DNA strand breaks were observed in MTX-sensitive cell lines (JAR, JEG-3 and MG63 but not in MTX-resistant cancer cells (A2780 and Hela after 48 h of MTX treatment. Only in the choriocarcinoma cells, the expression of homologous recombination (HR repair gene RAD51 was dramatically suppressed by MTX in a dose- and time-dependent manner, accompanied with the increase in p53. Conclusion: The MTX-induced DNA strand breaks accompanied by deficiencies in HR repair may contribute to the hypersensitivity to chemotherapy in choriocarcinoma. Keywords: choriocarcinoma, chemotherapy hypersensitivity, DNA double-strand break, RAD51, p53

  3. Regulation of homologous recombination in eukaryotes

    OpenAIRE

    Heyer, Wolf-Dietrich; Ehmsen, Kirk T.; Liu, Jie

    2010-01-01

    Homologous recombination is required for accurate chromosome segregation during the first meiotic division and constitutes a key repair and tolerance pathway for complex DNA damage including DNA double-stranded breaks, interstrand crosslinks, and DNA gaps. In addition, recombination and replication are inextricably linked, as recombination recovers stalled and broken replication forks enabling the evolution of larger genomes/replicons. Defects in recombination lead to genomic instability and ...

  4. SV40 host-substituted variants: a new look at the monkey DNA inserts and recombinant junctions.

    Science.gov (United States)

    Singer, Maxine; Winocour, Ernest

    2011-04-10

    The available monkey genomic data banks were examined in order to determine the chromosomal locations of the host DNA inserts in 8 host-substituted SV40 variant DNAs. Five of the 8 variants contained more than one linked monkey DNA insert per tandem repeat unit and in all cases but one, the 19 monkey DNA inserts in the 8 variants mapped to different locations in the monkey genome. The 50 parental DNAs (32 monkey and 18 SV40 DNA segments) which spanned the crossover and flanking regions that participated in monkey/monkey and monkey/SV40 recombinations were characterized by substantial levels of microhomology of up to 8 nucleotides in length; the parental DNAs also exhibited direct and inverted repeats at or adjacent to the crossover sequences. We discuss how the host-substituted SV40 variants arose and the nature of the recombination mechanisms involved. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Toxicity evaluation of e-juice and its soluble aerosols generated by electronic cigarettes using recombinant bioluminescent bacteria responsive to specific cellular damages.

    Science.gov (United States)

    Bharadwaj, Shiv; Mitchell, Robert J; Qureshi, Anjum; Niazi, Javed H

    2017-04-15

    Electronic-cigarettes (e-cigarette) are widely used as an alternative to traditional cigarettes but their safety is not well established. Herein, we demonstrate and validate an analytical method to discriminate the deleterious effects of e-cigarette refills (e-juice) and soluble e-juice aerosol (SEA) by employing stress-specific bioluminescent recombinant bacterial cells (RBCs) as whole-cell biosensors. These RBCs carry luxCDABE-operon tightly controlled by promoters that specifically induced to DNA damage (recA), superoxide radicals (sodA), heavy metals (copA) and membrane damage (oprF). The responses of the RBCs following exposure to various concentrations of e-juice/SEA was recorded in real-time that showed dose-dependent stress specific-responses against both the e-juice and vaporized e-juice aerosols produced by the e-cigarette. We also established that high doses of e-juice (4-folds diluted) lead to cell death by repressing the cellular machinery responsible for repairing DNA-damage, superoxide toxicity, ion homeostasis and membrane damage. SEA also caused the cellular damages but the cells showed enhanced bioluminescence expression without significant growth inhibition, indicating that the cells activated their global defense system to repair these damages. DNA fragmentation assay also revealed the disintegration of total cellular DNA at sub-toxic doses of e-juice. Despite their state of matter, the e-juice and its aerosols induce cytotoxicity and alter normal cellular functions, respectively that raises concerns on use of e-cigarettes as alternative to traditional cigarette. The ability of RBCs in detecting both harmful effects and toxicity mechanisms provided a fundamental understanding of biological response to e-juice and aerosols. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae) provide evidence for pervasive mitochondrial DNA recombination.

    Science.gov (United States)

    Sammler, Svenja; Bleidorn, Christoph; Tiedemann, Ralph

    2011-01-14

    Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA) may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle". Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni) and 22,737 bp (P. panini), they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation. The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB), which has been described from the chicken mitochondrial genome. As this RFB

  7. Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae provide evidence for pervasive mitochondrial DNA recombination

    Directory of Open Access Journals (Sweden)

    Bleidorn Christoph

    2011-01-01

    Full Text Available Abstract Background Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle". Results Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni and 22,737 bp (P. panini, they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation. Conclusions The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB, which has been

  8. BCR/ABL downregulates DNA-PK(CS)-dependent and upregulates backup non-homologous end joining in leukemic cells.

    Science.gov (United States)

    Poplawski, Tomasz; Blasiak, Janusz

    2010-06-01

    Non-homologous end joining (NHEJ) and homologous recombination repair (HRR) are the main mechanisms involved in the processing of DNA double strand breaks (DSBs) in humans. We showed previously that the oncogenic tyrosine kinase BCR/ABL stimulated DSBs repair by HRR. To evaluate the role of BCR/ABL in DSBs repair by NHEJ we examined the ability of leukemic BCR/ABL-expressing cell line BV173 to repair DNA damage induced by two DNA topoisomerase II inhibitors: etoposide and sobuzoxane. DNA lesions induced by sobuzoxane are repaired by a NHEJ pathway which is dependent on the catalytic subunit of protein kinase dependent on DNA (DNA-PK(CS); D-NHEJ), whereas damage evoked by etoposide are repaired by two distinct NHEJ pathways, dependent on or independent of DNA-PK(CS) (backup NHEJ, B-NHEJ). Cells incubated with STI571, a highly specific inhibitor of BCR/ABL, displayed resistance to these agents associated with an accelerated kinetics of DSBs repair, as measured by the neutral comet assay and pulsed field gel electrophoresis. However, in a functional NHEJ assay, cells preincubated with STI571 repaired DSBs induced by a restriction enzyme with a lower efficacy than without the preincubation and addition of wortmannin, a specific inhibitor of DNA-PK(CS), did not change efficacy of the NHEJ reaction. We suggest that BCR/ABL switch on B-NHEJ which is more error-prone then D-NHEJ and in such manner contribute to the increase of the genomic instability of leukemic cells.

  9. Induction of genetic recombination in the lambda bacteriophage by ultraviolet radiation of the Escherichia Coli cells

    International Nuclear Information System (INIS)

    Alcantara D, D.

    1986-12-01

    In this work there are reported the results that show that although the stimulation of the recombination of the Lambda bacteriophage, by UV irradiation of the cells of Escherichia Coli, it looks to be the result of the high expression of the functions of the SOS system, doesn't keep some relationship with the high concentration of protein reached RecA. (Author)

  10. PRKDC mutations associated with immunodeficiency, granuloma, and autoimmune regulator-dependent autoimmunity

    NARCIS (Netherlands)

    A.-L. Mathieu (Anne-Laure); E. Verronese (Estelle); G.I. Rice (Gillian I.); F. Fouyssac (Fanny); Y. Bertrand (Yves); C. Picard (Capucine); M. Chansel (Marie); J.E. Walter (Jolan E.); L.D. Notarangelo (Luigi Daniele); M.J. Butte (Manish J.); K.C. Nadeau (Kari Christine); K. Csomos (Krisztian); D.J. Chen (David); K. Chen (Karin); A. Delgado (Ana); C. Rigal (Chantal); C. Bardin (Christine); C. Schuetz (Catharina); D. Moshous (Despina); H. Reumaux (Héloïse); F. Plenat (François); A. Phan (Alice); M.-T. Zabot (Marie-Thérèse); B. Balme (Brigitte); S. Viel (Sébastien); J. Bienvenu (Jacques); P. Cochat (Pierre); M. van der Burg (Mirjam); C. Caux (Christophe); E.H. Kemp (E. Helen); I. Rouvet (Isabelle); C. Malcus (Christophe); J.-F. Méritet (Jean-Francois); A. Lim (Annick); Y.J. Crow (Yanick J.); N. Fabien (Nicole); C. Ménétrier-Caux (Christine); J.-P. De Villartay (Jean-Pierre); T. Walzer (Thierry); A. Belot (Alexandre)

    2015-01-01

    textabstractBackground PRKDC encodes for DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a kinase that forms part of a complex (DNA-dependent protein kinase [DNA-PK]) crucial for DNA double-strand break repair and V(D)J recombination. In mice DNA-PK also interacts with the transcription

  11. Immune responses elicited by Mycoplasma hyopneumoniae recombinant antigens and DNA constructs with potential for use in vaccination against porcine enzootic pneumonia.

    Science.gov (United States)

    Virginio, Veridiana Gomes; Gonchoroski, Taylor; Paes, Jéssica Andrade; Schuck, Desirée Cigaran; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

    2014-10-07

    Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (PEP) and causes major economic losses to the pig industry worldwide. Commercially available vaccines provide only partial protection and are relatively expensive. In this study, we assessed the humoral and cellular immune responses to three recombinant antigens of M. hyopneumoniae. Immune responses to selected domains of the P46, HSP70 and MnuA antigens (P46102-253, HSP70212-601 and MnuA182-378), delivered as recombinant subunit or DNA vaccines, were evaluated in BALB/c mice. All purified recombinant antigens and two DNA vaccines, pcDNA3.1(+)/HSP70212-601 and pcDNA3.1(+)/MnuA182-378, elicited a strong humoral immune response, indicated by high IgG levels in the serum. The cellular immune response was assessed by detection of IFN-γ, IL-10 and IL-4 in splenocyte culture supernatants. The recombinant subunit and DNA vaccines induced Th1-polarized immune responses, as evidenced by increased levels of IFN-γ. All recombinant subunit vaccines and the pcDNA3.1(+)/MnuA182-378 vaccine also induced the secretion of IL-10, a Th2-type cytokine, in large quantities. The mixed Th1/Th2-type response may elicit an effective immune response against M. hyopneumoniae, suggesting that P46102-253, HSP70212-601 and MnuA182-378 are potential novel and promising targets for the development of vaccines against PEP. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination.

    Science.gov (United States)

    Masani, Shahnaz; Han, Li; Meek, Katheryn; Yu, Kefei

    2016-02-02

    Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.

  13. Rev1 Recruits Ung to Switch Regions and Enhances dU Glycosylation for Immunoglobulin Class Switch DNA Recombination

    Directory of Open Access Journals (Sweden)

    Hong Zan

    2012-11-01

    Full Text Available By diversifying the biological effector functions of antibodies, class switch DNA recombination (CSR plays a critical role in the maturation of the immune response. It is initiated by activation-induced cytidine deaminase (AID-mediated deoxycytosine deamination, yielding deoxyuridine (dU, and dU glycosylation by uracil DNA glycosylase (Ung in antibody switch (S region DNA. Here we showed that the translesion DNA synthesis polymerase Rev1 directly interacted with Ung and targeted in an AID-dependent and Ung-independent fashion the S regions undergoing CSR. Rev1−/− Ung+/+ B cells reduced Ung recruitment to S regions, DNA-dU glycosylation, and CSR. Together with an S region spectrum of mutations similar to that of Rev1+/+ Ung−/− B cells, this suggests that Rev1 operates in the same pathway as Ung, as emphasized by further decreased CSR in Rev1−/− Msh2−/− B cells. Rescue of CSR in Rev1−/− B cells by a catalytically inactive Rev1 mutant shows that the important role of Rev1 in CSR is mediated by Rev1’s scaffolding function, not its enzymatic function.

  14. Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spore DNA

    International Nuclear Information System (INIS)

    Setlow, B.; Hand, A.R.; Setlow, P.

    1991-01-01

    Small, acid-soluble proteins (SASP) of the alpha/beta-type are associated with DNA in spores of Bacillus subtilis. Induction of synthesis of alpha/beta-type SASP in Escherichia coli resulted in rapid cessation of DNA synthesis, followed by a halt in RNA and then protein accumulation, although significant mRNA and protein synthesis continued. There was a significant loss in viability associated with SASP synthesis in E. coli: recA+ cells became extremely long filaments, whereas recA mutant cells became less filamentous. The nucleoids of cells with alpha/beta-type SASP were extremely condensed, as viewed in both light and electron microscopes, and immunoelectron microscopy showed that the alpha/beta-type SASP were associated with the cell DNA. Induction of alpha/beta-type SASP synthesis in E. coli increased the negative superhelical density of plasmid DNA by approximately 20%; UV irradiation of E. coli with alpha/beta-type SASP gave reduced yields of thymine dimers but significant amounts of the spore photoproduct. These changes in E. coli DNA topology and photochemistry due to alpha/beta-type SASP are similar to the effects of alpha/beta-type SASP on the DNA in Bacillus spores, further suggesting that alpha/beta-type SASP are a major factor determining DNA properties in bacterial spores

  15. Excess single-stranded DNA inhibits meiotic double-strand break repair.

    Directory of Open Access Journals (Sweden)

    Rebecca Johnson

    2007-11-01

    Full Text Available During meiosis, self-inflicted DNA double-strand breaks (DSBs are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE, in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects

  16. Recombinant cells and organisms having persistent nonstandard amino acid dependence and methods of making them

    Science.gov (United States)

    Church, George M.; Mandell, Daniel J.; Lajoie, Marc J.

    2017-12-05

    Recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

  17. Yeast Tdh3 (glyceraldehyde 3-phosphate dehydrogenase is a Sir2-interacting factor that regulates transcriptional silencing and rDNA recombination.

    Directory of Open Access Journals (Sweden)

    Alison E Ringel

    Full Text Available Sir2 is an NAD(+-dependent histone deacetylase required to mediate transcriptional silencing and suppress rDNA recombination in budding yeast. We previously identified Tdh3, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH, as a high expression suppressor of the lethality caused by Sir2 overexpression in yeast cells. Here we show that Tdh3 interacts with Sir2, localizes to silent chromatin in a Sir2-dependent manner, and promotes normal silencing at the telomere and rDNA. Characterization of specific TDH3 alleles suggests that Tdh3's influence on silencing requires nuclear localization but does not correlate with its catalytic activity. Interestingly, a genetic assay suggests that Tdh3, an NAD(+-binding protein, influences nuclear NAD(+ levels; we speculate that Tdh3 links nuclear Sir2 with NAD(+ from the cytoplasm.

  18. Effects of radiations on DNA and repair of the damage. Progress report, May 1, 1976--March 31, 1977

    International Nuclear Information System (INIS)

    Hutchinson, F.

    1977-01-01

    Last year's report that repair of DNA double-strand breaks from gamma rays occurs in E. coli was verified by additional experiments. Such repair requires recA function and the presence of another DNA molecule of the same base sequence, so it may involve a recombination-like event. Ultraviolet light acting on DNA containing bromouracil produces double-strand breaks by single photochemical events, and a single model can explain this as well as other results. Strains of E. coli which are unusually mutable by bromouracil--uvrE, mutL, mutR, mutS, are defective in mismatch repair. This strengthens the suggestion in last year's report that such mutagenesis occurs when enzymes responsible for the removal of mismatched bases are unable to remove all the mismatches. Ultraviolet mutagenesis of lambda phage may be a useful model for the study of mutagenesis in cells, because the effects of lesions in the gene mutated (i.e., in the phage) and changes in enzyme systems (by treating the host cells) can be examined separately. Quantitative data support this approach

  19. Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

    International Nuclear Information System (INIS)

    Billen, D.

    1987-01-01

    When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed

  20. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    Science.gov (United States)

    Hirsch, Matthew L; Fagan, B Matthew; Dumitru, Raluca; Bower, Jacquelyn J; Yadav, Swati; Porteus, Matthew H; Pevny, Larysa H; Samulski, R Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  1. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Matthew L Hirsch

    Full Text Available Human embryonic stem cells (hESCs are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  2. Effect of recB21, uvrD3, lexA101 and recF143 mutations on ultraviolet radiation sensitivity and genetic recombination in ΔuvrB strains of Escherichia coli K-12

    International Nuclear Information System (INIS)

    Wang, T.V.; Smith, K.C.

    1981-01-01

    The interaction of the recB21, uvrD3, lexA101, and recF143 mutations on UV radiation sensitization and genetic recombination was studied in isogenic strains containing all possible combinations of these mutations in a ΔuvrB genetic background. The relative UV radiation sensitivities of the multiply mutant strains in the ΔuvrB background were: recF recB lexA > recF recB uvrD lexA, recF recB uvrD > recA > recF uvrD lexA > recF recB, recF uvrD > recF lexA > recB uvrD lexA > recB uvrD > recB lexA, lexA uvrD > recB > lexA, uvrD > recF; three of these strains were more UV radiation sensitive than the uvrB recA strain. There was no correlation between the degree of radiation sensitivity and the degree of deficiency in genetic recombination. An analysis of the survival curves revealed that the recF mutation interacts synergistically with the recB, uvrD, and lexA mutations in UV radiation sensitization, while the recB, uvrD, and lexA mutations appear to interact additively with each other. We interpret these data to suggest that there are two major independent pathways for postreplication repair; one is dependent on the recF gene, and the other is dependent on the recB, uvrD, and lexA genes. (orig.)

  3. Deficiency of the Arabidopsis helicase RTEL1 triggers a SOG1-dependent replication checkpoint in response to DNA cross-links.

    Science.gov (United States)

    Hu, Zhubing; Cools, Toon; Kalhorzadeh, Pooneh; Heyman, Jefri; De Veylder, Lieven

    2015-01-01

    To maintain genome integrity, DNA replication is executed and regulated by a complex molecular network of numerous proteins, including helicases and cell cycle checkpoint regulators. Through a systematic screening for putative replication mutants, we identified an Arabidopsis thaliana homolog of human Regulator of Telomere Length 1 (RTEL1), which functions in DNA replication, DNA repair, and recombination. RTEL1 deficiency retards plant growth, a phenotype including a prolonged S-phase duration and decreased cell proliferation. Genetic analysis revealed that rtel1 mutant plants show activated cell cycle checkpoints, specific sensitivity to DNA cross-linking agents, and increased homologous recombination, but a lack of progressive shortening of telomeres, indicating that RTEL1 functions have only been partially conserved between mammals and plants. Surprisingly, RTEL1 deficiency induces tolerance to the deoxynucleotide-depleting drug hydroxyurea, which could be mimicked by DNA cross-linking agents. This resistance does not rely on the essential replication checkpoint regulator WEE1 but could be blocked by a mutation in the SOG1 transcription factor. Taken together, our data indicate that RTEL1 is required for DNA replication and that its deficiency activates a SOG1-dependent replication checkpoint. © 2015 American Society of Plant Biologists. All rights reserved.

  4. DNA hybrids suggesting a recombination process repairing radiation-induced DNA double-strand breaks in Ehrlich Ascites tumor cells

    International Nuclear Information System (INIS)

    Barthel, H.R.

    1984-01-01

    The results presented suggest the possibility of repair of DNA double-strand breaks by recombination, at least in the S and G 2 -phases of the cell cycle, in mammalian cells. Further experiments with synchronized cell cultures will have to show whether this process may also occur in the G 1 -phase of the cell cycle. (orig./AJ) [de

  5. GANP regulates the choice of DNA repair pathway by DNA-PKcs interaction in AID-dependent IgV region diversification.

    Science.gov (United States)

    Eid, Mohammed Mansour Abbas; Maeda, Kazuhiko; Almofty, Sarah Ameen; Singh, Shailendra Kumar; Shimoda, Mayuko; Sakaguchi, Nobuo

    2014-06-15

    RNA export factor germinal center-associated nuclear protein (GANP) interacts with activation-induced cytidine deaminase (AID) and shepherds it from the cytoplasm to the nucleus and toward the IgV region loci in B cells. In this study, we demonstrate a role for GANP in the repair of AID-initiated DNA damage in chicken DT40 B cells to generate IgV region diversity by gene conversion and somatic hypermutation. GANP plays a positive role in IgV region diversification of DT40 B cells in a nonhomologous end joining-proficient state. DNA-PKcs physically interacts with GANP, and this interaction is dissociated by dsDNA breaks induced by a topoisomerase II inhibitor, etoposide, or AID overexpression. GANP affects the choice of DNA repair mechanism in B cells toward homologous recombination rather than nonhomologous end joining repair. Thus, GANP presumably plays a critical role in protection of the rearranged IgV loci by favoring homologous recombination of the DNA breaks under accelerated AID recruitment. Copyright © 2014 by The American Association of Immunologists, Inc.

  6. Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli

    DEFF Research Database (Denmark)

    Williamson, M R; Doherty, J P; Woodcock, D M

    1993-01-01

    .HpaII plus M.HhaI) or (2) methylated with M.SssI which methylates at all CpG dinucleotides. These two protocols generated theoretical levels of DNA methylation in the central fragment of 10.5% and 33%, respectively. The construct was transformed into a series of isogenic (recA+) bacterial strains that were...

  7. 75 FR 31795 - Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting

    Science.gov (United States)

    2010-06-04

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting ACTION: Notice of cancellation of... information. Dated: May 26, 2010. Jacqueline Corrigan-Curay, Acting Director, Office of Biotechnology...

  8. Genetic recombination induced by DNA double-strand break in bacteriophage T4: nature of the left/right bias.

    Science.gov (United States)

    Shcherbakov, Victor P; Shcherbakova, Tamara; Plugina, Lidiya; Sizova, Svetlana; Kudryashova, Elena; Granovsky, Igor

    2008-06-01

    The experimental system combining double-strand breaks (DSBs), produced site-specifically by SegC endonuclease, with the famous advantages of the bacteriophage T4 rII mutant recombination analysis was used here to elucidate the origin of the recombination bias on two sides of the DSB, especially pronounced in gene 39 (topoisomerase II) and gene 59 (41-helicase loader) mutants. Three sources were found to contribute to the bias: (1) the SegC endonuclease may remain bound to the end of the broken DNA and thus protect it from exonuclease degradation; (2) in heteroduplex heterozygotes (HHs), arising as the recombinant products in the left-hand crosses, the transcribed strands are of rII mutant phenotype, so they, in contrast to the right-hand HHs, do not produce plaques on the lawn of the lambda-lysogenic host; and (3) the intrinsic polarity of T4 chromosome, reflected in transcription, may be a cause for discrimination of promoter-proximal and promoter-distal DNA sequences. It is shown that the apparent recombination bias does not imply one-sidedness of the DSB repair but just reflects a different depth of the end processing. It is inferred that the cause, underlying the "intrinsic" bias, might be interference between strand exchange and transcription. Topoisomerase and helicase functions are necessary to turn the process in favor of strand exchange. The idea is substantiated that the double-stranded to single-stranded DNA transition edge (not ss-DNA tip) serves as an actual recombinogenic element.

  9. Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

    International Nuclear Information System (INIS)

    Atchley, S.V.; Chen, D.J.C.; Strniste, G.F.; Walters, R.A.; Moyzis, R.K.

    1984-01-01

    We are developing a new recombinant DNA system for the detection and measurement of genetic change in humans caused by exposure to low level ionizing radiation. A unique feature of the method is the use of cloned repetitive DNA probes to assay human DNA for structural changes during or after irradiation. Repetitive sequences exist in different families. Collectively they constitute over 25% of the DNA in a human cell. Repeat families have between 10 and 500,000 members. We have constructed repetitive DNA sequence libraries using recombinant DNA techniques. From these libraries we have isolated and characterized individual repeats comprising 75 to 90% of the mass of human repetitive DNA. Repeats used in our assay system exist in tandem arrays in the genome. Perturbation of these sequences in a cell, followed by detection with a repeat probe, produces a new, multimeric ''ladder'' pattern on an autoradiogram. The repeat probe used in our initial study is complementary to 1% of human DNA. Therefore, the sensitivity of this method is several orders of magnitude better than existing assays. Preliminary evidence from human skin cells exposed to acute, low-dose x-ray treatments indicates that DNA is affected at a dose as low as 5R. The radiation doses used in this system are well within the range of doses received by astronauts during spaceflight missions. Due to its small material requirements, this technique could easily be adapted for use in space. 16 refs., 1 fig

  10. Temperature and carrier-density dependence of Auger and radiative recombination in nitride optoelectronic devices

    International Nuclear Information System (INIS)

    Kioupakis, Emmanouil; Yan, Qimin; Steiauf, Daniel; Van de Walle, Chris G

    2013-01-01

    Nitride light-emitting diodes are a promising solution for efficient solid-state lighting, but their performance at high power is affected by the efficiency-droop problem. Previous experimental and theoretical work has identified Auger recombination, a three-particle nonradiative carrier recombination mechanism, as the likely cause of the droop. In this work, we use first-principles calculations to elucidate the dependence of the radiative and Auger recombination rates on temperature, carrier density and quantum-well confinement. Our calculated data for the temperature dependence of the recombination coefficients are in good agreement with experiment and provide further validation on the role of Auger recombination in the efficiency reduction. Polarization fields and phase-space filling negatively impact device efficiency because they increase the operating carrier density at a given current density and increase the fraction of carriers lost to Auger recombination. (paper)

  11. Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale

    Science.gov (United States)

    Yu, Xiong; Egelman, Edward H.

    2010-01-01

    Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double stranded DNA with ∼ 6.4 subunits per turn, and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy (STEM) that the Dmc1 filament formed on single stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions, and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or STEM to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. PMID:20600108

  12. Multiple aspects of ATP-dependent nucleosome translocation by RSC and Mi-2 are directed by the underlying DNA sequence.

    Directory of Open Access Journals (Sweden)

    Joke J F A van Vugt

    Full Text Available BACKGROUND: Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted. METHODOLOGY/PRINCIPAL FINDINGS: We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type and native RSC from yeast (SWI/SNF-type repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps. CONCLUSIONS/SIGNIFICANCE: Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase

  13. Obtaining classical swine fever virus E2 recombinant protein and DNA-vaccine on the basis of one subunit

    International Nuclear Information System (INIS)

    Deryabin, O.; Deryabina, O.; Verbitskiy, P.; Kordyum, V.

    2005-01-01

    Three forms of E2 recombinant protein were expressed in E. coli. Swine sera obtained against different forms of the recombinant protein were cross-studied with indirect ELISA. Using individual proteins as an antigen, only 15% of sera against other forms of protein reacted positively, while 100% of heterologous sera showed positive reaction with fused protein. Challenge experiments showed the existence of protective action only from the individual protein. Specificity and activity of sera obtained from the animals after control challenge was confirmed in a blocking variant of ELISA. Genetic construction used a eukaryotic vector that contained the E2 protein gene. Immunization of mice with the resulting DNA induced synthesis of specific antibodies, the titre of which increased considerably after additional single immunization with the E2 recombinant protein, expressed in E. coli. This demonstrated the effectiveness of animal priming by DNA vaccine, and the possibility of using the E2 recombinant protein in E. coli for booster vaccination. (author)

  14. Evaluation of the General Atomic codes TAP and RECA for HTGR accident analyses

    International Nuclear Information System (INIS)

    Ball, S.J.; Cleveland, J.C.; Sanders, J.P.

    1978-01-01

    The General Atomic codes TAP (Transient Analysis Program) and RECA (Reactor Emergency Cooling Analysis) are evaluated with respect to their capability for predicting the dynamic behavior of high-temperature gas-cooled reactors (HTGRs) for postulated accident conditions. Several apparent modeling problems are noted, and the susceptibility of the codes to misuse and input errors is discussed. A critique of code verification plans is also included. The several cases where direct comparisons could be made between TAP/RECA calculations and those based on other independently developed codes indicated generally good agreement, thus contributing to the credibility of the codes

  15. [Improvement of thermal adaptability and fermentation of industrial ethanologenic yeast by genomic DNA mutagenesis-based genetic recombination].

    Science.gov (United States)

    Liu, Xiuying; He, Xiuping; Lu, Ying; Zhang, Borun

    2011-07-01

    Ethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, in the process of industrial production of ethanol, both cell growth and fermentation of ethanologenic S. cerevisiae are dramatically affected by environmental stresses, such as thermal stress. In this study, we improved both the thermotolerance and fermentation performance of industrial ethanologenic S. cerevisiae by combined usage of chemical mutagenesis and genomic DNA mutagenesis-based genetic recombination method. The recombinant S. cerevisiae strain T44-2 could grow at 44 degrees C, 3 degrees C higher than that of the original strain CE6. The survival rate of T44-2 was 1.84 and 1.87-fold of that of CE6 when heat shock at 48 degrees C and 52 degrees C for 1 h respectively. At temperature higher than 37 degrees C, recombinant strain T44-2 always gave higher cell growth and ethanol production than those of strain CE6. Meanwhile, from 30 degrees C to 40 degrees C, recombinant strain T44-2 produces 91.2-83.8 g/L of ethanol from 200 g/L of glucose, which indicated that the recombinant strain T44-2 had both thermotolerance and broad thermal adaptability. The work offers a novel method, called genomic DNA mutagenesis-based genetic recombination, to improve the physiological functions of S. cerevisiae.

  16. Evaluation of the effectiveness and safety of the thermo-treatment process to dispose of recombinant DNA waste from biological research laboratories.

    Science.gov (United States)

    Li, Meng-Nan; Zheng, Guang-Hong; Wang, Lei; Xiao, Wei; Fu, Xiao-Hua; Le, Yi-Quan; Ren, Da-Ming

    2009-01-01

    The discharge of recombinant DNA waste from biological laboratories into the eco-system may be one of the pathways resulting in horizontal gene transfer or "gene pollution". Heating at 100 degrees C for 5-10 min is a common method for treating recombinant DNA waste in biological research laboratories in China. In this study, we evaluated the effectiveness and the safety of the thermo-treatment method in the disposal of recombinant DNA waste. Quantitative PCR, plasmid transformation and electrophoresis technology were used to evaluate the decay/denaturation efficiency during the thermo-treatment process of recombinant plasmid, pET-28b. Results showed that prolonging thermo-treatment time could improve decay efficiency of the plasmid, and its decay half-life was 2.7-4.0 min during the thermo-treatment at 100 degrees C. However, after 30 min of thermo-treatment some transforming activity remained. Higher ionic strength could protect recombinant plasmid from decay during the treatment process. These results indicate that thermo-treatment at 100 degrees C cannot decay and inactivate pET-28b completely. In addition, preliminary results showed that thermo-treated recombinant plasmids were not degraded completely in a short period when they were discharged into an aquatic environment. This implies that when thermo-treated recombinant DNAs are discharged into the eco-system, they may have enough time to re-nature and transform, thus resulting in gene diffusion.

  17. Evaluation of the effectiveness and safety of the thermo-treatment process to dispose of recombinant DNA waste from biological research laboratories

    International Nuclear Information System (INIS)

    Li Mengnan; Zheng Guanghong; Wang Lei; Xiao Wei; Fu Xiaohua; Le Yiquan; Ren Daming

    2009-01-01

    The discharge of recombinant DNA waste from biological laboratories into the eco-system may be one of the pathways resulting in horizontal gene transfer or 'gene pollution'. Heating at 100 deg. C for 5-10 min is a common method for treating recombinant DNA waste in biological research laboratories in China. In this study, we evaluated the effectiveness and the safety of the thermo-treatment method in the disposal of recombinant DNA waste. Quantitative PCR, plasmid transformation and electrophoresis technology were used to evaluate the decay/denaturation efficiency during the thermo-treatment process of recombinant plasmid, pET-28b. Results showed that prolonging thermo-treatment time could improve decay efficiency of the plasmid, and its decay half-life was 2.7-4.0 min during the thermo-treatment at 100 deg. C. However, after 30 min of thermo-treatment some transforming activity remained. Higher ionic strength could protect recombinant plasmid from decay during the treatment process. These results indicate that thermo-treatment at 100 deg. C cannot decay and inactivate pET-28b completely. In addition, preliminary results showed that thermo-treated recombinant plasmids were not degraded completely in a short period when they were discharged into an aquatic environment. This implies that when thermo-treated recombinant DNAs are discharged into the eco-system, they may have enough time to re-nature and transform, thus resulting in gene diffusion

  18. Purification, crystallization and preliminary crystallographic analysis of a multiple cofactor-dependent DNA ligase from Sulfophobococcus zilligii

    International Nuclear Information System (INIS)

    Supangat, Supangat; An, Young Jun; Sun, Younguk; Kwon, Suk-Tae; Cha, Sun-Shin

    2010-01-01

    A recombinant multiple cofactor-dependent DNA ligase from S. zilligii has been purified and crystallized. X-ray diffraction data were collected to 2.9 Å resolution and the crystals belonged to space group P1. A recombinant DNA ligase from Sulfophobococcus zilligii that shows multiple cofactor specificity (ATP, ADP and GTP) was expressed in Escherichia coli and purified under reducing conditions. Crystals were obtained by the microbatch crystallization method at 295 K in a drop containing 1 µl protein solution (10 mg ml −1 ) and an equal volume of mother liquor [0.1 M HEPES pH 7.5, 10%(w/v) polyethylene glycol 10 000]. A data set was collected to 2.9 Å resolution using synchrotron radiation. The crystals belonged to space group P1, with unit-cell parameters a = 63.7, b = 77.1, c = 77.8 Å, α = 83.4, β = 82.4, γ = 74.6°. Assuming the presence of two molecules in the unit cell, the solvent content was estimated to be about 53.4%

  19. Mechanisms and Regulation of Mitotic Recombination in Saccharomyces cerevisiae

    Science.gov (United States)

    Symington, Lorraine S.; Rothstein, Rodney; Lisby, Michael

    2014-01-01

    Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell. PMID:25381364

  20. Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics.

    Science.gov (United States)

    Díaz-Mejía, J Javier; Celaj, Albi; Mellor, Joseph C; Coté, Atina; Balint, Attila; Ho, Brandon; Bansal, Pritpal; Shaeri, Fatemeh; Gebbia, Marinella; Weile, Jochen; Verby, Marta; Karkhanina, Anna; Zhang, YiFan; Wong, Cassandra; Rich, Justin; Prendergast, D'Arcy; Gupta, Gaurav; Öztürk, Sedide; Durocher, Daniel; Brown, Grant W; Roth, Frederick P

    2018-05-28

    Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1 , SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

  1. Effects of nuclear mutations for recombination and repair functions and of caffeine on mitochondrial recombination

    International Nuclear Information System (INIS)

    Fraenkel, A.H.M.

    1974-01-01

    Studies of both prokaryotic and eukaryotic organisms indicate that pathways governing repair of damage to nuclear DNA caused by x-ray or ultraviolet irradiation overlap with those controlling recombination. Fourteen nuclear mutants of Saccharomyces cerevisiae were tested in order to determine whether these mutant genes affected mitochondrial recombination. None of the mutations studied significantly affected mitochondrial recombination. The nuclear recombination and repair pathways studied do not overlap with the nuclear pathway which controls recombination of mitochondrial DNA. A second set of experiments was designed to test the effect of caffeine on both nuclear and mitochondrial recombination in Saccharomyces cerevisiae. (U.S.)

  2. Spin dependent surface recombination in silicon p-n junctions: the effect of irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kaplan, D [Laboratoire Central de Recherches, 91 - Corbeville par Orsay (France); Pepper, M [Cambridge Univ. (UK). Cavendish Lab.

    1980-06-01

    The results are presented of an investigation of spin dependent recombination in (100) oriented, gate controlled Si diodes irradiated by 30 keV electrons. After irradiation, recombination at the Si-SiO/sub 2/ interface is increased, and saturation of the spin resonance increases the diode forward current by 5 parts in 10/sup 4/. The results cannot be described by a conventional Shockley-Read recombination model. An alternative picture is proposed involving recombination between trapped electrons and trapped holes.

  3. DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli

    International Nuclear Information System (INIS)

    Dorson, J.W.; Moses, R.E.

    1978-01-01

    DNA synthesis after ultraviolet irradiation is low in wild type toluene-treated cells. The level of repair incorporation is greater in strains deficient in DNA polymerase I. The low level of repair synthesis is attributable to the concerted action of DNA polymerase I and polynucleotide ligase. Repair synthesis is stimulated by blocking ligase activity with the addition of nicotinamide mononucleotide (NMN) or the use of a ligase temperature-sensitive mutant. NMN stimulation is specific for DNA polymerase I-mediated repair synthesis, as it is absent in isogenic strains deficient in the polymerase function or the 5' yields 3' exonuclease function associated with DNA polymerase I. DNA synthesis that is stimulated by NMN is proportional to the ultraviolet exposure at low doses, nonconservative in nature, and is dependent on the uvrA gene product but is independent of the recA gene product. These criteria place this synthesis in the excision repair pathway. The NMN-stimulated repair synthesis requires ATP and is N-ethylmaleimide-resistant. The use of NMN provides a direct means for evaluating the involvement of DNA polymerase I in excision repair

  4. Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

    Science.gov (United States)

    Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

    2014-01-01

    High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

  5. The ATM signaling cascade promotes recombination-dependent pachytene arrest in mouse spermatocytes.

    Directory of Open Access Journals (Sweden)

    Sarai Pacheco

    2015-03-01

    Full Text Available Most mutations that compromise meiotic recombination or synapsis in mouse spermatocytes result in arrest and apoptosis at the pachytene stage of the first meiotic prophase. Two main mechanisms are thought to trigger arrest: one independent of the double-strand breaks (DSBs that initiate meiotic recombination, and another activated by persistent recombination intermediates. Mechanisms underlying the recombination-dependent arrest response are not well understood, so we sought to identify factors involved by examining mutants deficient for TRIP13, a conserved AAA+ ATPase required for the completion of meiotic DSB repair. We find that spermatocytes with a hypomorphic Trip13 mutation (Trip13mod/mod arrest with features characteristic of early pachynema in wild type, namely, fully synapsed chromosomes without incorporation of the histone variant H1t into chromatin. These cells then undergo apoptosis, possibly in response to the arrest or in response to a defect in sex body formation. However, TRIP13-deficient cells that additionally lack the DSB-responsive kinase ATM progress further, reaching an H1t-positive stage (i.e., similar to mid/late pachynema in wild type despite the presence of unrepaired DSBs. TRIP13-deficient spermatocytes also progress to an H1t-positive stage if ATM activity is attenuated by hypomorphic mutations in Mre11 or Nbs1 or by elimination of the ATM-effector kinase CHK2. These mutant backgrounds nonetheless experience an apoptotic block to further spermatogenic progression, most likely caused by failure to form a sex body. DSB numbers are elevated in Mre11 and Nbs1 hypomorphs but not Chk2 mutants, thus delineating genetic requirements for the ATM-dependent negative feedback loop that regulates DSB numbers. The findings demonstrate for the first time that ATM-dependent signaling enforces the normal pachytene response to persistent recombination intermediates. Our work supports the conclusion that recombination defects trigger

  6. Diversification of DnaA dependency for DNA replication in cyanobacterial evolution.

    Science.gov (United States)

    Ohbayashi, Ryudo; Watanabe, Satoru; Ehira, Shigeki; Kanesaki, Yu; Chibazakura, Taku; Yoshikawa, Hirofumi

    2016-05-01

    Regulating DNA replication is essential for all living cells. The DNA replication initiation factor DnaA is highly conserved in prokaryotes and is required for accurate initiation of chromosomal replication at oriC. DnaA-independent free-living bacteria have not been identified. The dnaA gene is absent in plastids and some symbiotic bacteria, although it is not known when or how DnaA-independent mechanisms were acquired. Here, we show that the degree of dependency of DNA replication on DnaA varies among cyanobacterial species. Deletion of the dnaA gene in Synechococcus elongatus PCC 7942 shifted DNA replication from oriC to a different site as a result of the integration of an episomal plasmid. Moreover, viability during the stationary phase was higher in dnaA disruptants than in wild-type cells. Deletion of dnaA did not affect DNA replication or cell growth in Synechocystis sp. PCC 6803 or Anabaena sp. PCC 7120, indicating that functional dependency on DnaA was already lost in some nonsymbiotic cyanobacterial lineages during diversification. Therefore, we proposed that cyanobacteria acquired DnaA-independent replication mechanisms before symbiosis and such an ancestral cyanobacterium was the sole primary endosymbiont to form a plastid precursor.

  7. Bacterial Artificial Chromosome Mutagenesis Using Recombineering

    Directory of Open Access Journals (Sweden)

    Kumaran Narayanan

    2011-01-01

    Full Text Available Gene expression from bacterial artificial chromosome (BAC clones has been demonstrated to facilitate physiologically relevant levels compared to viral and nonviral cDNA vectors. BACs are large enough to transfer intact genes in their native chromosomal setting together with flanking regulatory elements to provide all the signals for correct spatiotemporal gene expression. Until recently, the use of BACs for functional studies has been limited because their large size has inherently presented a major obstacle for introducing modifications using conventional genetic engineering strategies. The development of in vivo homologous recombination strategies based on recombineering in E. coli has helped resolve this problem by enabling facile engineering of high molecular weight BAC DNA without dependence on suitably placed restriction enzymes or cloning steps. These techniques have considerably expanded the possibilities for studying functional genetics using BACs in vitro and in vivo.

  8. DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

    Science.gov (United States)

    Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

  9. Mycobacterium smegmatis SftH exemplifies a distinctive clade of superfamily II DNA-dependent ATPases with 3' to 5' translocase and helicase activities.

    Science.gov (United States)

    Yakovleva, Lyudmila; Shuman, Stewart

    2012-08-01

    Bacterial DNA helicases are nucleic acid-dependent NTPases that play important roles in DNA replication, recombination and repair. We are interested in the DNA helicases of Mycobacteria, a genus of the phylum Actinobacteria, which includes the human pathogen Mycobacterium tuberculosis and its avirulent relative Mycobacterium smegmatis. Here, we identify and characterize M. smegmatis SftH, a superfamily II helicase with a distinctive domain structure, comprising an N-terminal NTPase domain and a C-terminal DUF1998 domain (containing a putative tetracysteine metal-binding motif). We show that SftH is a monomeric DNA-dependent ATPase/dATPase that translocates 3' to 5' on single-stranded DNA and has 3' to 5' helicase activity. SftH homologs are found in bacteria representing 12 different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis). SftH homologs are evident in more than 30 genera of Archaea. Among eukarya, SftH homologs are present in plants and fungi.

  10. Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

    Science.gov (United States)

    Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

    2017-11-26

    Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

  11. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); D.C. van Gent (Dik); J.H.J. Hoeijmakers (Jan)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  12. Cloning of an E. coli RecA and yeast RAD51 homolog, radA, an allele of the uvsC in Aspergillus nidulans and its mutator effects.

    Science.gov (United States)

    Seong, K Y; Chae, S K; Kang, H S

    1997-04-30

    An E. coli RecA and yeast RAD51 homolog from Aspergillus nidulans, radA, has been cloned by screening genomic and cDNA libraries with a PCR-amplified probe. This probe was generated using primers carrying the conserved sequences of eukaryotic RecA homologs. The deduced amino acid sequence revealed two conserved Walker-A and -B type nucleotide-binding domains and exhibited 88%, 60%, and 53% identity with Mei-3 of Neurospora crassa, rhp51+ of Schizosaccharomyces pombe, and Rad51 of Saccharomyces cerevisiae, respectively. radA null mutants constructed by replacing the whole coding region with a selection marker showed high methyl methanesulfonate (MMS) sensitivity. Heterozygous diploids of radA disruptant with the uvsC114 mutant failed to complement with respect to MMS-sensitivity, indicating that radA is an allele of uvsC. In selecting spontaneous forward selenate resistant mutations, mutator effects were observed in radA null mutants similarly to those shown in uvsC114 mutant strains.

  13. RNA-Mediated cis Regulation in Acinetobacter baumannii Modulates Stress-Induced Phenotypic Variation.

    Science.gov (United States)

    Ching, Carly; Gozzi, Kevin; Heinemann, Björn; Chai, Yunrong; Godoy, Veronica G

    2017-06-01

    In the nosocomial opportunistic pathogen Acinetobacter baumannii , RecA-dependent mutagenesis, which causes antibiotic resistance acquisition, is linked to the DNA damage response (DDR). Notably, unlike the Escherichia coli paradigm, recA and DDR gene expression in A. baumannii is bimodal. Namely, there is phenotypic variation upon DNA damage, which may provide a bet-hedging strategy for survival. Thus, understanding recA gene regulation is key to elucidate the yet unknown DDR regulation in A. baumannii Here, we identify a structured 5' untranslated region (UTR) in the recA transcript which serves as a cis -regulatory element. We show that a predicted stem-loop structure in this 5' UTR affects mRNA half-life and underlies bimodal gene expression and thus phenotypic variation in response to ciprofloxacin treatment. We furthermore show that the stem-loop structure of the recA 5' UTR influences intracellular RecA protein levels and, in vivo , impairing the formation of the stem-loop structure of the recA 5' UTR lowers cell survival of UV treatment and decreases rifampin resistance acquisition from DNA damage-induced mutagenesis. We hypothesize that the 5' UTR allows for stable recA transcripts during stress, including antibiotic treatment, enabling cells to maintain suitable RecA levels for survival. This innovative strategy to regulate the DDR in A. baumannii may contribute to its success as a pathogen. IMPORTANCE Acinetobacter baumannii is an opportunistic pathogen quickly gaining antibiotic resistances. Mutagenesis and antibiotic resistance acquisition are linked to the DNA damage response (DDR). However, how the DDR is regulated in A. baumannii remains unknown, since unlike most bacteria, A. baumannii does not follow the regulation of the Escherichia coli paradigm. In this study, we have started to uncover the mechanisms regulating the novel A. baumannii DDR. We have found that a cis -acting 5' UTR regulates recA transcript stability, RecA protein levels, and DNA

  14. Dynamic translocation of ligand-complexed DNA through solid-state nanopores with optical tweezers

    International Nuclear Information System (INIS)

    Sischka, Andy; Spiering, Andre; Anselmetti, Dario; Khaksar, Maryam; Laxa, Miriam; Koenig, Janine; Dietz, Karl-Josef

    2010-01-01

    We investigated the threading and controlled translocation of individual lambda-DNA (λ-DNA) molecules through solid-state nanopores with piconewton force sensitivity, millisecond time resolution and picoampere ionic current sensitivity with a set-up combining quantitative 3D optical tweezers (OT) with electrophysiology. With our virtually interference-free OT set-up the binding of RecA and single peroxiredoxin protein molecules to λ-DNA was quantitatively investigated during dynamic translocation experiments where effective forces and respective ionic currents of the threaded DNA molecule through the nanopore were measured during inward and outward sliding. Membrane voltage-dependent experiments of reversible single protein/DNA translocation scans yield hysteresis-free, asymmetric single-molecule fingerprints in the measured force and conductance signals that can be attributed to the interplay of optical trap and electrostatic nanopore potentials. These experiments allow an exact localization of the bound protein along the DNA strand and open fascinating applications for label-free detection of DNA-binding ligands, where structural and positional binding phenomena can be investigated at a single-molecule level.

  15. Defective processing of methylated single-stranded DNA by E. coli alkB mutants

    Science.gov (United States)

    Dinglay, Suneet; Trewick, Sarah C.; Lindahl, Tomas; Sedgwick, Barbara

    2000-01-01

    Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells. PMID:10950872

  16. Oligonucleotide recombination enabled site-specific mutagenesis in bacteria

    Science.gov (United States)

    Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligo recombination is one type of recombineering that uses ssDNA olig...

  17. DNA damage, homology-directed repair, and DNA methylation.

    Directory of Open Access Journals (Sweden)

    Concetta Cuozzo

    2007-07-01

    Full Text Available To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP genes (DR-GFP. A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

  18. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as w...

  19. Construction of C35 gene bait recombinants and T47D cell cDNA library.

    Science.gov (United States)

    Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

    2017-11-20

    C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

  20. Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

    Science.gov (United States)

    Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

    2001-04-03

    Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

  1. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) for directed enzyme evolution.

    Science.gov (United States)

    Gonzalez-Perez, David; Molina-Espeja, Patricia; Garcia-Ruiz, Eva; Alcalde, Miguel

    2014-01-01

    Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.

  2. Chromosomal Fragmentation in "Escherichia Coli": Its Absence in "mutT" Mutants and Its Mechanisms in "seqA" Mutants

    Science.gov (United States)

    Rotman, Ella Rose

    2009-01-01

    Chromosomal fragmentation in "Escherichia coli" is a lethal event for the cell unless mended by the recombinational repair proteins RecA, RecBCD, and RuvABC. Certain mutations exacerbate problems that cause the cell to be dependent on the recombinational repair proteins for viability. We tested whether the absence of the MutT protein caused…

  3. Induction of intrachromosomal homologous recombination in whole plants

    International Nuclear Information System (INIS)

    Puchta, H.; Swoboda, P.; Hohn, B.

    1995-01-01

    The influence of different factors on frequencies of intrachromosomal homologous recombination in whole Arabidopsis thaliana and tobacco plants was analyzed using a disrupted β-glucuronidase marker gene. Recombination frequencies were enhanced several fold by DNA damaging agents like UV-light or MMS (methyl methanesulfonate). Applying 3-methoxybenzamide (3-MB), an inhibitor of poly(ADP)ribose polymerase (PARP), an enzyme that is postulated to be involved in DNA repair, enhanced homologous recombination frequencies strongly. These findings indicate that homologous recombination is involved in DNA repair and can (at least partially) compensate for other DNA repair pathways. Indications that recombination in plants can be induced by environmental stress factors that are not likely to be involved in DNA metabolism were also found; Arabidopsis plants growing in a medium containing 0.1 M NaCl exhibited elevated recombination frequencies. The possible general effects of ‘environmental’ challenges on genome flexibility are discussed. (author)

  4. Factores asociados a recaídas por tuberculosis en Lima este - Perú

    Directory of Open Access Journals (Sweden)

    María Ríos Hipólito

    2002-01-01

    Full Text Available Objetivo: Determinar los factores de riesgo asociados a recaídas por tuberculosis en Lima Este - Perú, entre marzo y diciembre del 2000. Materiales y métodos: estudio caso-control. Se definió a los casos (184 como los pacientes que recibieron tratamiento con el esquema I alguna vez, egresaron como curados y volvieron a presentar otro episodio de tuberculosis BK positivo durante 1999. Los controles (368 fueron los pacientes nuevos con tuberculosis BK positivo tratados en 1998 que no recayeron. Resultados: se asociaron significativamente a las recaídas el sexo masculino, la edad mayor de 50 años, el consumo de drogas, la residencia en un área urbana, el hacinamiento, la percepción errada de la enfermedad (PEE y la desocupación, no así el contacto con un paciente tuberculoso. Luego del análisis multivariado, sólo se asociaron el área urbana, el hacinamiento, la PEE y el tratamiento irregular. Conclusiones: la residencia en un área urbana, el hacinamiento, la PEE y la irregularidad en el tratamiento son factores asociados significativamente a recaídas en pacientes con TBC pulmonar BK(+ de Lima Este, Perú.

  5. Immunoglobulin class-switch recombination deficiencies.

    Science.gov (United States)

    Durandy, Anne; Kracker, Sven

    2012-07-30

    Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.

  6. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick

    2016-03-01

    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  7. Suppression of the E. coli SOS response by dNTP pool changes.

    Science.gov (United States)

    Maslowska, Katarzyna H; Makiela-Dzbenska, Karolina; Fijalkowska, Iwona J; Schaaper, Roel M

    2015-04-30

    The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  8. IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

    Science.gov (United States)

    Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

    2017-03-01

    Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. © 2017 American Society of Plant Biologists. All rights reserved.

  9. Dependence of the quasiparticle recombination rate on the superconducting gap and TC

    Science.gov (United States)

    Carr, G. L.; Xi, Xiaoxiang; Hwang, J.; Tashiro, H.; Reitze, D. H.; Tanner, D. B.

    2010-03-01

    The relaxation of excess quasiparticles in a BCS superconductor is known to depend on quantities such as the quasiparticle & phonon density of states, and their coupling (Kaplan et al, Phys. Rev. B 14 4854, 1976). Disorder or an applied field can disrupt superconductivity, as evidenced by a reduced TC. We consider some simple modifications to the quasiparticle density of states consistent with a suppressed energy gap and TC, leading to changes in the intrinsic and effective (measured) rates for excess quasiparticles to recombine into pairs. We review some results for disordered MoGe and discuss the magnetic-field dependence of the recombination process.

  10. DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

    Directory of Open Access Journals (Sweden)

    Yuxin Feng

    Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

  11. Molecular cloning of a recA-like gene from the cyanobacterium Anabaena variabilis

    International Nuclear Information System (INIS)

    Owttrim, G.W.; Coleman, J.R.

    1987-01-01

    A recA-like gene isolated from the cyanobacterium Anabaena variabilis was cloned and partially characterized. When introduced into Escherichia coli recA mutants, the 7.5-kilobase-pair plasmid-borne DNA insert restored resistance to methyl methanesulfonate and UV irradiation, as well as recombination proficiency when measured by Hfr-mediated conjugation. The cyanobacterial recA gene restored spontaneous but not mitomycin C-induced prophage production. Restriction analysis and subcloning yielded a 1.5-kilobase-pair Sau3A fragment which also restored methylmethane sulfonate resistance and coded for a 38- to 40-kilodalton polypeptide when expressed in an in vitro transcription-translation system

  12. Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Derek L Lindstrom

    2011-03-01

    Full Text Available Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array. As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

  13. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot

    DEFF Research Database (Denmark)

    Albers, Eliene; Sbroggiò, Mauro; Martin Gonzalez, Javier

    2017-01-01

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific...

  14. Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

    KAUST Repository

    Fornander, Louise H

    2012-02-22

    Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

  15. Contextos de abstinência e de recaída na recuperação da dependência química

    Directory of Open Access Journals (Sweden)

    Rigotto Simone Demore

    2002-01-01

    Full Text Available Foram entrevistados nove homens e três mulheres, residentes na região da cidade de Caxias do Sul - RS, todos diagnosticados como dependentes de substâncias, segundo critérios do DSM-IV, para que descrevessem suas experiências de abstinência e recaída nas tentativas de recuperação da dependência química. A análise qualitativa das entrevistas orientou-se pelos movimentos reflexivos de descrição, redução e interpretação fenomenológica. A experiência da abstinência foi atribuída aos seguintes constituintes e contextos experienciais: consciência do problema aditivo por parte do dependente, resgate de vínculos familiares, recomposição de auto-estima, afastamento de ambientes favorecedores da adição, e envolvimento em práticas religiosas. A ausência dos constituintes e contextos identificados na experiência de abstinência caracterizou a manutenção do consumo. Os elos experienciais interpretados como essenciais à experiência de abstinência foram as redes interpessoais de apoio - constituídas por profissionais, familiares e novos amigos - e o envolvimento como colaboradores na recuperação de outros dependentes químicos.

  16. Dual recombinant Lactococcus lactis for enhanced delivery of DNA vaccine reporter plasmid pPERDBY.

    Science.gov (United States)

    Yagnik, Bhrugu; Sharma, Drashya; Padh, Harish; Desai, Priti

    2017-04-01

    Food grade Lactococcus lactis has been widely used as an antigen and DNA delivery vehicle. We have previously reported the use of non-invasive L. lactis to deliver the newly constructed immunostimulatory DNA vaccine reporter plasmid, pPERDBY. In the present report, construction of dual recombinant L. lactis expressing internalin A of Listeria monocytogenes and harboring pPERDBY (LL InlA + pPERDBY) to enhance the efficiency of delivery of DNA by L. lactis is outlined. After confirmation and validation of LL InlA + pPERDBY, its DNA delivery potential was compared with previously developed non-invasive r- L. lactis::pPERDBY. The use of invasive L. lactis resulted in around threefold increases in the number of enhanced green fluorescent protein-expressing Caco-2 cells. These findings reinforce the prospective application of invasive strain of L. lactis for delivery of DNA/RNA and antigens. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

  17. Single-strand breaks in the DNA of the uvrA and uvrB strains of Escherichia coli K-12 after ultraviolet irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Youngs, D A; Smith, K C [Stanford Univ., Calif. (USA). Dept. of Radiology

    1976-12-01

    DNA single-strand breaks were produced in uvrA and uvrB strains of E.coli K-12 after UV (254 nm) irradiation. These breaks appeared to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appeared to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA101 or uvrD gene products. It is hypothesized that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA, uvrB-independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.

  18. The use of recombinant DNA techniques to study radiation-induced damage, repair and genetic change in mammalian cells

    International Nuclear Information System (INIS)

    Thacker, J.

    1986-01-01

    A brief introduction is given to appropriate elements of recombinant DNA techniques and applications to problems in radiobiology are reviewed with illustrative detail. Examples are included of studies with both 254 nm ultraviolet light and ionizing radiation and the review progresses from the molecular analysis of DNA damage in vitro through to the nature of consequent cellular responses. The review is dealt with under the following headings: Molecular distribution of DNA damage, The use of DNA-mediated gene transfer to assess damage and repair, The DNA double strand break: use of restriction endonucleases to model radiation damage, Identification and cloning of DNA repair genes, Analysis of radiation-induced genetic change. (UK)

  19. RTEL1 maintains genomic stability by suppressing homologous recombination.

    Science.gov (United States)

    Barber, Louise J; Youds, Jillian L; Ward, Jordan D; McIlwraith, Michael J; O'Neil, Nigel J; Petalcorin, Mark I R; Martin, Julie S; Collis, Spencer J; Cantor, Sharon B; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C; Rose, Ann M; Boulton, Simon J

    2008-10-17

    Homologous recombination (HR) is an important conserved process for DNA repair and ensures maintenance of genome integrity. Inappropriate HR causes gross chromosomal rearrangements and tumorigenesis in mammals. In yeast, the Srs2 helicase eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has been elusive. Here, we identify C. elegans RTEL-1 as a functional analog of Srs2 and describe its vertebrate counterpart, RTEL1, which is required for genome stability and tumor avoidance. We find that rtel-1 mutant worms and RTEL1-depleted human cells share characteristic phenotypes with yeast srs2 mutants: lethality upon deletion of the sgs1/BLM homolog, hyperrecombination, and DNA damage sensitivity. In vitro, purified human RTEL1 antagonizes HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control after deregulation of RTEL1 may be a critical event that drives genome instability and cancer.

  20. Temperature dependence of acceptor-hole recombination in germanium

    International Nuclear Information System (INIS)

    Darken, L.S.; Jellison, G.E. Jr.

    1989-01-01

    The recombination kinetics of several centers (Zn - , Cu - , B - , CuH - 2 , CuH - x , Zn = , Cu = , and CuH = x ) in high-purity Ge have been measured as a function of temperature from 8 to 160 K by transient capacitance techniques and are significantly faster than expected from cascade theory. The cascade theory also gives the wrong temperature dependence, and the wrong z dependence. Instead, the data are generally fit by the expression N v /4pτ c congruent kT/h (p and τ c are, respectively, the free-hole concentration in the sample and the experimental mean capture time for a center)

  1. Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response.

    Science.gov (United States)

    Mori, Tetsuya; Nakamura, Tatsuro; Okazaki, Naoto; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

    2012-01-01

    The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

  2. Density dependence of dielectronic recombination in selenium

    International Nuclear Information System (INIS)

    Hagelstein, P.L.; Rosen, M.D.; Jacobs, V.L.

    1986-01-01

    Dielectronic recombination has been found to be the dominant recombination process in the determination of the ionization balance of selenium near the Ne-like sequence under conditions relevant to the exploding-foil EUV laser plasmas. The dielectronic recombination process tends to populate excited levels, and these levels in turn are more susceptible to subsequent excitation and ionization than are the ground-state ions. If one defines an effective recombination rate which includes, in addition to the primary recombination, the subsequent excitation and ionization of the additional excited-state population due to the primary recombination, then this effective recombination rate can be density-sensitive at relatively low electron density. We present results for this effective dielectronic recombination rate at an electron density of 3 x 10/sup 20/ electrons/cm 3 for recombination from Ne-like to Na-like selenium and from F-like to Ne-like selenium. In the former case, the effective recombination rate coefficient is found to be 1.8 x 10/sup -11/ cm 3 /sec at 1.0 keV, which is to be compared with the zero-density value of 2.8 x 10/sup -11/ cm 3 /sec. In the latter case (F-like to Ne-like), the effective recombination rate coefficient is found to be 1.3 x 10/sup -11/ cm 3 /sec, which is substantially reduced from the zero-density result of 3.3 x 10/sup -11/ cm 3 /sec. We have examined the effects of dielectronic recombination on the laser gain of the dominant Ne-like 3p-3s transitions and have compared our results with those presented by Whitten et al. [Phys. Rev. A 33, 2171 (1986)

  3. Zinc blocks SOS-induced antibiotic resistance via inhibition of RecA in Escherichia coli.

    Science.gov (United States)

    Bunnell, Bryan E; Escobar, Jillian F; Bair, Kirsten L; Sutton, Mark D; Crane, John K

    2017-01-01

    Zinc inhibits the virulence of diarrheagenic E. coli by inducing the envelope stress response and inhibiting the SOS response. The SOS response is triggered by damage to bacterial DNA. In Shiga-toxigenic E. coli, the SOS response strongly induces the production of Shiga toxins (Stx) and of the bacteriophages that encode the Stx genes. In E. coli, induction of the SOS response is accompanied by a higher mutation rate, called the mutator response, caused by a shift to error-prone DNA polymerases when DNA damage is too severe to be repaired by canonical DNA polymerases. Since zinc inhibited the other aspects of the SOS response, we hypothesized that zinc would also inhibit the mutator response, also known as hypermutation. We explored various different experimental paradigms to induce hypermutation triggered by the SOS response, and found that hypermutation was induced not just by classical inducers such as mitomycin C and the quinolone antibiotics, but also by antiviral drugs such as zidovudine and anti-cancer drugs such as 5-fluorouracil, 6-mercaptopurine, and azacytidine. Zinc salts inhibited the SOS response and the hypermutator phenomenon in E. coli as well as in Klebsiella pneumoniae, and was more effective in inhibiting the SOS response than other metals. We then attempted to determine the mechanism by which zinc, applied externally in the medium, inhibits hypermutation. Our results show that zinc interferes with the actions of RecA, and protects LexA from RecA-mediated cleavage, an early step in initiation of the SOS response. The SOS response may play a role in the development of antibiotic resistance and the effect of zinc suggests ways to prevent it.

  4. Zinc blocks SOS-induced antibiotic resistance via inhibition of RecA in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Bryan E Bunnell

    Full Text Available Zinc inhibits the virulence of diarrheagenic E. coli by inducing the envelope stress response and inhibiting the SOS response. The SOS response is triggered by damage to bacterial DNA. In Shiga-toxigenic E. coli, the SOS response strongly induces the production of Shiga toxins (Stx and of the bacteriophages that encode the Stx genes. In E. coli, induction of the SOS response is accompanied by a higher mutation rate, called the mutator response, caused by a shift to error-prone DNA polymerases when DNA damage is too severe to be repaired by canonical DNA polymerases. Since zinc inhibited the other aspects of the SOS response, we hypothesized that zinc would also inhibit the mutator response, also known as hypermutation. We explored various different experimental paradigms to induce hypermutation triggered by the SOS response, and found that hypermutation was induced not just by classical inducers such as mitomycin C and the quinolone antibiotics, but also by antiviral drugs such as zidovudine and anti-cancer drugs such as 5-fluorouracil, 6-mercaptopurine, and azacytidine. Zinc salts inhibited the SOS response and the hypermutator phenomenon in E. coli as well as in Klebsiella pneumoniae, and was more effective in inhibiting the SOS response than other metals. We then attempted to determine the mechanism by which zinc, applied externally in the medium, inhibits hypermutation. Our results show that zinc interferes with the actions of RecA, and protects LexA from RecA-mediated cleavage, an early step in initiation of the SOS response. The SOS response may play a role in the development of antibiotic resistance and the effect of zinc suggests ways to prevent it.

  5. Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites.

    Directory of Open Access Journals (Sweden)

    Xiao P Peng

    2018-01-01

    Full Text Available Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA array. Each rDNA repeat contains a programmed replication fork barrier (RFB established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1∆ and fob1∆ similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.

  6. Induction of homologous recombination in Saccharomyces cerevisiae.

    Science.gov (United States)

    Simon, J R; Moore, P D

    1988-09-01

    We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

  7. Total DNA of Glycyrrhiza uralensis transformed into Hansenula anomala by ion implantation:Preparing Glycyrrhizic acid in recombined yeasts

    International Nuclear Information System (INIS)

    Jin Xiang; Mao Peihong; Lu Jie; Ma Yuan

    2010-01-01

    Glycyrrhizic acid (GA) in Glycyrrhiza uralensis (G. uralensis) is physiologically active. In this study, the total DNA of wild G. uralensis was randomly transformed into Hansenula anomaly by implantation of low-energy Ar + and N + , to produce five recombinant yeast strains relating to biological synthesis of the GA or Glycyrrhetinic acid (GAs). After culturing in liquid medium for 96 h, the resultant GA, 18α-GAs and 18β-Gas were determined by reversed-phase high performance liquid chromatography (RP-HPLC), and the corresponding concentrations were 114.49, 0.56, and 0.81 mg·L -1 . After one hundred primers were analyzed with random amplified polymorphic DNA (RAPD), the seven different DNA fragments were produced by the N7059 strain of recombined yeasts, and, the polymerase chain reaction (PCR) verified that one of them came from the genome of G. uralensis, indicating a successful transfer of genetic information by ion implantation. (authors)

  8. The use of recombinant DNA technology for the development of a bluetongue virus subunit vaccine

    International Nuclear Information System (INIS)

    Huismans, H.

    1985-01-01

    The double-standed RNA gene coding for the surface antigen responsible for inducing neutralising anti-bodies has been isolated, converted to DNA, and cloned in the plasmid pBR322. So far, only plasmids containing inserts smaller than the gene have been obtained. The recombinant plasmids were isolated by screening for specific antibiotic resistance markers and characterized by size, restriction enzymes and hybridization with a 32 P-labelled DNA probe made with BTV-m RNA as template. Possible strategies for the development of a bluetongue virus submit vaccine are discussed

  9. Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold

    Directory of Open Access Journals (Sweden)

    Wittinghofer Alfred

    2005-03-01

    Full Text Available Abstract Background aaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown. Results Here we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 Å resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (α/β/α-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the β-sheet consisting of four additional β-strands. Conclusion We conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.

  10. Therapeutic Recombinant Monoclonal Antibodies

    Science.gov (United States)

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  11. Use of a ring chromosome and pulsed-field gels to study interhomolog recombination, double-strand DNA breaks and sister-chromatid exchange in yeast

    International Nuclear Information System (INIS)

    Game, J.C.; Sitney, K.C.; Cook, V.E.; Mortimer, R.K.

    1989-01-01

    The authors describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome II (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. They demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints. They present data on the timing of commitment to meiotic recombination scored genetically. They have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-size circles originating in part from sister-chromatid exchange, which they find to be frequent during meiosis

  12. Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast.

    Directory of Open Access Journals (Sweden)

    Bilge Argunhan

    Full Text Available Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs. The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI whereas no significant reduction was found in smaller chromosomes (III and VI. On the other hand, the absence of Rad17 (a critical component of the ATR pathway lead to an increase in DSB formation (chromosomes VII and II were tested. We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

  13. Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination.

    Science.gov (United States)

    Thakur, Roshan S; Basavaraju, Shivakumar; Somyajit, Kumar; Jain, Akshatha; Subramanya, Shreelakshmi; Muniyappa, Kalappa; Nagaraju, Ganesh

    2013-04-01

    In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M. tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichia coli ∆recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions. © 2013 The Authors Journal compilation © 2013 FEBS.

  14. Homologous Recombination as a Replication Fork Escort: Fork-Protection and Recovery

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    Audrey Costes

    2012-12-01

    Full Text Available Homologous recombination is a universal mechanism that allows DNA repair and ensures the efficiency of DNA replication. The substrate initiating the process of homologous recombination is a single-stranded DNA that promotes a strand exchange reaction resulting in a genetic exchange that promotes genetic diversity and DNA repair. The molecular mechanisms by which homologous recombination repairs a double-strand break have been extensively studied and are now well characterized. However, the mechanisms by which homologous recombination contribute to DNA replication in eukaryotes remains poorly understood. Studies in bacteria have identified multiple roles for the machinery of homologous recombination at replication forks. Here, we review our understanding of the molecular pathways involving the homologous recombination machinery to support the robustness of DNA replication. In addition to its role in fork-recovery and in rebuilding a functional replication fork apparatus, homologous recombination may also act as a fork-protection mechanism. We discuss that some of the fork-escort functions of homologous recombination might be achieved by loading of the recombination machinery at inactivated forks without a need for a strand exchange step; as well as the consequence of such a model for the stability of eukaryotic genomes.

  15. On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage.

    Science.gov (United States)

    Shcherbakov, Victor P; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Kudryashova, Elena

    2011-01-02

    The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase. Copyright © 2010 Elsevier B.V. All rights reserved.

  16. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

    Science.gov (United States)

    Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

    2016-01-01

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

  17. Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.

    Directory of Open Access Journals (Sweden)

    José-Daniel Aroca-Aguilar

    Full Text Available Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc. affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.

  18. Sequence-dependent DNA deformability studied using molecular dynamics simulations.

    Science.gov (United States)

    Fujii, Satoshi; Kono, Hidetoshi; Takenaka, Shigeori; Go, Nobuhiro; Sarai, Akinori

    2007-01-01

    Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein-DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein-DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.

  19. BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice.

    Directory of Open Access Journals (Sweden)

    Yulong Liang

    2010-01-01

    Full Text Available BRIT1 protein (also known as MCPH1 contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1(-/- mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1(-/- mice and mouse embryonic fibroblasts (MEFs were hypersensitive to gamma-irradiation. BRIT1(-/- MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1(-/- mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2's function and as a result leads to infertility and genomic instability in mice.

  20. Probing Temperature-Dependent Recombination Kinetics in Polymer:Fullerene Solar Cells by Electric Noise Spectroscopy

    Directory of Open Access Journals (Sweden)

    Giovanni Landi

    2017-09-01

    Full Text Available The influence of solvent additives on the temperature behavior of both charge carrier transport and recombination kinetics in bulk heterojunction solar cells has been investigated by electric noise spectroscopy. The observed differences in charge carrier lifetime and mobility are attributed to a different film ordering and donor-acceptor phase segregation in the blend. The measured temperature dependence indicates that bimolecular recombination is the dominant loss mechanism in the active layer, affecting the device performance. Blend devices prepared with a high-boiling-point solvent additive show a decreased recombination rate at the donor-acceptor interface as compared to the ones prepared with the reference solvent. A clear correlation between the device performance and the morphological properties is discussed in terms of the temperature dependence of the mobility-lifetime product.

  1. Assembly and dynamics of the bacteriophage T4 homologous recombination machinery

    Directory of Open Access Journals (Sweden)

    Morrical Scott W

    2010-12-01

    Full Text Available Abstract Homologous recombination (HR, a process involving the physical exchange of strands between homologous or nearly homologous DNA molecules, is critical for maintaining the genetic diversity and genome stability of species. Bacteriophage T4 is one of the classic systems for studies of homologous recombination. T4 uses HR for high-frequency genetic exchanges, for homology-directed DNA repair (HDR processes including DNA double-strand break repair, and for the initiation of DNA replication (RDR. T4 recombination proteins are expressed at high levels during T4 infection in E. coli, and share strong sequence, structural, and/or functional conservation with their counterparts in cellular organisms. Biochemical studies of T4 recombination have provided key insights on DNA strand exchange mechanisms, on the structure and function of recombination proteins, and on the coordination of recombination and DNA synthesis activities during RDR and HDR. Recent years have seen the development of detailed biochemical models for the assembly and dynamics of presynaptic filaments in the T4 recombination system, for the atomic structure of T4 UvsX recombinase, and for the roles of DNA helicases in T4 recombination. The goal of this chapter is to review these recent advances and their implications for HR and HDR mechanisms in all organisms.

  2. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

    Directory of Open Access Journals (Sweden)

    Sandra Arenhart

    Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

  3. The role of DNA repair processes in the biological efficiency of heavy ions

    International Nuclear Information System (INIS)

    Krasavin, E.A.; Amirtaev, K.G.; Kozubek, S.; Tokarova, B.; Tcherevatenko, A.P.

    1988-01-01

    Survival curves of E. coli wild type, recA, and Gam r 444 strains for different types of radiation were measured. The dependence of the radiosensitivity (D 0 -1 ) on LET (L) in the case of recA mutant is continuously decreasing. The dependence D 0 -1 (L) in the case of wild type and superresistant mutant cells has a local maximum for L=100 keV/μm. For L > 100 keV/μm the sensitives of all three bacterial strains do not differ significantly

  4. DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

    International Nuclear Information System (INIS)

    Urushihara, Yusuke; Kobayashi, Junya; Matsumoto, Yoshihisa; Komatsu, Kenshi; Oda, Shoji; Mitani, Hiroshi

    2012-01-01

    Highlights: ► We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. ► A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. ► DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. ► DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. ► DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after γ-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of γH2AX foci after γ-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of γH2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after γ-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the number of 53BP1 foci compared with wild-type cells. These results suggest that low level of DNA-PK activity causes aberrant DNA-PKcs autophosphorylation

  5. Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

    Science.gov (United States)

    Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

    2016-03-04

    Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. The Effect of Basepair Mismatch on DNA Strand Displacement

    OpenAIRE

    Broadwater, D.?W.?Bo; Kim, Harold?D.

    2016-01-01

    DNA strand displacement is a key reaction in DNA homologous recombination and DNA mismatch repair and is also heavily utilized in DNA-based computation and locomotion. Despite its ubiquity in science and engineering, sequence-dependent effects of displacement kinetics have not been extensively characterized. Here, we measured toehold-mediated strand displacement kinetics using single-molecule fluorescence in the presence of a single base pair mismatch. The apparent displacement rate varied si...

  7. V(D)J recombination process and the Pre-B to immature B-cells transition are altered in Fanca ?/? mice

    OpenAIRE

    Nguyen, Thuy Vy; Pawlikowska, Patrycja; Firlej, Virginie; Rosselli, Filippo; Aoufouchi, Sa?d

    2016-01-01

    B-lymphocytes in the bone marrow (BM) must generate a functional B-cell receptor and overcome the negative selection induced by reactivity with autoantigens. Two rounds of DNA recombination are required for the production of functional immunoglobulin heavy (Ig-HCs) and light (LCs) chains necessary for the continuation of B-lymphocyte development in the BM. Both rounds depend on the joint action of recombination activating gene-1 (RAG-1) and RAG-2 endonucleases with the DNA non-homologous end-...

  8. Role of DNA-PK in cellular responses to DNA double-strand breaks

    International Nuclear Information System (INIS)

    Chen, D.J.

    2003-01-01

    DNA double-strand breaks (DSBs) are probably the most dangerous of the many different types of DNA damage that occur within the cell. DSBs are generated by exogenous agents such as ionizing radiation (IR) or by endogenously generated reactive oxygen species and occur as intermediates during meiotic and V(D)J recombination. The repair of DSBs is of paramount importance to the cell as misrepair of DSBs can lead to cell death or promote tumorigenesis. In eukaryotes there exists two distinct mechanisms for DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, however, it is clear that nonhomologous repair of DSBs is highly active and plays a major role in conferring radiation resistance to the cell. The NHEJ machinery minimally consists of the DNA-dependent Protein Kinase (DNA-PK) and a complex of XRCC4 and DNA Ligase IV. The DNA-PK complex is composed of a 470 kDa catalytic subunit (DNA-PKcs), and the heterodimeric Ku70 and Ku80 DNA end-binding complex. DNA-PKcs is a PI-3 kinase with homology to ATM and ATR in its C-terminal kinase domain. The DNA-PK complex protects and tethers the ends, and directs assembly and, perhaps, the activation of other NHEJ proteins. We have previously demonstrated that the kinase activity of DNA-PK is essential for DNA DSB repair and V(D)J recombination. It is, therefore, of immense interest to determine the in vivo targets of DNA-PKcs and the mechanisms by which phosphorylation of these targets modulates NHEJ. Recent studies have resulted in the identification of a number of protein targets that are phosphorylated by and/or interact with DNA-PKcs. Our laboratory has recently identified autophosphorylation site(s) on DNA-PKcs. We find that phosphorylation at these sites in vivo is an early and essential response to DSBs and demonstrate, for the first time, the localization of DNA-PKcs to the sites of DNA damage in vivo. Furthermore, mutation of these phosphorylation sites in mammalian

  9. Recombinant DNA in Cambridge: lessons for nuclear energy

    International Nuclear Information System (INIS)

    Federow, H.

    1977-09-01

    The 1976 experience of Cambridge, Massachusetts, in settling the recombinant DNA research issue is unique in recent history as the first instance of essentially lay panels judging the conduct of scientific research. Furthermore, because the panel was composed of citizens who would be affected by the research, the experience suggests a model for conflict resolution in other areas of public controversy. With one of these, nuclear energy, the controversy has two important points in common: although the primary burden of any accident would be borne by the local community, benefits of the DNA research or reactor operation accrue to a much broader range of people; and in both issues there is a need to resolve the question, ''How safe is safe enough.'' It is therefore proposed that a panel similar to the Cambridge one could be established to deal with the controversy surrounding a proposed nuclear plant. In any community where there was such controversy, a panel could be convened to assess whether the plant was acceptable to that community. Such a panel would be composed of members of the community who were not affected directly by the plant. It would also have to have a restricted range of inquiry, oriented toward the specifics of the proposed plant. Such a plant review panel, under properly designed procedures, could change the licensing process to one concerned solely with safety and provide an appropriate forum for issues concerning the acceptability of nuclear power

  10. Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates.

    Science.gov (United States)

    Jung, Hana; Lee, Jin A; Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

    2014-01-01

    Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

  11. recA+-dependent inactivation of the lambda repressor in Escherichia coli lysogens by γ-radiation and by tif expression

    International Nuclear Information System (INIS)

    West, S.C.; Powell, K.A.; Emmerson, P.T.

    1975-01-01

    When lambda lysogens of E. coli are induced by γ-radiation the lambda repressor, as measured by its specific binding to lambda DNA, is rapidly inactivated by a recA + -dependent process which does not require new protein synthesis. This rapid inactivation is similar to inactivation of repressor by expression of the temperature sensitive E. coli mutation tif. In contrast, induction by UV irradiation or mitomycin C treatment requires new protein synthesis and there is a lag before the repressor is inactivated (Tomizawa and Ogawa, 1967; Shinagawa and Itoh, 1973). (orig.) [de

  12. Evidence for a Chk2-BRCA1-BRCA2 pathway in controlling homologous recombination

    International Nuclear Information System (INIS)

    Powell, S.N.

    2003-01-01

    The BRCA2 protein is thought to play a role as a supportive protein for the assembly of Rad51 filaments at the sites of DNA damage or stalled DNA replication, and thereby facilitates the process of homologous recombination (HR). We provide direct evidence that the interaction of BRCA2 and Rad51, via the BRC repeat motifs of BRCA2, is the key to its function in HR. Furthermore, the BRCA2's role to facilitate HR is dependent on a replicating DNA template, closely linking the process of HR to DNA replication. To date, no other role for BRCA2 has been elucidated in-vivo. BRCA1, by contrast, has a complex series of functions including a supportive role in HR, a possible role in non-homologous recombination (NHR), transcriptional co-activation and E3 ubiquitin ligase activity. The protein undergoes extensive post-translational modification, principally by phosphorylation, in both S-phase and in response to DNA damage. We show that ATM-dependent modifications of BRCA1 are important for S-phase and G2/M checkpoints, but have no direct impact on DNA repair. However, a chk2 dependent modification of BRCA1 at serine-988, appears critical for the promotion of Rad51-dependent HR and the inhibition of Mre11/Rad50/NBS1- dependent repair. Direct modification of chk2 kinase activity, by over-expression of a kinase-dead chk2, results in an identical phenotype as seen with the S988A mutation of BRCA1. Taken together, these results suggest that a chk2-BRCA1-BRCA2 dependent pathway promotes error-free HR, suppresses error-prone NHR and thereby maintains genomic stability

  13. Spin-dependent recombination involving oxygen-vacancy complexes in silicon

    Science.gov (United States)

    Franke, David P.; Hoehne, Felix; Vlasenko, Leonid S.; Itoh, Kohei M.; Brandt, Martin S.

    2014-05-01

    Spin-dependent relaxation and recombination processes in γ-irradiated n-type Czochralski-grown silicon are studied using continuous wave (cw) and pulsed electrically detected magnetic resonance (EDMR). Two processes involving the SL1 center, the neutral excited triplet state of the oxygen-vacancy complex, are observed which can be separated by their different dynamics. One of the processes is the relaxation of the excited SL1 state to the ground state of the oxygen-vacancy complex, the other a charge transfer between 31P donors and SL1 centers forming close pairs, as indicated by electrically detected electron double resonance. For both processes, the recombination dynamics is studied with pulsed EDMR techniques. We demonstrate the feasibility of true zero-field cw and pulsed EDMR for spin-1 systems and use this to measure the lifetimes of the different spin states of SL1 also at vanishing external magnetic field.

  14. Recombinant DNA production of spider silk proteins.

    Science.gov (United States)

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-11-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  15. Study of UV-induced mutagenesis in Bacillus subtilis

    International Nuclear Information System (INIS)

    Filippov, V.D.; Lotareva, O.V.

    1978-01-01

    The mechanism of UV-induced mutagenesis was studied in Bacillus subtilis departing from the assumption that a lower yield of UV-induced mutations should be found in mutants deficient in the recombination if production of mutations is coupled with the recombination process. Three recombination-deficient strains were used: two (recA and recF) with defects in different recombination pathways and the third (recB) has a block at a stage common for both of them. UV light induced reversions to prototrophy in recB cells and did not in recA and recF strains. Direct mutations, which confer to the cell additional growth requirements, were induced by UV light in recA and recF mutants. It is concluded that UV-induced mutagenesis in B subtilis is independent of the two known recombination mechanisms

  16. In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A

    NARCIS (Netherlands)

    Azevedo, de M.; Karczewski, J.; Lefevre, F.; Azevedo, V.; Miyoshi, A.; Wells, J.; Langella, P.; Chatel, J.M.

    2012-01-01

    Background The use of food-grade Lactic Acid Bacteria (LAB) as DNA delivery vehicles represents an attractive strategy to deliver DNA vaccines at the mucosal surfaces as they are generally regarded as safe (GRAS). We previously showed that either native Lactococcus lactis (LL) or recombinant

  17. Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination.

    Science.gov (United States)

    Longerich, Simonne; Meira, Lisiane; Shah, Dharini; Samson, Leona D; Storb, Ursula

    2007-12-01

    Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T>G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag-/- mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A>C:G transition mutations in Aag-/- animals, suggesting that Aag may be involved in creating the SHM A>T bias seen in wild type mice.

  18. Correlated motion of protein subdomains and large-scale conformational flexibility of RecA protein filament

    Science.gov (United States)

    Yu, Garmay; A, Shvetsov; D, Karelov; D, Lebedev; A, Radulescu; M, Petukhov; V, Isaev-Ivanov

    2012-02-01

    Based on X-ray crystallographic data available at Protein Data Bank, we have built molecular dynamics (MD) models of homologous recombinases RecA from E. coli and D. radiodurans. Functional form of RecA enzyme, which is known to be a long helical filament, was approximated by a trimer, simulated in periodic water box. The MD trajectories were analyzed in terms of large-scale conformational motions that could be detectable by neutron and X-ray scattering techniques. The analysis revealed that large-scale RecA monomer dynamics can be described in terms of relative motions of 7 subdomains. Motion of C-terminal domain was the major contributor to the overall dynamics of protein. Principal component analysis (PCA) of the MD trajectories in the atom coordinate space showed that rotation of C-domain is correlated with the conformational changes in the central domain and N-terminal domain, that forms the monomer-monomer interface. Thus, even though C-terminal domain is relatively far from the interface, its orientation is correlated with large-scale filament conformation. PCA of the trajectories in the main chain dihedral angle coordinate space implicates a co-existence of a several different large-scale conformations of the modeled trimer. In order to clarify the relationship of independent domain orientation with large-scale filament conformation, we have performed analysis of independent domain motion and its implications on the filament geometry.

  19. DNA Binding in High Salt: Analysing the Salt Dependence of Replication Protein A3 from the Halophile Haloferax volcanii

    Directory of Open Access Journals (Sweden)

    Jody A. Winter

    2012-01-01

    Full Text Available Halophilic archaea maintain intracellular salt concentrations close to saturation to survive in high-salt environments and their cellular processes have adapted to function under these conditions. Little is known regarding halophilic adaptation of the DNA processing machinery, particularly intriguing since protein-DNA interactions are classically salt sensitive. To investigate such adaptation, we characterised the DNA-binding capabilities of recombinant RPA3 from Haloferax volcanii (HvRPA3. Under physiological salt conditions (3 M KCl, HvRPA3 is monomeric, binding 18 nucleotide ssDNA with nanomolar affinity, demonstrating that RPAs containing the single OB-fold/zinc finger architecture bind with broadly comparable affinity to two OB-fold/zinc finger RPAs. Reducing the salt concentration to 1 M KCl induces dimerisation of the protein, which retains its ability to bind DNA. On circular ssDNA, two concentration-dependent binding modes are observed. Conventionally, increased salt concentration adversely affects DNA binding but HvRPA3 does not bind DNA in 0.2 M KCl, although multimerisation may occlude the binding site. The single N-terminal OB-fold is competent to bind DNA in the absence of the C-terminal zinc finger, albeit with reduced affinity. This study represents the first quantitative characterisation of DNA binding in a halophilic protein in extreme salt concentrations.

  20. Histone dosage regulates DNA damage sensitivity in a checkpoint-independent manner by the homologous recombination pathway

    Science.gov (United States)

    Liang, Dun; Burkhart, Sarah Lyn; Singh, Rakesh Kumar; Kabbaj, Marie-Helene Miquel; Gunjan, Akash

    2012-01-01

    In eukaryotes, multiple genes encode histone proteins that package genomic deoxyribonucleic acid (DNA) and regulate its accessibility. Because of their positive charge, ‘free’ (non-chromatin associated) histones can bind non-specifically to the negatively charged DNA and affect its metabolism, including DNA repair. We have investigated the effect of altering histone dosage on DNA repair in budding yeast. An increase in histone gene dosage resulted in enhanced DNA damage sensitivity, whereas deletion of a H3–H4 gene pair resulted in reduced levels of free H3 and H4 concomitant with resistance to DNA damaging agents, even in mutants defective in the DNA damage checkpoint. Studies involving the repair of a HO endonuclease-mediated DNA double-strand break (DSB) at the MAT locus show enhanced repair efficiency by the homologous recombination (HR) pathway on a reduction in histone dosage. Cells with reduced histone dosage experience greater histone loss around a DSB, whereas the recruitment of HR factors is concomitantly enhanced. Further, free histones compete with the HR machinery for binding to DNA and associate with certain HR factors, potentially interfering with HR-mediated repair. Our findings may have important implications for DNA repair, genomic stability, carcinogenesis and aging in human cells that have dozens of histone genes. PMID:22850743

  1. Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

    Institute of Scientific and Technical Information of China (English)

    王恒樑; 冯尔玲; 林云; 廖翔; 金明; 黄留玉; 苏国富; 黄翠芬

    2002-01-01

    In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.

  2. Loss of DNA topoisomerase I activity alters many cellular functions in Salmonella typhimurium

    International Nuclear Information System (INIS)

    Overbye, K.M.; Basu, S.K.; Margolin, P.

    1983-01-01

    In this paper is reported the absence of DNA topoisomerase I in S. typhimurium results in an increased level of the recBC DNase (exonuclease V) enzyme, an almost total abolition of both direct and indirect mutagenesis by alkylating agents, and altered characteristics in the formation of chromosomal tandem duplications. We also present evidence that modifications in DNA superhelicity may strongly affect the pattern of DNA degrafation initiated by treatment of recA mutant cells with bleomycin and mitomycin C. 43 references, 3 figures, 3 tables

  3. Interdependence of the kinetics of NTP hydrolysis and the stability of the RecA-ssDNA complex.

    Science.gov (United States)

    Katz, F S; Bryant, F R

    2001-09-18

    The ssDNA-dependent NTP hydrolysis activity of the RecA protein was examined using a series of dTn oligomers ranging in size from dT10 to dT2000 as the ssDNA effector. There were three distinct manifestations of the dTn-dependent NTP hydrolysis reaction, depending on the length of the dTn effector that was used. With longer dTn oligomers, NTP hydrolysis occurred with a turnover number of 20-25 min(-1) and the observed S0.5 value for the NTP was independent of the concentration of the dTn oligomer (DNA concentration-independent hydrolysis). With dTn oligomers of intermediate length, NTP hydrolysis still occurred with a turnover number of 20-25 min(-1), but the observed S0.5 for the NTP decreased with increasing dTn concentration until reaching a value similar to that obtained with the longer dTn oligomers (DNA concentration-dependent hydrolysis). With shorter dTn oligomers, the NTP hydrolysis activity was effectively eliminated. Although this general progression of kinetic behavior was observed for the three structurally related NTPs (dATP, ATP, and GTP), the dTn oligomer length at which DNA concentration-independent, DNA concentration-dependent, and no NTP hydrolysis was observed depended on the NTP being considered. For example, dATP (S0.5 = 35 microM) was hydrolyzed in the presence of dT20, whereas ATP (S0.5 = 70 microM) and GTP (S0.5 = 1200 microM) required at least dT50 and dT200 for hydrolysis, respectively. These results are discussed in terms of a kinetic model in which the stability of the RecA-ssDNA-NTP complex is dependent on the intrinsic S0.5 value of the NTP being hydrolyzed.

  4. Spin-dependent recombination involving oxygen-vacancy complexes in silicon

    OpenAIRE

    Franke, David P.; Hoehne, Felix; Vlasenko, Leonid S.; Itoh, Kohei M.; Brandt, Martin S.

    2014-01-01

    Spin-dependent relaxation and recombination processes in $\\gamma$-irradiated $n$-type Czochralski-grown silicon are studied using continuous wave (cw) and pulsed electrically detected magnetic resonance (EDMR). Two processes involving the SL1 center, the neutral excited triplet state of the oxygen-vacancy complex, are observed which can be separated by their different dynamics. One of the processes is the relaxation of the excited SL1 state to the ground state of the oxygen-vacancy complex, t...

  5. The role of DNA dependent protein kinase in synapsis of DNA ends.

    Science.gov (United States)

    Weterings, Eric; Verkaik, Nicole S; Brüggenwirth, Hennie T; Hoeijmakers, Jan H J; van Gent, Dik C

    2003-12-15

    DNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks the action of exonucleases and ligases. The DNA termini become accessible after autophosphorylation of DNA-PK(CS), which we demonstrate to require synapsis of DNA ends. Interestingly, the presence of DNA-PK prevents ligation of the two synapsed termini, but allows ligation to another DNA molecule. This alteration of the ligation route is independent of the type of ligase that we used, indicating that the intrinsic architecture of the DNA-PK complex itself is not able to support ligation of the synapsed DNA termini. We present a working model in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA-PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. We infer that joining of synapsed DNA termini would require an additional protein factor.

  6. Spin-dependent tunneling recombination in heterostructures with a magnetic layer

    Energy Technology Data Exchange (ETDEWEB)

    Denisov, K. S., E-mail: denisokonstantin@gmail.com; Rozhansky, I. V.; Averkiev, N. S. [Russian Academy of Sciences, Ioffe Physical–Technical Institute (Russian Federation); Lähderanta, E. [Lappeenranta University of Technology (Finland)

    2017-01-15

    We propose a mechanism for the generation of spin polarization in semiconductor heterostructures with a quantum well and a magnetic impurity layer spatially separated from it. The spin polarization of carriers in a quantum well originates from spin-dependent tunneling recombination at impurity states in the magnetic layer, which is accompanied by a fast linear increase in the degree of circular polarization of photoluminescence from the quantum well. Two situations are theoretically considered. In the first case, resonant tunneling to the spin-split sublevels of the impurity center occurs and spin polarization is caused by different populations of resonance levels in the quantum well for opposite spin projections. In the second, nonresonant case, the spin-split impurity level lies above the occupied states of electrons in the quantum well and plays the role of an intermediate state in the two-stage coherent spin-dependent recombination of an electron from the quantum well and a hole in the impurity layer. The developed theory allows us to explain both qualitatively and quantitatively the kinetics of photoexcited electrons in experiments with photoluminescence with time resolution in Mn-doped InGaAs heterostructures.

  7. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.

    Science.gov (United States)

    Zheng, Wenjun; Wang, Qingsong; Bi, Qun

    2016-04-01

    Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.

  8. The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

    Science.gov (United States)

    Knoll, Alexander; Higgins, James D; Seeliger, Katharina; Reha, Sarah J; Dangel, Natalie J; Bauknecht, Markus; Schröpfer, Susan; Franklin, F Christopher H; Puchta, Holger

    2012-04-01

    The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

  9. Recombinant protein production from stable mammalian cell lines and pools.

    Science.gov (United States)

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

    Science.gov (United States)

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-01-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

  11. Controlled Release from Recombinant Polymers

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  12. A nuclear mutation defective in mitochondrial recombination in yeast.

    Science.gov (United States)

    Ling, F; Makishima, F; Morishima, N; Shibata, T

    1995-08-15

    Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

  13. DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

    Directory of Open Access Journals (Sweden)

    Adam J L Cook

    2007-04-01

    Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

  14. Laboratory of Mutagenesis and DNA Repair

    International Nuclear Information System (INIS)

    2000-01-01

    Full text: Two main lines of research were continued: the first one concerned the mechanisms controlling the fidelity of DNA replication in Escherichia coli; the second concerned cellular responses of Saccharomyces cerevisiae to DNA damaging agents. We have been investigating the question whether during chromosomal DNA replication in Escherichia coli the two DNA strands may be replicated with differential accuracy. To address this question we set up a new system that allows the examination of mutagenesis either of the leading strand or the lagging strand. Our results suggest that the lagging strand replication of the E. coli chromosome may be more accurate than leading strand replication. More recently, we studied mutagenesis of the two strands in recA730 strains which exhibit constitutive expression of the SOS system. Our results clearly indicate that in recA730 strains there is a significant difference in the fidelity of replication between the two replicating strands. Based on our data we propose a model describing a possible mechanism of SOS mutagenesis. To get more insight into cellular responses to DNA damage we have isolated several novel genes of S. cerevisiae, the transcription of which is induced by DNA lesions. Main effort was concentrated on the characterization of the DIN7 gene. We found that Din7p specifically affects the metabolism of mitochondrial DNA (mtDNA). The elevated level of Din7p results in an increased frequency of mitochondrial petite mutants, as well as in a higher frequency of mitochondrial point mutations. Din7p affects also the stability of microsatellite sequences present in the mitochondrial genome. As expected, Din7p was found to be located in mitochondria. In another project, we found that the DIN8 gene isolated in our laboratory is identical with the UMP1 gene encoding a chaperone-like protein involved in 20S proteasome maturation. Interestingly, induction of UMP1 expression in response to DNA damage is subject to regulation

  15. Sequence dependence of electron-induced DNA strand breakage revealed by DNA nanoarrays

    DEFF Research Database (Denmark)

    Keller, Adrian; Rackwitz, Jenny; Cauët, Emilie

    2014-01-01

    The electronic structure of DNA is determined by its nucleotide sequence, which is for instance exploited in molecular electronics. Here we demonstrate that also the DNA strand breakage induced by low-energy electrons (18 eV) depends on the nucleotide sequence. To determine the absolute cross sec...

  16. Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks.

    Science.gov (United States)

    Stephanou, Nicolas C; Gao, Feng; Bongiorno, Paola; Ehrt, Sabine; Schnappinger, Dirk; Shuman, Stewart; Glickman, Michael S

    2007-07-01

    Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis Delta recA Delta ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.

  17. Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase.

    Directory of Open Access Journals (Sweden)

    Lakshmikanth Mariyanna

    Full Text Available Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR-specific recombinase (Tre-recombinase has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD from the HIV-1 Tat trans-activator or the translocation motif (TLM of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.

  18. Time-dependent wave-packet study of the direct low-energy dissociative recombination of HD+

    International Nuclear Information System (INIS)

    Orel, A. E.

    2000-01-01

    Wave-packet methods involving the numerical solution of the time-dependent Schroedinger equation have been used with great success in the calculation of cross sections for dissociative recombination of molecular ions by electron impact in the high energy region where the ''boomerang'' model [L. Dube and A. Herzenberg, Phys. Rev. A 11, 1314 (1975)] is valid. We extend this method to study low-energy dissociative recombination where this approximation is no longer appropriate. We apply the method to the ''direct'' low-energy dissociative recombination of HD + . Our results are in excellent agreement with calculations using the multichannel quantum defect method. (c) 2000 The American Physical Society

  19. The role of Candida albicans homologous recombination factors Rad54 and Rdh54 in DNA damage sensitivity

    Directory of Open Access Journals (Sweden)

    White Theodore C

    2011-09-01

    Full Text Available Abstract Background The fungal pathogen Candida albicans is frequently seen in immune suppressed patients, and resistance to one of the most widely used antifungals, fluconazole (FLC, can evolve rapidly. In recent years it has become clear that plasticity of the Candida albicans genome contributes to drug resistance through loss of heterozygosity (LOH at resistance genes and gross chromosomal rearrangements that amplify gene copy number of resistance associated genes. This study addresses the role of the homologous recombination factors Rad54 and Rdh54 in cell growth, DNA damage and FLC resistance in Candida albicans. Results The data presented here support a role for homologous recombination in cell growth and DNA damage sensitivity, as Candida albicans rad54Δ/rad54Δ mutants were hypersensitive to MMS and menadione, and had an aberrant cell and nuclear morphology. The Candida albicans rad54Δ/rad54Δ mutant was defective in invasion of Spider agar, presumably due to the altered cellular morphology. In contrast, mutation of the related gene RDH54 did not contribute significantly to DNA damage resistance and cell growth, and deletion of either Candida albicans RAD54 or Candida albicans RDH54 did not alter FLC susceptibility. Conclusions Together, these results support a role for homologous recombination in genome stability under nondamaging conditions. The nuclear morphology defects in the rad54Δ/rad54Δ mutants show that Rad54 performs an essential role during mitotic growth and that in its absence, cells arrest in G2. The viability of the single mutant rad54Δ/rad54Δ and the inability to construct the double mutant rad54Δ/rad54Δ rdh54Δ/rdh54Δ suggests that Rdh54 can partially compensate for Rad54 during mitotic growth.

  20. Genetic exchanges caused by ultraviolet photoproducts in phage lamda DNA molecules: the role of DNA replication

    International Nuclear Information System (INIS)

    Lin, P.F.; Howard-Flanders, P.; Yale Univ., New Haven, Conn.

    1976-01-01

    Genetic recombination induced by structural damage in DNA molecules was investigated in E. coli K12(lamda) lysogens infected with genetically marked phage lamda. Photoproducts were induced in the phage DNA before infection by exposing them either to 313 nm light in the presence of acetophenone or to 254 nm light. To test the role of the replication of the damage phage DNA on the frequency of the induced recombination , both heteroimmune and homoimmune crosses were performed, and scored for P + recombinants. In heteroimmune crosses, recombination was less frequent in infected cells exposed to visible light and in wild type cells able to perform excision repair than in excision-defective lysogens. Therefore, much of the induced recombination can be attributed to the pyrimidine dimers in the phage DNA. In homoimmune crosses, replication of the phage DNA containing ultraviolet photoproducts was represented by lamda immunity, and was further blocked by the lack of the P gene product needed for replication. The 254 nm photoproducts increased the frequency of recombination in these homoimmune crosses, even though phage DNA replication was blocked. Irradiation with 313 nm light and acetophenone M, which produces dimers and unknown photoproducts, was not as effective per dimer as the 254 nm light. It is concluded from these results that certain unidentified 254 nm photoproducts can cause recombination even in the absence of DNA replication. They are not pyrimidine dimers, as they are not susceptible to excision repair or photoreactivation. In contrast, pyrimidine dimers appear to cause recombination only when the DNA containing them undergoes replication. (orig./MG) [de

  1. Time-dependent mobility and recombination of the photoinduced charge carriers in conjugated polymer/fullerene bulk heterojunction solar cells

    Science.gov (United States)

    Mozer, A. J.; Dennler, G.; Sariciftci, N. S.; Westerling, M.; Pivrikas, A.; Österbacka, R.; Juška, G.

    2005-07-01

    Time-dependent mobility and recombination in the blend of poly[2-methoxy-5-(3,7-dimethyloctyloxy)-phenylene vinylene] (MDMO-PPV) and 1-(3-methoxycarbonyl)propyl-1-phenyl-(6,6)- C61 (PCBM) is studied simultaneously using the photoinduced charge carrier extraction by linearly increasing voltage technique. The charge carriers are photogenerated by a strongly absorbed, 3 ns laser flash, and extracted by the application of a reverse bias voltage pulse after an adjustable delay time (tdel) . It is found that the mobility of the extracted charge carriers decreases with increasing delay time, especially shortly after photoexcitation. The time-dependent mobility μ(t) is attributed to the energy relaxation of the charge carriers towards the tail states of the density of states distribution. A model based on a dispersive bimolecular recombination is formulated, which properly describes the concentration decay of the extracted charge carriers at all measured temperatures and concentrations. The calculated bimolecular recombination coefficient β(t) is also found to be time-dependent exhibiting a power law dependence as β(t)=β0t-(1-γ) with increasing slope (1-γ) with decreasing temperatures. The temperature dependence study reveals that both the mobility and recombination of the photogenerated charge carriers are thermally activated processes with activation energy in the range of 0.1 eV. Finally, the direct comparison of μ(t) and β(t) shows that the recombination of the long-lived charge carriers is controlled by diffusion.

  2. DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

    Science.gov (United States)

    Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan P

    2017-09-01

    Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25bp dsDNA or 25bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35bp blunt ended dsDNA) or 25bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Molecular anatomy of the recombination mediator function of Saccharomyces cerevisiae Rad52

    DEFF Research Database (Denmark)

    Seong, C.; Sehorn, M.G.; Plate, Iben

    2008-01-01

    A helical filament of Rad51 on single-strand DNA (ssDNA), called the presynaptic filament, catalyzes DNA joint formation during homologous recombination. Rad52 facilitates presynaptic filament assembly, and this recombination mediator activity is thought to rely on the interactions of Rad52...... with Rad51, the ssDNA-binding protein RPA, and ssDNA. The N-terminal region of Rad52, which has DNA binding activity and an oligomeric structure, is thought to be crucial for mediator activity and recombination. Unexpectedly, we find that the C-terminal region of Rad52 also harbors a DNA binding function....... Importantly, the Rad52 C-terminal portion alone can promote Rad51 presynaptic filament assembly. The middle portion of Rad52 associates with DNA-bound RPA and contributes to the recombination mediator activity. Accordingly, expression of a protein species that harbors the middle and C-terminal regions of Rad...

  4. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Kenyon, C.J.; Walker, G.C.

    1988-05-01

    Operon fusions in Escherichia coli were obtained that showed increased beta-galactosidase expression in response to treatment with the DNA-damaging agent mitomycin C. These fusions were generated by using the Mud(ApR, lac) vector to insert the lactose structural genes randomly into the bacterial chromosome. Induction of beta-galactosidase in these strains, which carried fusions of lac to these din (damage-inducible) loci, was (i) triggered by UV light as well as by mitomycin C and (ii) abolished by either a recA- or a lexA- mutation. Similar characteristics of induction were observed when the lactose genes were fused to a prophage lambda promoter by using Mud(ApR, lac). These results indicate that E. coli contains a set of genes that, like prophage lambda genes, are expressed in response to DNA-damaging agents and regulated by the recA and lexA gene products. These din genes map at five bacterial loci. One din::Mud(ApR, lac) insertion results in a UV-sensitive phenotype and may be within the uvrA transcriptional unit.

  5. Physical analyses of E. coli heteroduplex recombination products in vivo: on the prevalence of 5' and 3' patches.

    Directory of Open Access Journals (Sweden)

    Laura M Gumbiner-Russo

    Full Text Available BACKGROUND: Homologous recombination in Escherichia coli creates patches (non-crossovers or splices (half crossovers, each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE repair specify that only the 3'-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands. METHODOLOGY/PRINCIPLE FINDINGS: This paper re-examines heteroduplex-patch-strand polarity using phage lambda and the lambdadv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for lambda recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5' or 3' DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation. CONCLUSIONS/SIGNIFICANCE: These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the

  6. Recombinant organisms for production of industrial products

    OpenAIRE

    Adrio, Jose-Luis; Demain, Arnold L

    2009-01-01

    A revolution in industrial microbiology was sparked by the discoveries of ther double-stranded structure of DNA and the development of recombinant DNA technology. Traditional industrial microbiology was merged with molecular biology to yield improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals and industrial enzymes. Novel genetic techniques such as metabolic engineering, combinatorial biosynthesis and molecular breeding...

  7. Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

    Science.gov (United States)

    Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

    2017-01-05

    Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Equilibrious Strand Exchange Promoted by DNA Conformational Switching

    Science.gov (United States)

    Wu, Zhiguo; Xie, Xiao; Li, Puzhen; Zhao, Jiayi; Huang, Lili; Zhou, Xiang

    2013-01-01

    Most of DNA strand exchange reactions in vitro are based on toehold strategy which is generally nonequilibrium, and intracellular strand exchange mediated by proteins shows little sequence specificity. Herein, a new strand exchange promoted by equilibrious DNA conformational switching is verified. Duplexes containing c-myc sequence which is potentially converted into G-quadruplex are designed in this strategy. The dynamic equilibrium between duplex and G4-DNA is response to the specific exchange of homologous single-stranded DNA (ssDNA). The SER is enzyme free and sequence specific. No ATP is needed and the displaced ssDNAs are identical to the homologous ssDNAs. The SER products and exchange kenetics are analyzed by PAGE and the RecA mediated SER is performed as the contrast. This SER is a new feature of G4-DNAs and a novel strategy to utilize the dynamic equilibrium of DNA conformations.

  9. Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Kalpana Mujoo

    2017-11-01

    Full Text Available The nitric oxide (NO-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1. Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18, which causes stem cell differentiation has no effect on double-strand break (DSB repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

  10. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    Science.gov (United States)

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-02-17

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair.

    Directory of Open Access Journals (Sweden)

    Corentin Claeys Bouuaert

    2017-05-01

    Full Text Available Mlh1-Mlh3 (MutLγ is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ and, surprisingly, single-stranded DNA (ssDNA, which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced

  12. Prevalence of Germline Mutations in Genes Engaged in DNA Damage Repair by Homologous Recombination in Patients with Triple-Negative and Hereditary Non-Triple-Negative Breast Cancers.

    Directory of Open Access Journals (Sweden)

    Pawel Domagala

    Full Text Available This study sought to assess the prevalence of common germline mutations in several genes engaged in the repair of DNA double-strand break by homologous recombination in patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers. Tumors deficient in this type of DNA damage repair are known to be especially sensitive to DNA cross-linking agents (e.g., platinum drugs and to poly(ADP-ribose polymerase (PARP inhibitors.Genetic testing was performed for 36 common germline mutations in genes engaged in the repair of DNA by homologous recombination, i.e., BRCA1, BRCA2, CHEK2, NBN, ATM, PALB2, BARD1, and RAD51D, in 202 consecutive patients with triple-negative breast cancers and hereditary non-triple-negative breast cancers.Thirty five (22.2% of 158 patients in the triple-negative group carried mutations in genes involved in DNA repair by homologous recombination, while 10 (22.7% of the 44 patients in the hereditary non-triple-negative group carried such mutations. Mutations in BRCA1 were most frequent in patients with triple-negative breast cancer (18.4%, and mutations in CHEK2 were most frequent in patients with hereditary non-triple-negative breast cancers (15.9%. In addition, in the triple-negative group, mutations in CHEK2, NBN, and ATM (3.8% combined were found, while mutations in BRCA1, NBN, and PALB2 (6.8% combined were identified in the hereditary non-triple-negative group.Identifying mutations in genes engaged in DNA damage repair by homologous recombination other than BRCA1/2 can substantially increase the proportion of patients with triple-negative breast cancer and hereditary non-triple-negative breast cancer who may be eligible for therapy using PARP inhibitors and platinum drugs.

  13. GroEL and dnaK genes of Escherichia coli are induced by UV irradiation and nalidixic acid in an htpR+-dependent fashion

    International Nuclear Information System (INIS)

    Krueger, J.H.; Walker, G.C.

    1984-01-01

    Two proteins with molecular weights of 61,000 and 73,000 were found to be induced by UV light in Escherichia coli mutants in which the SOS responses are constitutively expressed. The induction of these proteins by UV light and nalidixic acid was shown to be independent of the recA + lexA + regulatory system. Analysis of these proteins by two-dimensional gel electrophoresis and comparison with the heat-shock proteins of E. coli revealed that the M/sub r/ 61,000 protein comigrated with the groEL gene product, that the M/sub r/ 73,000 protein comigrated with the dnaK gene product, and that other heat-shock proteins were also induced. The induction of groEL and dnaK by UV light and nalidixic acid is controlled by the htpR locus. The results suggest that the regulatory response of E. coli to agents such as UV light and nalidixic acid is more complex than previously thought. 35 references, 6 figures, 1 table

  14. The contribution of alu elements to mutagenic DNA double-strand break repair.

    Science.gov (United States)

    Morales, Maria E; White, Travis B; Streva, Vincent A; DeFreece, Cecily B; Hedges, Dale J; Deininger, Prescott L

    2015-03-01

    Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both

  15. The unconventional xer recombination machinery of Streptococci/Lactococci

    NARCIS (Netherlands)

    Le Bourgeois, Pascal; Bugarel, Marie; Campo, Nathalie; Daveran-Mingot, Marie-Line; Labonte, Jessica; Lanfranchi, Daniel; Lautier, Thomas; Pages, Carine; Ritzenthaler, Paul

    Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving

  16. Genome-Wide Analysis of Heteroduplex DNA in Mismatch Repair–Deficient Yeast Cells Reveals Novel Properties of Meiotic Recombination Pathways

    Science.gov (United States)

    Martini, Emmanuelle; Borde, Valérie; Legendre, Matthieu; Audic, Stéphane; Regnault, Béatrice; Soubigou, Guillaume; Dujon, Bernard; Llorente, Bertrand

    2011-01-01

    Meiotic DNA double-strand breaks (DSBs) initiate crossover (CO) recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs). Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR) protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs). First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR–proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans–hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs) during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates. PMID

  17. Genome-wide analysis of heteroduplex DNA in mismatch repair-deficient yeast cells reveals novel properties of meiotic recombination pathways.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Martini

    2011-09-01

    Full Text Available Meiotic DNA double-strand breaks (DSBs initiate crossover (CO recombination, which is necessary for accurate chromosome segregation, but DSBs may also repair as non-crossovers (NCOs. Multiple recombination pathways with specific intermediates are expected to lead to COs and NCOs. We revisited the mechanisms of meiotic DSB repair and the regulation of CO formation, by conducting a genome-wide analysis of strand-transfer intermediates associated with recombination events. We performed this analysis in a SK1 × S288C Saccharomyces cerevisiae hybrid lacking the mismatch repair (MMR protein Msh2, to allow efficient detection of heteroduplex DNAs (hDNAs. First, we observed that the anti-recombinogenic activity of MMR is responsible for a 20% drop in CO number, suggesting that in MMR-proficient cells some DSBs are repaired using the sister chromatid as a template when polymorphisms are present. Second, we observed that a large fraction of NCOs were associated with trans-hDNA tracts constrained to a single chromatid. This unexpected finding is compatible with dissolution of double Holliday junctions (dHJs during repair, and it suggests the existence of a novel control point for CO formation at the level of the dHJ intermediate, in addition to the previously described control point before the dHJ formation step. Finally, we observed that COs are associated with complex hDNA patterns, confirming that the canonical double-strand break repair model is not sufficient to explain the formation of most COs. We propose that multiple factors contribute to the complexity of recombination intermediates. These factors include repair of nicks and double-stranded gaps, template switches between non-sister and sister chromatids, and HJ branch migration. Finally, the good correlation between the strand transfer properties observed in the absence of and in the presence of Msh2 suggests that the intermediates detected in the absence of Msh2 reflect normal intermediates.

  18. RNase H and replication of ColE1 DNA in Escherichia coli.

    OpenAIRE

    Naito, S; Uchida, H

    1986-01-01

    Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase ...

  19. Breakthrough of ultraviolet light from various brands of fluorescent lamps: Lethal effects on DNA repair-defective bacteria

    International Nuclear Information System (INIS)

    Hartman, P.E.; Biggley, W.H.

    1996-01-01

    In a comparative study of 17 pairs of 15 W fluorescent lamps intended for use in homes and purchased in local stores, we detect over 10-fold differences in UVB + UVC emissions between various lamps. This breakthrough of ultraviolet (UV) light is in part correlated with ability of lamps to kill DNA repair-defective recA - uvrB - Salmonella. Relative proficiency of lamps in eliciting photoreactivation of UV-induced DNA lesions also plays a prominent role in the relative rates of bacterial inactivation by emissions from different lamps. Lamps made in Chile, such as Phillips brand lamps and one type of General Electric lamp, produce far less UVB + UVC and fail to kill recA - uvrB - bacteria. In contrast, all tested lamps manufactured in the USA, Hungary, and Japan exhibit readily observed deleterious biological effects. When an E. coli recA - uvrB - phr - (photolyase-negative) triple mutant is used for assay, lethal radiations are detected from all lamps, and single-hit exponential inactivation rates rather closely correlate to amount of directly measured UVB + UVC output of each pair of lamps. Although all lamps tested may meet international and Unite States standards for radiation safely, optimal practices in lamp manufacture are clearly capable of decreasing human exposure to indoor UV light. 38 refs., 3 figs., 1 tab

  20. Gene conversion at the gray locus of Sordaria fimicola: fit of the experimental data to a hybrid DNA model of recombination.

    Science.gov (United States)

    Kalogeropoulos, A; Thuriaux, P

    1985-03-01

    A hybrid DNA (hDNA) model of recombination has been algebraically formulated, which allows the prediction of frequencies of postmeiotic segregation and conversion of a given allele and their probability of being associated with a crossing over. The model considered is essentially the "Aviemore model." In contrast to some other interpretations of recombination, it states that gene conversion can only result from the repair of heteroduplex hDNA, with postmeiotic segregation resulting from unrepaired heteroduplexes. The model also postulates that crossing over always occurs distally to the initiation site of the hDNA. Eleven types of conversion and postmeiotic segregation with or without associated crossover were considered. Their theoretical frequencies are given by 11 linear equations with ten variables, four describing heteroduplex repair, four giving the probability of hDNA formation and its topological properties and two giving the probability that crossing over occurs at the left or right of the converting allele. Using the experimental data of Kitani and coworkers on conversion at the six best studied gray alleles of Sordaria fimicola, we found that the model considered fit the data at a P level above or very close (allele h4) to the 5% level of sampling error provided that the hDNA is partly asymmetric. The best fitting solutions are such that the hDNA has an equal probability of being formed on either chromatid or, alternatively, that both DNA strands have the same probability of acting as the invading strand during hDNA formation. The two mismatches corresponding to a given allele are repaired with different efficiencies. Optimal solutions are found if one allows for repair to be more efficient on the asymmetric hDNA than on the symmetric one. In the case of allele g1, our data imply that the direction of repair is nonrandom with respect to the strand on which it occurs.

  1. Hyper-recombinogenity of the chimeric protein RecAX53 (Esherichia coli/Pseudomonas aeruginosa is caused by its increased dynamics

    Directory of Open Access Journals (Sweden)

    Daria B Chervyakova

    2008-12-01

    Full Text Available RecAX53 is the most recombinogenic protein among the chimeric RecA proteins composed ofEsherichia coli RecA (RecAEc and Pseudomonas aeruginosa RecA (RecAPa protein fragments. We found out that RecAX53 protein is more rapid in ATP hydrolysis, dissociation from single-stranded DNA (ssDNA, SSB protein displacement from ssDNA and in association with doublestranded DNA (dsDNA, as compared with RecAEc and RecAPa proteins. These results indicate that the RecAX53 hyper-recombinogenity is caused by high dynamics of this protein - by its rapid association with and dissociation from ssDNA. The ability of RecAX53 to bind actively with dsDNA accounts for the SOS-independent mechanism of hyper-recombination used by this protein.

  2. Effect of NaN3 on oxygen-dependent lethality of UV-A in Escherichia coli mutants lacking active oxygen-defence and DNA-repair systems

    International Nuclear Information System (INIS)

    Yamada, Kazumasa; Ono, Tetsuyoshi; Nishioka, Hajime

    1996-01-01

    Escherichia coli mutants which lack defence systems against such active oxygen forms as OxyR (ΔoxyR), superoxide dismutase (SOD) (sodA and sodB) and catalase (katE and katG) are sensitive to UV-A lethality under aerobic conditions, whereas OxyR- and SOD-mutants have resistance under anaerobic conditions and in the presence of sodium azide (NaN 3 ) during irradiation. UV-A induces lipid peroxidation in the ΔoxyR mutant, which is suppressed by NaN 3 . These results suggest that UV-A generates 1 O 2 or the hydroxyl radical to produce lipid peroxides intracellularly in the ΔoxyR mutant and that O 2 - stress may be generated in the sodAB mutant after 8 hr of exposure to UV-A. The sensitivities of such DNA repair-deficient mutants as recA ind- and uvrA to UV-A also were examined and compared. These mutants are sensitive to UV-A lethality under aerobic conditions but show only slight resistance under anaerobic conditions or in the presence of NaN 3 during irradiation. We conclude that NaN 3 protects these mutant cells from oxygen-dependent UV-A lethality. (author)

  3. Induction of genetic recombination in the lambda bacteriophage by ultraviolet radiation of the Escherichia Coli cells; Induccion de recombinacion genetica en el bacteriofago lambda por irradiacion ultravioleta de las cellulas de Escherichia Coli

    Energy Technology Data Exchange (ETDEWEB)

    Alcantara D, D [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    1986-12-15

    In this work there are reported the results that show that although the stimulation of the recombination of the Lambda bacteriophage, by UV irradiation of the cells of Escherichia Coli, it looks to be the result of the high expression of the functions of the SOS system, doesn't keep some relationship with the high concentration of protein reached RecA. (Author)

  4. Induction of genetic recombination in the lambda bacteriophage by ultraviolet radiation of the Escherichia Coli cells; Induccion de recombinacion genetica en el bacteriofago lambda por irradiacion ultravioleta de las cellulas de Escherichia Coli

    Energy Technology Data Exchange (ETDEWEB)

    Alcantara D, D. [ININ, 52045 Ocoyoacac, Estado de Mexico (Mexico)

    1986-12-15

    In this work there are reported the results that show that although the stimulation of the recombination of the Lambda bacteriophage, by UV irradiation of the cells of Escherichia Coli, it looks to be the result of the high expression of the functions of the SOS system, doesn't keep some relationship with the high concentration of protein reached RecA. (Author)

  5. Factores psicosociales en la recaída de la dependencia al alcohol: Un análisis de ruta

    Directory of Open Access Journals (Sweden)

    Fabiola Alejandra Useche Torrealba

    2017-12-01

    Full Text Available Introducción: El consumo de alcohol causa alrededor del 6% de muertes a nivel mundial y se espera que el 70% de los pacientes en recuperación recaigan en los primeros seis meses de tratamiento. Esto, aunado a la aceptación social de esta sustancia y su fácil acceso, hacen necesario estudiar las variables asociadas a la recaída. Objetivo: Estudiar la influencia de variables sociodemográficas y psicosociales, sobre la recaída de la dependencia al alcohol. Métodos: Se realizó una investigación de campo, transversal y explicativa-correlacional, con la participación de 180 miembros de Alcohólicos Anónimos de Venezuela. Resultados: Participaron 87 mujeres y 93 hombres, con un promedio de 50 años. El índice de recaída fue de 18%, con un promedio de 130 meses de abstinencia. Se encontraron altos niveles de apoyo social, autoeficacia y autoestima, así como valores intermedios de estrés, impulsividad y resiliencia. Las variables estudiadas se asociaron con el tiempo en abstinencia (R = 0.615; p < 0.05 explicando el 35% de la varianza total. La edad (β = 0.57; p = 0.00, autoestima (β = -0.156; p = 0.02, apoyo social (β = 0.148; p = 0.02 y sexo (β = -0.135; p = 0.03 fueron las variables con mayor eficacia en la ruta principal. Conclusiones: El modelo propuesto se cumplió parcialmente. Es necesario profundizar el estudio de relaciones no planteadas entre las variables moderadoras, y si estas tienen otras que las modifiquen. Se recomienda enfatizar el abordaje de las variables que resultaron relevantes en la prevención de la recaída.

  6. A Small-Molecule Inducible Synthetic Circuit for Control of the SOS Gene Network without DNA Damage.

    Science.gov (United States)

    Kubiak, Jeffrey M; Culyba, Matthew J; Liu, Monica Yun; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

    2017-11-17

    The bacterial SOS stress-response pathway is a pro-mutagenic DNA repair system that mediates bacterial survival and adaptation to genotoxic stressors, including antibiotics and UV light. The SOS pathway is composed of a network of genes under the control of the transcriptional repressor, LexA. Activation of the pathway involves linked but distinct events: an initial DNA damage event leads to activation of RecA, which promotes autoproteolysis of LexA, abrogating its repressor function and leading to induction of the SOS gene network. These linked events can each independently contribute to DNA repair and mutagenesis, making it difficult to separate the contributions of the different events to observed phenotypes. We therefore devised a novel synthetic circuit to unlink these events and permit induction of the SOS gene network in the absence of DNA damage or RecA activation via orthogonal cleavage of LexA. Strains engineered with the synthetic SOS circuit demonstrate small-molecule inducible expression of SOS genes as well as the associated resistance to UV light. Exploiting our ability to activate SOS genes independently of upstream events, we further demonstrate that the majority of SOS-mediated mutagenesis on the chromosome does not readily occur with orthogonal pathway induction alone, but instead requires DNA damage. More generally, our approach provides an exemplar for using synthetic circuit design to separate an environmental stressor from its associated stress-response pathway.

  7. R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata

    Energy Technology Data Exchange (ETDEWEB)

    Youvan, D C [Univ. of California, Berkeley; Elder, J T; Sandlin, D E; Zsebo, K; Alder, D P; Panopoulos, N J; Marrs, B L; Hearst, J E

    1982-01-01

    Site-directed mutagenesis of the photosynthetic apparatus (PSA) genes in Rhodopseudomonas capsulata is presented utilizing a transposon Tn7 mutagenized R-prime. The R-prime, pRPS404, bears most of the genes necessary for the differentiation of the photosynthetic apparatus. Mutagenesis of the R-prime with Tn7 in Escherichia coli, conjugation into R. capsulata, and homologous recombination with the wild-type alleles efficiently generates photosynthetic apparatus lesions. Wild-type alleles are lost spontaneously and the Tn7-induced lesions are revealed by subsequent intramolecular recombination between IS21 insertion elements that bracket the prime sequences in direct repeat. The molecular nature of the intermediates involved in the transposition, recombination and deletion have been investigated by Southern hybridization analysis. The spontaneous loss of wild-type alleles after homologous recombination with the chromosome may be of general use to other prokaryotic site-directed transposon mutagenesis schemes. The IS21-mediated deletion of the prime DNA is dependent on the RecA protein in E. coli, generating the parental R-factor bearing one IS21 element. A genetic-physical map exists for a portion of the prime photosynthetic apparatus DNA. When Tn7 is inserted into a bacteriochlorophyll gene in the R-prime and then crossed into R. capsulata, mutants are produced that accumulate a bacteriochlorophyll precursor, which is in excellent agreement with the existing genetic-physical map. This corroborates the mutagenesis scheme.

  8. Characterization of recombinant human HBP/CAP37/azurocidin, a pleiotropic mediator of inflammation-enhancing LPS-induced cytokine release from monocytes.

    Science.gov (United States)

    Rasmussen, P B; Bjørn, S; Hastrup, S; Nielsen, P F; Norris, K; Thim, L; Wiberg, F C; Flodgaard, H

    1996-07-15

    Neutrophil-derived heparin-binding protein (HBP) is a strong chemoattractant for monocytes. We report here for the first time the expression of recombinant HBP. A baculovirus containing the human HBP cDNA mediated in insect cells the secretion of a 7-residue N-terminally extended HBP form (pro-HBP). Deletion of the pro-peptide-encoding cDNA sequence resulted in correctly processed HBP at the N-terminus. Electrospray mass spectrum analysis of recombinant HBP yielded a molecular weight of 27.237 +/- 3 amu. Consistent with this mass is a HBP form of 225 amino acids (mature part +3 amino acid C-terminal extension). The biological activity of recombinant HBP was confirmed by its chemotactic action towards monocytes. Furthermore, we have shown that recombinant HBP stimulates in a dose-dependent manner the lipopolysaccharide (LPS)-induced cytokine release from human monocytes.

  9. Research and development of basic technologies for the next generation industries, 'recombinant DNA utilizing technology'. Evaluation on the research and development; Jisedai sangyo kiban gijutsu kenkyu kaihatsu 'kumikae DNA riyo gijutsu'. Kenkyu kaihatsu hyoka

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1991-03-01

    Research, development and evaluation were performed with an objective of establishing the basic technology related to the recombinant DNA utilizing technology to create new microorganisms for processes in the chemical industry. The major achievements of the present research and development include establishment of the P450 gene manifestation system attributed from microsomes and mitochondria, and the success of the world's first simultaneous manifestation of P450 and reduction enzyme. Furthermore, the fused enzyme combining P450 and the reduction enzyme genetically was successfully manufactured ahead of the other countries, opening the way to industrializing the recombinant enzymes for use in bio-processes in the chemical industry. In creating a high-efficiency secretion recombinant bacillus subtilis stock, a bacillus subtilis host whose protease activity has been noticeably decreased was created. As an achievement of the research on the 'basic recombinant DNA technology', high-efficiency manifestation vector of medium level thermophile was created, and its usefulness was demonstrated. In addition, a host and vector system for high level thermophile was developed for the first time in the world. These achievements have opened the way to industrial utilization of the thermophilic bacteria. (NEDO)

  10. DNA end resection by CtIP and exonuclease 1 prevents genomic instability

    DEFF Research Database (Denmark)

    Eid, Wassim; Steger, Martin; El-Shemerly, Mahmoud

    2010-01-01

    End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1). In this s......End resection of DNA-which is essential for the repair of DNA double-strand breaks (DSBs) by homologous recombination-relies first on the partnership between MRE11-RAD50-NBS1 (MRN) and CtIP, followed by a processive step involving helicases and exonucleases such as exonuclease 1 (EXO1...... of DNA-PK-dependent radial chromosome formation. Thus, our study identifies new functions of CtIP and EXO1 in DNA end resection and provides new information on the regulation of DSB repair pathways, which is a key factor in the maintenance of genome integrity....

  11. Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

    KAUST Repository

    Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Corniè re, Axelle; Takahashi, Masayuki; Nordé n, Bengt

    2012-01-01

    Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated

  12. Meiotic recombination hotspots - a comparative view.

    Science.gov (United States)

    Choi, Kyuha; Henderson, Ian R

    2015-07-01

    During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover. Meiotic recombination has a profound effect on patterns of genetic variation and is an important tool during crop breeding. Crossovers initiate from programmed DNA double-stranded breaks that are processed to form single-stranded DNA, which can invade a homologous chromosome. Strand invasion events mature into double Holliday junctions that can be resolved as crossovers. Extensive variation in the frequency of meiotic recombination occurs along chromosomes and is typically focused in narrow hotspots, observed both at the level of DNA breaks and final crossovers. We review methodologies to profile hotspots at different steps of the meiotic recombination pathway that have been used in different eukaryote species. We then discuss what these studies have revealed concerning specification of hotspot locations and activity and the contributions of both genetic and epigenetic factors. Understanding hotspots is important for interpreting patterns of genetic variation in populations and how eukaryotic genomes evolve. In addition, manipulation of hotspots will allow us to accelerate crop breeding, where meiotic recombination distributions can be limiting. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  13. Effect of UV-irradiation on DNA-membrane complex of Bacillus subtilis

    International Nuclear Information System (INIS)

    Chefranova, O.A.; Gaziev, A.I.

    1979-01-01

    The UV radiation effect on DNA membrane complex of Bacillus subtilis has been studied. Increase of DNA content in the DNA membrane complex in two strains of 168 and recA - and its decrease in the polA - strain are shown. The above effect in the first two stamms is suppressed with caffeine and correlates with the change in protein content in the DNA membrane complex, determined by a radioactive label, but not lipids in other words, fixation of DNA and membrane goes through proteins. Capability of DNA content increase in the DNA membrane complex after UV irradiation and subsequent bacteria incubation in a total medium correlates with the relative sensitivity of stamm UV sensitivity. It is suggested, that the reparation synthesis goes in cells on the membrane and that binding of DNA and the membrane is necessary for the normal DNA reparation process

  14. DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

    Directory of Open Access Journals (Sweden)

    Elzbieta Pawłowska

    2017-07-01

    Full Text Available The human DNA2 (DNA replication helicase/nuclease 2 protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER, interacting with the replication protein A (RPA and the flap endonuclease 1 (FEN1. DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN and Bloom syndrome protein (BLM. In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.

  15. Metal binding proteins, recombinant host cells and methods

    Science.gov (United States)

    Summers, Anne O.; Caguiat, Jonathan J.

    2004-06-15

    The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

  16. Mechanism of replication of ultraviolet-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli. Implications for SOS mutagenesis

    International Nuclear Information System (INIS)

    Livneh, Z.

    1986-01-01

    Replication of UV-irradiated oligodeoxynucleotide-primed single-stranded phi X174 DNA with Escherichia coli DNA polymerase III holoenzyme in the presence of single-stranded DNA-binding protein was investigated. The extent of initiation of replication on the primed single-stranded DNA was not altered by the presence of UV-induced lesions in the DNA. The elongation step exhibited similar kinetics when either unirradiated or UV-irradiated templates were used. Inhibition of the 3'----5' proofreading exonucleolytic activity of the polymerase by dGMP or by a mutD mutation did not increase bypass of pyrimidine photodimers, and neither did purified RecA protein influence the extent of photodimer bypass as judged by the fraction of full length DNA synthesized. Single-stranded DNA-binding protein stimulated bypass since in its absence the fraction of full length DNA decreased 5-fold. Termination of replication at putative pyrimidine dimers involved dissociation of the polymerase from the DNA, which could then reinitiate replication at other available primer templates. Based on these observations a model for SOS-induced UV mutagenesis is proposed

  17. A nuclear mutation defective in mitochondrial recombination in yeast.

    OpenAIRE

    Ling, F; Makishima, F; Morishima, N; Shibata, T

    1995-01-01

    Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homolo...

  18. A prime/boost strategy by DNA/fowlpox recombinants expressing a mutant E7 protein for the immunotherapy of HPV-associated cancers.

    Science.gov (United States)

    Radaelli, Antonia; De Giuli Morghen, Carlo; Zanotto, Carlo; Pacchioni, Sole; Bissa, Massimiliano; Franconi, Rosella; Massa, Silvia; Paolini, Francesca; Muller, Antonio; Venuti, Aldo

    2012-12-01

    Development of effective therapeutic vaccines against human papilloma virus (HPV) infections remains a priority, considering the high number of new cases of cervical cancer each year by high-risk HPVs, in particular by HPV-16. Vaccines expressing the E7 oncoprotein, which is detectable in all HPV-positive pre-cancerous and cancer cells, might clear already established tumors and support the treatment of HPV-related lesions. In this study, DNA or fowlpox virus recombinants expressing the harmless variant E7GGG of the HPV-16 E7 oncoprotein (DNA(E7GGG) and FP(E7GGG)) were generated. Two immunization regimens were tested in a pre-clinical mouse model by homologous (FP/FP) or heterologous (DNA/FP) prime-boost protocols to evaluate the immune response and therapeutic efficacy of the proposed HPV-16 vaccine. Low levels of anti-E7-specific antibodies were elicited after immunization, and in vivo experiments resulted in a higher number of tumor-free mice after the heterologous immunization. These results establish a preliminary indication for therapy of HPV-related tumors by the combined use of DNA and avipox recombinants, which might represent safer immunogens than vaccinia-based vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Either non-homologous ends joining or homologous recombination is required to repair double-strand breaks in the genome of macrophage-internalized Mycobacterium tuberculosis.

    Science.gov (United States)

    Brzostek, Anna; Szulc, Izabela; Klink, Magdalena; Brzezinska, Marta; Sulowska, Zofia; Dziadek, Jaroslaw

    2014-01-01

    The intracellular pathogen Mycobacterium tuberculosis (Mtb) is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs). These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR) and non-homologous ends joining (NHEJ), in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA), NHEJ [Δ(ku,ligD)], or both DSB repair systems [Δ(ku,ligD,recA)]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA) mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA) mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.

  20. Either non-homologous ends joining or homologous recombination is required to repair double-strand breaks in the genome of macrophage-internalized Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Anna Brzostek

    Full Text Available The intracellular pathogen Mycobacterium tuberculosis (Mtb is constantly exposed to a multitude of hostile conditions and is confronted by a variety of potentially DNA-damaging assaults in vivo, primarily from host-generated antimicrobial toxic radicals. Exposure to reactive nitrogen species and/or reactive oxygen species causes different types of DNA damage, including oxidation, depurination, methylation and deamination, that can result in single- or double-strand breaks (DSBs. These breaks affect the integrity of the whole genome and, when left unrepaired, can lead to cell death. Here, we investigated the role of the DSB repair pathways, homologous recombination (HR and non-homologous ends joining (NHEJ, in the survival of Mtb inside macrophages. To this end, we constructed Mtb strains defective for HR (ΔrecA, NHEJ [Δ(ku,ligD], or both DSB repair systems [Δ(ku,ligD,recA]. Experiments using these strains revealed that either HR or NHEJ is sufficient for the survival and propagation of tubercle bacilli inside macrophages. Inhibition of nitric oxide or superoxide anion production with L-NIL or apocynin, respectively, enabled the Δ(ku,ligD,recA mutant strain lacking both systems to survive intracellularly. Complementation of the Δ(ku,ligD,recA mutant with an intact recA or ku-ligD rescued the ability of Mtb to propagate inside macrophages.

  1. A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks

    Science.gov (United States)

    Schär, Primo; Herrmann, Gernot; Daly, Graham; Lindahl, Tomas

    1997-01-01

    Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis. PMID:9271115

  2. Recovery of arrested replication forks by homologous recombination is error-prone.

    Directory of Open Access Journals (Sweden)

    Ismail Iraqui

    Full Text Available Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.

  3. Recombinant vaccines and the development of new vaccine strategies

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, I.P.; Leite, L.C.C. [Centro de Biotecnologia, Instituto Butantan, São Paulo, SP (Brazil)

    2012-09-07

    Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

  4. Recombinant vaccines and the development of new vaccine strategies

    Directory of Open Access Journals (Sweden)

    I.P. Nascimento

    2012-12-01

    Full Text Available Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks.

  5. Recombinant vaccines and the development of new vaccine strategies

    International Nuclear Information System (INIS)

    Nascimento, I.P.; Leite, L.C.C.

    2012-01-01

    Vaccines were initially developed on an empirical basis, relying mostly on attenuation or inactivation of pathogens. Advances in immunology, molecular biology, biochemistry, genomics, and proteomics have added new perspectives to the vaccinology field. The use of recombinant proteins allows the targeting of immune responses focused against few protective antigens. There are a variety of expression systems with different advantages, allowing the production of large quantities of proteins depending on the required characteristics. Live recombinant bacteria or viral vectors effectively stimulate the immune system as in natural infections and have intrinsic adjuvant properties. DNA vaccines, which consist of non-replicating plasmids, can induce strong long-term cellular immune responses. Prime-boost strategies combine different antigen delivery systems to broaden the immune response. In general, all of these strategies have shown advantages and disadvantages, and their use will depend on the knowledge of the mechanisms of infection of the target pathogen and of the immune response required for protection. In this review, we discuss some of the major breakthroughs that have been achieved using recombinant vaccine technologies, as well as new approaches and strategies for vaccine development, including potential shortcomings and risks

  6. Regulation of rDNA stability by sumoylation

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine; Lisby, Michael

    2009-01-01

    Repair of DNA lesions by homologous recombination relies on the copying of genetic information from an intact homologous sequence. However, many eukaryotic genomes contain repetitive sequences such as the ribosomal gene locus (rDNA), which poses a risk for illegitimate recombination. Therefore, t......6 complex and sumoylation of Rad52, which directs DNA double-strand breaks in the rDNA to relocalize from within the nucleolus to the nucleoplasm before association with the recombination machinery. The relocalization before repair is important for maintaining rDNA stability. The focus...

  7. Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Moore, Destaye M; Karlin, Justin; González-Barrera, Sergio

    2009-01-01

    In the yeast Saccharomyces cerevisiae, the Rad1-Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1-Rad10 endonuclease cleaves 3' branches of DNA and aberrant 3' DNA ends that are refractory to other 3' processing enzymes. ...

  8. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  9. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Xiong Hairong [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); State Key Laboratory of Virology, Institute of Medical Virology, Wuhan University School of Medicine, Wuhan (China); Lee, Robert J. [Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus, OH (United States); Haura, Eric B. [Thoracic Oncology and Experimental Therapeutics Programs, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Edwards, John G. [Apeliotus Technologies, Inc., Atlanta, GA (United States); Dynan, William S. [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Li Shuyi, E-mail: sli@georgiahealth.edu [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Apeliotus Technologies, Inc., Atlanta, GA (United States)

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

  10. Recombination Phenotypes of Escherichia coli greA Mutants

    Directory of Open Access Journals (Sweden)

    Poteete Anthony R

    2011-03-01

    Full Text Available Abstract Background The elongation factor GreA binds to RNA polymerase and modulates transcriptional pausing. Some recent research suggests that the primary role of GreA may not be to regulate gene expression, but rather, to promote the progression of replication forks which collide with RNA polymerase, and which might otherwise collapse. Replication fork collapse is known to generate dsDNA breaks, which can be recombinogenic. It follows that GreA malfunction could have consequences affecting homologous recombination. Results Escherichia coli mutants bearing substitutions of the active site acidic residues of the transcription elongation factor GreA, D41N and E44K, were isolated as suppressors of growth inhibition by a toxic variant of the bacteriophage lambda Red-beta recombination protein. These mutants, as well as a D41A greA mutant and a greA deletion, were tested for proficiency in recombination events. The mutations were found to increase the efficiency of RecA-RecBCD-mediated and RecA-Red-mediated recombination, which are replication-independent, and to decrease the efficiency of replication-dependent Red-mediated recombination. Conclusion These observations provide new evidence for a role of GreA in resolving conflicts between replication and transcription.

  11. Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

    International Nuclear Information System (INIS)

    Shokri, Leila; Williams, Mark C; Rouzina, Ioulia

    2009-01-01

    Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork

  12. Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein.

    Science.gov (United States)

    Galvão, Carolina W; Pedrosa, Fábio O; Souza, Emanuel M; Yates, M Geoffrey; Chubatsu, Leda S; Steffens, Maria Berenice R

    2004-06-01

    The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role. The RecX protein of the H. seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein. DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA. The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions. The ability of RecX to bind DNA may be relevant to its function in the SOS response.

  13. Lambda bacteriophage gene products and x-ray sensitivity of Escherichia coli: comparison of red-dependent and gam-dependent radioresistance

    International Nuclear Information System (INIS)

    Trgovcevic, Z.; Rupp, W.D.

    1975-01-01

    When gene products of lambda bacteriophage are introduced into a cell by transient induction of a lysogen, increased resistance of the cells to x rays results. This phenomenon has been called phage-induced radioresistance. Genetic studies show at least two classes of induced radioresistance. The first type depends on the products of the lambda red genes and is observed in bacteria that are mutated in the recB gene. It is thought that the lambda red products compensate for the missing RecBC nuclease in the repair of x-ray damage. An optimal effect is obtained even when the lambda red products are supplied 1 h after irradiation. The lesions that are affected by the red-dependent process are probably not deoxyribonucleic acid strand breaks because the extent of deoxyribonucleic acid strand rejoining is not altered by the red products. The second type of phage-induced radioresistance requires the gam product of lambda and is observed in wild-type and polA strains. The lambda gam + gene product must be present immediately after irradiation to exert its full effect. In its presence, DNA breakdown is decreased, and a greater fraction of DNA is converted back to high molecular weight. Strains carrying lex, recA, or certain other combinations of mutations do not show any detectable phage-induced radioresistance. (U.S.)

  14. Diffusion-driven and excitation-dependent recombination rate in blue InGaN/GaN quantum well structures

    International Nuclear Information System (INIS)

    Aleksiejūnas, R.; Gelžinytė, K.; Nargelas, S.; Jarašiūnas, K.; Vengris, M.; Armour, E. A.; Byrnes, D. P.; Arif, R. A.; Lee, S. M.; Papasouliotis, G. D.

    2014-01-01

    We report on diffusion-driven and excitation-dependent carrier recombination rate in multiple InGaN/GaN quantum wells by using photoluminescence, light-induced absorption, and diffraction techniques. We demonstrate gradually increasing with excitation carrier diffusivity and its correlation with the recombination rate. At low carrier densities, an increase in radiative emission and carrier lifetime was observed due to partial saturation of non-radiative recombination centers. However, at carrier densities above ∼5 × 10 18  cm −3 , a typical value of photoluminescence efficiency droop, a further increase of diffusivity forces the delocalized carriers to face higher number of fast non-radiative recombination centers leading to an increase of non-radiative losses

  15. Diffusion-driven and excitation-dependent recombination rate in blue InGaN/GaN quantum well structures

    Energy Technology Data Exchange (ETDEWEB)

    Aleksiejūnas, R.; Gelžinytė, K.; Nargelas, S., E-mail: saulius.nargelas@ff.vu.lt; Jarašiūnas, K. [Department of Semiconductor Optoelectronics, Institute of Applied Research, Vilnius University, Saulėtekio 9–III, 10222 Vilnius (Lithuania); Vengris, M. [Laser Research Center, Vilnius University, Saulėtekio 10, 10223 Vilnius (Lithuania); Armour, E. A.; Byrnes, D. P.; Arif, R. A.; Lee, S. M.; Papasouliotis, G. D. [Veeco Instruments, Turbodisc Operations, 394 Elizabeth Avenue, Somerset, New Jersey 08873 (United States)

    2014-01-13

    We report on diffusion-driven and excitation-dependent carrier recombination rate in multiple InGaN/GaN quantum wells by using photoluminescence, light-induced absorption, and diffraction techniques. We demonstrate gradually increasing with excitation carrier diffusivity and its correlation with the recombination rate. At low carrier densities, an increase in radiative emission and carrier lifetime was observed due to partial saturation of non-radiative recombination centers. However, at carrier densities above ∼5 × 10{sup 18} cm{sup −3}, a typical value of photoluminescence efficiency droop, a further increase of diffusivity forces the delocalized carriers to face higher number of fast non-radiative recombination centers leading to an increase of non-radiative losses.

  16. RTEL1: an essential helicase for telomere maintenance and the regulation of homologous recombination.

    Science.gov (United States)

    Uringa, Evert-Jan; Youds, Jillian L; Lisaingo, Kathleen; Lansdorp, Peter M; Boulton, Simon J

    2011-03-01

    Telomere maintenance and DNA repair are crucial processes that protect the genome against instability. RTEL1, an essential iron-sulfur cluster-containing helicase, is a dominant factor that controls telomere length in mice and is required for telomere integrity. In addition, RTEL1 promotes synthesis-dependent strand annealing to direct DNA double-strand breaks into non-crossover outcomes during mitotic repair and in meiosis. Here, we review the role of RTEL1 in telomere maintenance and homologous recombination and discuss models linking RTEL1's enzymatic activity to its function in telomere maintenance and DNA repair.

  17. Effect of deoxyribonucleic acid replication inhibitors on bacterial recombination

    International Nuclear Information System (INIS)

    Canosi, U.; Siccardi, A.G.; Falaschi, A.; Mazza, G.

    1976-01-01

    Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis. Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B. subtilis. The results reported in this work demonstrated that the drug was also without effect on the transfection SPP1 or SPO-1 phage DNA (a process that requires a recombination event). The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage. HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage. In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B. subtilis) seems to be involved also in recombination, but not in the excision repair process. The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction

  18. Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

    DEFF Research Database (Denmark)

    Moss, Jennifer; Tinline-Purvis, Helen; Walker, Carol A

    2010-01-01

    Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found...... the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed...

  19. Dialects of the DNA Uptake Sequence in Neisseriaceae

    Science.gov (United States)

    Frye, Stephan A.; Nilsen, Mariann; Tønjum, Tone; Ambur, Ole Herman

    2013-01-01

    In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS), which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS–dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5′-CTG-3′ is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS–dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic transformation

  20. Dialects of the DNA uptake sequence in Neisseriaceae.

    Directory of Open Access Journals (Sweden)

    Stephan A Frye

    2013-04-01

    Full Text Available In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS, which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS-dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5'-CTG-3' is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS-dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic

  1. Influence of a uvrD mutation on survival and repair of X-irradiated Escherichia coli K-12 cells

    International Nuclear Information System (INIS)

    Schueren, E. van der; Youngs, D.A.; Smith, K.C.

    1977-01-01

    The presence of a uvrD mutation increased the X-ray sensitivities of E.coli wild-type and polA strains, but had no effect on the sensitivities of recA and recB strains, and little effect on a lexA strain. Incubation of irradiated cells in medium containing 2,4-dinitrophenol or chloramphenicol decreased the survival of wild-type and uvrD cells, but had no effect on the survival of recA, recB and lexA strains. Alkaline sucrose gradient sedimentation studies indicated that the uvrD strain is deficient in the growth-medium-dependent (Type III) repair of DNA single-strand breaks. These results indicate that the uvrD mutation inhibits certain rec + lex + -dependent repair processes, including the growth-medium-dependent (Type III) repair of X-ray-induced DNA single-strand breaks, but does not inhibit other rec + lex + -dependent processes that are sensitive to 2,4-dinitrophenol and chloramphenicol. (author)

  2. Influence of a uvrD mutation on survival and repair of x-irradiated Escherichia coli K-12 cells

    Energy Technology Data Exchange (ETDEWEB)

    van der Schueren, E; Youngs, D A; Smith, K C [Stanford Univ., Calif. (USA). Dept. of Radiology

    1977-06-01

    The presence of a uvrD mutation increased the x-ray sensitivities of E.coli wild-type and polA strains, but had no effect on the sensitivities of recA and recB strains, and little effect on a lexA strain. Incubation of irradiated cells in medium containing 2,4-dinitrophenol or chloramphenicol decreased the survival of wild-type and uvrD cells, but had no effect on the survival of recA, recB and lexA strains. Alkaline sucrose gradient sedimentation studies indicated that the uvrD strain is deficient in the growth-medium-dependent (Type III) repair of DNA single-strand breaks. These results indicate that the uvrD mutation inhibits certain rec/sup +/lex/sup +/-dependent repair processes, including the growth-medium-dependent (Type III) repair of x-ray-induced DNA single-strand breaks, but does not inhibit other rec/sup +/lex/sup +/-dependent processes that are sensitive to 2,4-dinitrophenol and chloramphenicol.

  3. Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites

    International Nuclear Information System (INIS)

    Lenz, J.; Okenquist, S.A.; LoSardo, J.E.; Hamilton, K.K.; Doetsch, P.W.

    1990-01-01

    Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of β-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins

  4. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  5. Recombinant HT{sub m4} gene, protein and assays

    Science.gov (United States)

    Lim, B.; Adra, C.N.; Lelias, J.M.

    1996-09-03

    The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

  6. When two is not enough: a CtIP tetramer is required for DNA repair by Homologous Recombination.

    Science.gov (United States)

    Forment, Josep V; Jackson, Stephen P; Pellegrini, Luca

    2015-01-01

    Homologous recombination (HR) is central to the repair of double-strand DNA breaks that occur in S/G2 phases of the cell cycle. HR relies on the CtIP protein (Ctp1 in fission yeast, Sae2 in budding yeast) for resection of DNA ends, a key step in generating the 3'-DNA overhangs that are required for the HR strand-exchange reaction. Although much has been learned about the biological importance of CtIP in DNA repair, our mechanistic insight into its molecular functions remains incomplete. It has been recently discovered that CtIP and Ctp1 share a conserved tetrameric architecture that is mediated by their N-terminal domains and is critical for their function in HR. The specific arrangement of protein chains in the CtIP/Ctp1 tetramer indicates that an ability to bridge DNA ends might be an important feature of CtIP/Ctp1 function, establishing an intriguing similarity with the known ability of the MRE11-RAD50-NBS1 complex to link DNA ends. Although the exact mechanism of action remains to be elucidated, the remarkable evolutionary conservation of CtIP/Ctp1 tetramerisation clearly points to its crucial role in HR.

  7. Biotechnology and genetic engineering in the new drug development. Part I. DNA technology and recombinant proteins.

    Science.gov (United States)

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Pharmaceutical biotechnology has a long tradition and is rooted in the last century, first exemplified by penicillin and streptomycin as low molecular weight biosynthetic compounds. Today, pharmaceutical biotechnology still has its fundamentals in fermentation and bioprocessing, but the paradigmatic change affected by biotechnology and pharmaceutical sciences has led to an updated definition. The biotechnology revolution redrew the research, development, production and even marketing processes of drugs. Powerful new instruments and biotechnology related scientific disciplines (genomics, proteomics) make it possible to examine and exploit the behavior of proteins and molecules. Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. This technology, now approximately 25 years old, is becoming one of the most important technologies developed in the 20(th) century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances regarding rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we describe these processes.

  8. Deriving the coronal hole electron temperature: electron density dependent ionization / recombination considerations

    International Nuclear Information System (INIS)

    Doyle, John Gerard; Perez-Suarez, David; Singh, Avninda; Chapman, Steven; Bryans, Paul; Summers, Hugh; Savin, Daniel Wolf

    2010-01-01

    Comparison of appropriate theoretically derived line ratios with observational data can yield estimates of a plasma's physical parameters, such as electron density or temperature. The usual practice in the calculation of the line ratio is the assumption of excitation by electrons/protons followed by radiative decay. Furthermore, it is normal to use the so-called coronal approximation, i.e. one only considers ionization and recombination to and from the ground-state. A more accurate treatment is to include ionization/recombination to and from metastable levels. Here, we apply this to two lines from adjacent ionization stages, Mg IX 368 A and Mg X 625 A, which has been shown to be a very useful temperature diagnostic. At densities typical of coronal hole conditions, the difference between the electron temperature derived assuming the zero density limit compared with the electron density dependent ionization/recombination is small. This, however, is not the case for flares where the electron density is orders of magnitude larger. The derived temperature for the coronal hole at solar maximum is around 1.04 MK compared to just below 0.82 MK at solar minimum.

  9. SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair

    DEFF Research Database (Denmark)

    McCord, Ronald A; Michishita, Eriko; Hong, Tao

    2009-01-01

    -PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor......-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA......, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA...

  10. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

    Directory of Open Access Journals (Sweden)

    Johannes S Gach

    Full Text Available Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5 boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART. Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC. We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.

  11. Resident enhanced repair: novel repair process action on plasmid DNA transformed into Escherichia coli K-12

    International Nuclear Information System (INIS)

    Strike, P.; Roberts, R.J.

    1982-01-01

    The survival of UV-irradiated DNA of plasmid NTP16 was monitored after its transformation into recipient cells containing an essentially homologous undamaged plasmid, pLV9. The presence of pLV9 resulted in a substantial increase in the fraction of damaged NTP16 molecules which survived in the recipient cells. This enhanced survival requires the host uvrA + and uvrB + gene products, but not the host recA + gene product. The requirement for both homologous DNA and the uvrA + gene products suggests that a novel repair process may act on plasmid DNA. Possible mechanisms for this process are considered

  12. nuvA, an Aspergillus nidulans gene involved in DNA repair and recombination, is a homologue of Saccharomyces cerevisiae RAD18 and Neurospora crassa uvs-2.

    Science.gov (United States)

    Iwanejko, L; Cotton, C; Jones, G; Tomsett, B; Strike, P

    1996-03-01

    A 40 kb genomic clone and 2.3 kb EcoRI subclone that rescued the DNA repair and recombination defects of the Aspergillus nidulans nuvA11 mutant were isolated and the subclone sequenced. The subclone hybridized to a cosmid in a chromosome-specific library confirming the assignment of nuvA to linkage group IV and indicating its closeness to bimD. Amplification by PCR clarified the relative positions of nuvA and bimD. A region identified within the subclone, encoding a C3HC4 zinc finger motif, was used as a probe to retrieve a cDNA clone. Sequencing of this clone showed that the nuvA gene has an ORF of 1329 bp with two introns of 51 bp and 60 bp. Expression of nuvA appears to be extremely low. The putative NUVA polypeptide has two zinc finger motifs, a molecular mass of 48906 Da and has 39% identity with the Neurospora crassa uvs-2 and 25% identity with the Saccharomyces cerevisiae RAD18 translation products. Although mutations in nuvA, uvs-2 and RAD18 produce similar phenotypes, only the nuvA11 mutation affects meiotic recombination. A role for nuvA in both DNA repair and genetic recombination is proposed.

  13. Cell biology of homologous recombination in yeast

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Rothstein, Rodney; Lisby, Michael

    2011-01-01

    Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Saccharomyces...

  14. Involvement of the yeast DNA polymerase delta in DNA repair in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Giot, L. [State University of New York at Stony Brook, Stony Brook, NY. (United States); Chanet, R.; Simon, M.; Facca, C.; Faye, G.

    1997-08-15

    The POL3 encoded catalytic subunit of DNA polymerase delta possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae. Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain. The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination. These phenotypes are observed at 24 degrees, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication. Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways. Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11. SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase delta. SDP5 is most probably the p55 subunit of Pol delta of S. cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair. (author)

  15. DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism

    Science.gov (United States)

    Gubaev, Airat; Weidlich, Daniela; Klostermeier, Dagmar

    2016-01-01

    The topological state of DNA is important for replication, recombination and transcription, and is regulated in vivo by DNA topoisomerases. Gyrase introduces negative supercoils into DNA at the expense of ATP hydrolysis. It is the accepted view that gyrase achieves supercoiling by a strand passage mechanism, in which double-stranded DNA is cleaved, and a second double-stranded segment is passed through the gap, converting a positive DNA node into a negative node. We show here that gyrase with only one catalytic tyrosine that cleaves a single strand of its DNA substrate can catalyze DNA supercoiling without strand passage. We propose an alternative mechanism for DNA supercoiling via nicking and closing of DNA that involves trapping, segregation and relaxation of two positive supercoils. In contrast to DNA supercoiling, ATP-dependent relaxation and decatenation of DNA by gyrase lacking the C-terminal domains require both tyrosines and strand passage. Our results point towards mechanistic plasticity of gyrase and might pave the way for finding novel and specific mechanism-based gyrase inhibitors. PMID:27557712

  16. Underwound DNA under Tension: Structure, Elasticity, and Sequence-Dependent Behaviors

    Science.gov (United States)

    Sheinin, Maxim Y.; Forth, Scott; Marko, John F.; Wang, Michelle D.

    2011-09-01

    DNA melting under torsion plays an important role in a wide variety of cellular processes. In the present Letter, we have investigated DNA melting at the single-molecule level using an angular optical trap. By directly measuring force, extension, torque, and angle of DNA, we determined the structural and elastic parameters of torsionally melted DNA. Our data reveal that under moderate forces, the melted DNA assumes a left-handed structure as opposed to an open bubble conformation and is highly torsionally compliant. We have also discovered that at low forces melted DNA properties are highly dependent on DNA sequence. These results provide a more comprehensive picture of the global DNA force-torque phase diagram.

  17. Resistance to nitrofurantoin and UV-irradiation in recA; uvrA; and uvrA, lexA, Escherichia coli mutants conferred by an R-plasmid from an Escherichia coli clinical isolate

    Energy Technology Data Exchange (ETDEWEB)

    Obaseiki-Ebor, E.E. (Univ. of Benin, Benin City (Nigeria). Faculty of Pharmacy, Dept. of Pharmaceutical Microbiology)

    1984-01-01

    There have been some reports of R-plasmids conferring nitrofuran resistance by decreasing the reduction of nitrofurantoin. The mechanism by which these R-plasmids mediate nitrofurantoin resistance is still not properly understood. Since DNA repair mutants are very sensitive to nitrofurantoin, it was therefore of interest to see whether R-plasmids conferring nitrofurantoin resistance affected the nitrofurantoin sensitivity of recA; uvrA and uvrA, lexA strains of E. coli K-12. Protection against UV-irradiation was also estimated. The experiments showed that the nitrofurantoin resistance conferred by R-plasmid pBN105 was not due to defective nitrofurantoin reduction or altered permeability of the cell. Because it is known that repair-deficient bacteria have increased susceptibility to nitrofurantoin, it may be suggested that the mechanisms of UV and nitrofurantoin protection conferred by pBN105 to the DNA repair mutant strains are related.

  18. Resistance to nitrofurantoin and UV-irradiation in recA; uvrA; and uvrA, lexA, Escherichia coli mutants conferred by an R-plasmid from an Escherichia coli clinical isolate

    International Nuclear Information System (INIS)

    Obaseiki-Ebor, E.E.

    1984-01-01

    There have been some reports of R-plasmids conferring nitrofuran resistance by decreasing the reduction of nitrofurantoin. The mechanism by which these R-plasmids mediate nitrofurantoin resistance is still not properly understood. Since DNA repair mutants are very sensitive to nitrofurantoin, it was therefore of interest to see whether R-plasmids conferring nitrofurantoin resistance affected the nitrofurantoin sensitivity of recA; uvrA and uvrA, lexA strains of E. coli K-12. Protection against UV-irradiation was also estimated. The experiments showed that the nitrofurantoin resistance conferred by R-plasmid pBN105 was not due to defective nitrofurantoin reduction or altered permeability of the cell. Because it is known that repair-deficient bacteria have increased susceptibility to nitrofurantoin, it may be suggested that the mechanisms of UV and nitrofurantoin protection conferred by pBN105 to the DNA repair mutant strains are related. (Auth.)

  19. The Dependence of the Dose Response Supralinearity of Peak 5 in TLD-100 on Recombination Temperature

    International Nuclear Information System (INIS)

    Horowitz, Y.S.; Satinger, D.; Oster, L.

    1999-01-01

    Isothermal readout of LiF:Mg,Ti (TLD-700) has recently been used to study the dependence of the supralinearity of peak 5 on recombination temperature. The results were interpreted to be in conflict with earlier results which investigated the effect of readout heating rate on the supralinearity of peak 5 in TLD-100. In this work the two experiments are inspected in greater detail. It is illustrated that the isothermal decay data is not in conflict with the heating rate data. However, the heating rate results do apparently indicate a strong transition in the temperature dependence of the relative strengths of the recombination and competitive cross sections at approximately 235 deg. C, which requires further study and analysis. (author)

  20. Radiation induced asymmetries in mitotic recombination: evidence for a directional bias in the formation of asymmetric hybrid DNA in yeast

    International Nuclear Information System (INIS)

    Friedman, L.R.; Sobell, H.M.

    We have examined radiation-induced mitotic recombination using two alleles (his1-36, his1-49) in the his1 gene. When the haploid containing his1-36 is irradiated with varying doses of γ rays and then mated with the unirradiated strain containing his1-49, analyses of the selected prototrophs show them to be primarily + +/+ 49. If, on the other hand, the haploid strain containing his1-49 is the irradiated parent, the prototrophic diploids are primarily + +/36 +. In control experiments, where either both strains are irradiated or not irradiated, no such asymmetries are found. These data indicate that the irradiated haploid chromosome tends to be the recipient of genetic information. We interpret these results as indicating a directional bias in the formation of hybrid DNA in radiation-induced mitotic recombination, and discuss these results in terms of current models of genetic recombination

  1. Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis

    International Nuclear Information System (INIS)

    Marrero, R.; Yasbin, R.E.

    1988-01-01

    By use of the Bacillus subtilis bacteriophage cloning vehicle Phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages Phi 105Rec Phi1 (3.85-kilobase insert) and Phi 105Rec Phi4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE + strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage Phi105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either Phi 105Rec Phi 1 or Phi 105RecPhi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages Phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages Phi 105RecPhi 1 and Phi 105Rec Phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA + gene product antibodies. Collectively, these data demonstrate that the recE + gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination

  2. Research and development of basic technologies for the next generation industries, 'recombinant DNA utilizing technology'. Evaluation on the research and development; Jisedai sangyo kiban gijutsu kenkyu kaihatsu 'kumikae DNA riyo gijutsu'. Kenkyu kaihatsu hyoka

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1991-03-01

    Research, development and evaluation were performed with an objective of establishing the basic technology related to the recombinant DNA utilizing technology to create new microorganisms for processes in the chemical industry. The major achievements of the present research and development include establishment of the P450 gene manifestation system attributed from microsomes and mitochondria, and the success of the world's first simultaneous manifestation of P450 and reduction enzyme. Furthermore, the fused enzyme combining P450 and the reduction enzyme genetically was successfully manufactured ahead of the other countries, opening the way to industrializing the recombinant enzymes for use in bio-processes in the chemical industry. In creating a high-efficiency secretion recombinant bacillus subtilis stock, a bacillus subtilis host whose protease activity has been noticeably decreased was created. As an achievement of the research on the 'basic recombinant DNA technology', high-efficiency manifestation vector of medium level thermophile was created, and its usefulness was demonstrated. In addition, a host and vector system for high level thermophile was developed for the first time in the world. These achievements have opened the way to industrial utilization of the thermophilic bacteria. (NEDO)

  3. Relative contribution of homologous recombination and non-homologous end-joining to DNA double-strand break repair after oxidative stress in Saccharomyces cerevisiae.

    Science.gov (United States)

    Letavayová, Lucia; Marková, Eva; Hermanská, Katarína; Vlcková, Viera; Vlasáková, Danusa; Chovanec, Miroslav; Brozmanová, Jela

    2006-05-10

    Oxidative damage to DNA seems to be an important factor in developing many human diseases including cancer. It involves base and sugar damage, base-free sites, DNA-protein cross-links and DNA single-strand (SSB) and double-strand (DSB) breaks. Oxidative DSB can be formed in various ways such as their direct induction by the drug or their generation either through attempted and aborted repair of primary DNA lesions or through DNA replication-dependent conversion of SSB. In general, two main pathways are responsible for repairing DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ), with both of them being potential candidates for the repair of oxidative DSB. We have examined relative contribution of HR and NHEJ to cellular response after oxidative stress in Saccharomyces cerevisiae. Therefore, cell survival, mutagenesis and DSB induction and repair in the rad52, yku70 and rad52 yku70 mutants after hydrogen peroxide (H(2)O(2)), menadione (MD) or bleomycin (BLM) exposure were compared to those obtained for the corresponding wild type. We show that MD exposure does not lead to observable DSB induction in yeast, suggesting that the toxic effects of this agent are mediated by other types of DNA damage. Although H(2)O(2) treatment generates some DSB, their yield is relatively low and hence DSB may only partially be responsible for toxicity of H(2)O(2), particularly at high doses of the agent. On the other hand, the basis of the BLM toxicity resides primarily in DSB induction. Both HR and NHEJ act on BLM-induced DSB, although their relative participation in the process is not equal. Based on our results we suggest that the complexity and/or the quality of the BLM-induced DSB might represent an obstacle for the NHEJ pathway.

  4. A sequence-dependent rigid-base model of DNA

    Science.gov (United States)

    Gonzalez, O.; Petkevičiutė, D.; Maddocks, J. H.

    2013-02-01

    A novel hierarchy of coarse-grain, sequence-dependent, rigid-base models of B-form DNA in solution is introduced. The hierarchy depends on both the assumed range of energetic couplings, and the extent of sequence dependence of the model parameters. A significant feature of the models is that they exhibit the phenomenon of frustration: each base cannot simultaneously minimize the energy of all of its interactions. As a consequence, an arbitrary DNA oligomer has an intrinsic or pre-existing stress, with the level of this frustration dependent on the particular sequence of the oligomer. Attention is focussed on the particular model in the hierarchy that has nearest-neighbor interactions and dimer sequence dependence of the model parameters. For a Gaussian version of this model, a complete coarse-grain parameter set is estimated. The parameterized model allows, for an oligomer of arbitrary length and sequence, a simple and explicit construction of an approximation to the configuration-space equilibrium probability density function for the oligomer in solution. The training set leading to the coarse-grain parameter set is itself extracted from a recent and extensive database of a large number of independent, atomic-resolution molecular dynamics (MD) simulations of short DNA oligomers immersed in explicit solvent. The Kullback-Leibler divergence between probability density functions is used to make several quantitative assessments of our nearest-neighbor, dimer-dependent model, which is compared against others in the hierarchy to assess various assumptions pertaining both to the locality of the energetic couplings and to the level of sequence dependence of its parameters. It is also compared directly against all-atom MD simulation to assess its predictive capabilities. The results show that the nearest-neighbor, dimer-dependent model can successfully resolve sequence effects both within and between oligomers. For example, due to the presence of frustration, the model can

  5. A sequence-dependent rigid-base model of DNA.

    Science.gov (United States)

    Gonzalez, O; Petkevičiūtė, D; Maddocks, J H

    2013-02-07

    A novel hierarchy of coarse-grain, sequence-dependent, rigid-base models of B-form DNA in solution is introduced. The hierarchy depends on both the assumed range of energetic couplings, and the extent of sequence dependence of the model parameters. A significant feature of the models is that they exhibit the phenomenon of frustration: each base cannot simultaneously minimize the energy of all of its interactions. As a consequence, an arbitrary DNA oligomer has an intrinsic or pre-existing stress, with the level of this frustration dependent on the particular sequence of the oligomer. Attention is focussed on the particular model in the hierarchy that has nearest-neighbor interactions and dimer sequence dependence of the model parameters. For a Gaussian version of this model, a complete coarse-grain parameter set is estimated. The parameterized model allows, for an oligomer of arbitrary length and sequence, a simple and explicit construction of an approximation to the configuration-space equilibrium probability density function for the oligomer in solution. The training set leading to the coarse-grain parameter set is itself extracted from a recent and extensive database of a large number of independent, atomic-resolution molecular dynamics (MD) simulations of short DNA oligomers immersed in explicit solvent. The Kullback-Leibler divergence between probability density functions is used to make several quantitative assessments of our nearest-neighbor, dimer-dependent model, which is compared against others in the hierarchy to assess various assumptions pertaining both to the locality of the energetic couplings and to the level of sequence dependence of its parameters. It is also compared directly against all-atom MD simulation to assess its predictive capabilities. The results show that the nearest-neighbor, dimer-dependent model can successfully resolve sequence effects both within and between oligomers. For example, due to the presence of frustration, the model can

  6. Charge recombination process in X-ray irradiated pyrene-doped polystyrene as studied by optically detected electron spin resonance and magnetic field dependence of the recombination fluorescence

    International Nuclear Information System (INIS)

    Okazaki, Masaharu; Tai, Yutaka; Toriyama, Kazumi

    1993-01-01

    The optically-detected ESR (ODESR) spectrum and magnetic field dependence on recombination fluorescence were observed for X-ray irradiated pyrene-doped polystyrene at temperatures of 242-348 K. The ODESR intensity as a function of the pyrene concentration, 0.1-8.9 wt%, showed an unusual minimum at about 1.0%. Two phases were separated in the magnetic field dependence of the fluorescence: one was sharp and saturates at fields of over 50 mT, while the other was broad with a dip at around 60-150 mT. The cause of this dip was naturally attributed to the ST -1 level crossing. The sharp magnetic field effect also showed a minimum at around a concentration of 1.0 wt%. These novel findings have been interpreted using a recombination model modified from the previous one for pyrene-doped ethylene-propylene rubber and polyethylene. The essential points of the present model are: (1) although electron hopping within the polystyrene molecule is rapid, electron transfer at the last step of recombination between the polystyrene anion and the pyrene cation proceeds at a moderate rate; (2) the hole-transfer rate in the polymer chain is moderate; (3) electron hopping between the doped pyrene molecules is very much dependent on the concentration; (4) hole hopping between the pyrenes is inhibited. (author)

  7. Conformation-dependent DNA attraction

    Science.gov (United States)

    Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

    2014-05-01

    Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg2+ ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg2+ or Na+, benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg2+ bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by

  8. Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities.

    Science.gov (United States)

    Ordonez, Heather; Unciuleac, Mihaela; Shuman, Stewart

    2012-05-01

    The Escherichia coli RecQ DNA helicase participates in a pathway of DNA repair that operates in parallel to the recombination pathway driven by the multisubunit helicase-nuclease machine RecBCD. The model mycobacterium Mycobacterium smegmatis executes homologous recombination in the absence of its helicase-nuclease machine AdnAB, though it lacks a homolog of E. coli RecQ. Here, we identify and characterize M. smegmatis RqlH, a RecQ-like helicase with a distinctive domain structure. The 691-amino acid RqlH polypeptide consists of a RecQ-like ATPase domain (amino acids 1-346) and tetracysteine zinc-binding domain (amino acids 435-499), separated by an RqlH-specific linker. RqlH lacks the C-terminal HRDC domain found in E. coli RecQ. Rather, the RqlH C-domain resembles bacterial ComF proteins and includes a phosphoribosyltransferase-like module. We show that RqlH is a DNA-dependent ATPase/dATPase that translocates 3'-5' on single-stranded DNA and has 3'-5' helicase activity. These functions inhere to RqlH-(1-505), a monomeric motor unit comprising the ATPase, linker and zinc-binding domains. RqlH homologs are distributed widely among bacterial taxa. The mycobacteria that encode RqlH lack a classical RecQ, though many other Actinobacteria have both RqlH and RecQ. Whereas E. coli K12 encodes RecQ but lacks a homolog of RqlH, other strains of E. coli have both RqlH and RecQ.

  9. Generation of Modified Pestiviruses by Targeted Recombination

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Friis, Martin Barfred; Risager, Peter Christian

    involves targeted modification of viral cDNA genomes, cloned within BACs, by Red/ET recombination-mediated mutagenesis in E.coli DH10B cells. Using recombination-mediated mutagenesis for the targeted design, the work can be expedited and focused in principal on any sequence within the viral genome...

  10. Doping in the recombinant era: strategies and counterstrategies.

    Science.gov (United States)

    Azzazy, Hassan M E; Mansour, Mai M H; Christenson, Robert H

    2005-11-01

    Advances in recombinant DNA technology have created one of the most powerful weapons in the current doping arsenal: recombinant proteins [Sweeney HL. Gene doping. Sci Am 2004;291:62-9; Unal M, Ozer Unal D. Gene doping in sports. Sports Med 2004;34:357-62]. Recombinant erythropoietin (EPO) and human growth hormone (hGH) are currently being abused but are fortunately detectable either directly by employing isoelectric focusing and immunoassays or indirectly by assessing changes in selected hematopoietic parameters. The detection is technically demanding due to the extent of similarity between the recombinant proteins and their endogenous counterparts. Another issue facing detection efforts is the speed and conditions at which blood samples are collected and analyzed in a sports setting. Recently, gene doping, which stemmed out of legitimate gene therapy trials, has emerged as the next level of doping. Erythropoietin (EPO), human growth hormone (hGH), insulin-like growth factor-1 (IGF-1), peroxisome proliferator-activated receptor-delta (PPAR delta), and myostatin inhibitor genes have been identified as primary targets for doping. Sports clinical scientists today are racing against the clock because assuring the continued integrity of sports competition depends on their ability to outpace the efforts of dopers by developing new detection strategies.

  11. SPAR1/RTEL1 maintains genomic stability by suppressing homologous recombination

    Science.gov (United States)

    Barber, Louise J.; Youds, Jillian L.; Ward, Jordan D.; McIlwraith, Michael J.; O’Neil, Nigel J.; Petalcorin, Mark I.R.; Martin, Julie S.; Collis, Spencer J.; Cantor, Sharon B.; Auclair, Melissa; Tissenbaum, Heidi; West, Stephen C.; Rose, Ann M.; Boulton, Simon J.

    2013-01-01

    SUMMARY Inappropriate homologous recombination (HR) can cause gross chromosomal rearrangements that in mammalian cells may lead to tumorigenesis. In yeast, the Srs2 protein is an anti-recombinase that eliminates inappropriate recombination events, but the functional equivalent of Srs2 in higher eukaryotes has proven to be elusive. In this work, we identify C. elegans SPAR-1 as a functional analogue of Srs2 and describe its vertebrate counterpart, SPAR1/RTEL1, which is required for genome stability and tumour avoidance. We find that spar-1 mutant worms and SPAR1 knockdown human cells share characteristic phenotypes with yeast srs2 mutants, including inviability upon deletion of the sgs1/BLM homologue, hyper-recombination, and DNA damage sensitivity. In vitro, purified human SPAR1 antagonises HR by promoting the disassembly of D loop recombination intermediates in a reaction dependent upon ATP hydrolysis. We propose that loss of HR control following deregulation of SPAR1/RTEL1 may be a critical event that drives genome instability and cancer. PMID:18957201

  12. Nrf2 facilitates repair of radiation induced DNA damage through homologous recombination repair pathway in a ROS independent manner in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Jayakumar, Sundarraj; Pal, Debojyoti; Sandur, Santosh K., E-mail: sskumar@barc.gov.in

    2015-09-15

    Highlights: • Nrf2 inhibition in A549 cells led to attenuated DNA repair and radiosensitization. • Influence of Nrf2 on DNA repair is not linked to its antioxidant function. • Nrf2 influences DNA repair through homologous recombination (HR) repair pathway. • Many genes involved in HR pathway show ARE sequences in their upstream region. - Abstract: Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4 Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2

  13. Conformation-dependent DNA attraction.

    Science.gov (United States)

    Li, Weifeng; Nordenskiöld, Lars; Zhou, Ruhong; Mu, Yuguang

    2014-06-21

    Understanding how DNA molecules interact with other biomolecules is related to how they utilize their functions and is therefore critical for understanding their structure-function relationships. For a long time, the existence of Z-form DNA (a left-handed double helical version of DNA, instead of the common right-handed B-form) has puzzled the scientists, and the definitive biological significance of Z-DNA has not yet been clarified. In this study, the effects of DNA conformation in DNA-DNA interactions are explored by molecular dynamics simulations. Using umbrella sampling, we find that for both B- and Z-form DNA, surrounding Mg(2+) ions always exert themselves to screen the Coulomb repulsion between DNA phosphates, resulting in very weak attractive force. On the contrary, a tight and stable bound state is discovered for Z-DNA in the presence of Mg(2+) or Na(+), benefiting from their hydrophobic nature. Based on the contact surface and a dewetting process analysis, a two-stage binding process of Z-DNA is outlined: two Z-DNA first attract each other through charge screening and Mg(2+) bridges to phosphate groups in the same way as that of B-DNA, after which hydrophobic contacts of the deoxyribose groups are formed via a dewetting effect, resulting in stable attraction between two Z-DNA molecules. The highlighted hydrophobic nature of Z-DNA interaction from the current study may help to understand the biological functions of Z-DNA in gene transcription.

  14. Molecular Precision at Micrometer Length Scales: Hierarchical Assembly of DNA-Protein Nanostructures.

    Science.gov (United States)

    Schiffels, Daniel; Szalai, Veronika A; Liddle, J Alexander

    2017-07-25

    Robust self-assembly across length scales is a ubiquitous feature of biological systems but remains challenging for synthetic structures. Taking a cue from biology-where disparate molecules work together to produce large, functional assemblies-we demonstrate how to engineer microscale structures with nanoscale features: Our self-assembly approach begins by using DNA polymerase to controllably create double-stranded DNA (dsDNA) sections on a single-stranded template. The single-stranded DNA (ssDNA) sections are then folded into a mechanically flexible skeleton by the origami method. This process simultaneously shapes the structure at the nanoscale and directs the large-scale geometry. The DNA skeleton guides the assembly of RecA protein filaments, which provides rigidity at the micrometer scale. We use our modular design strategy to assemble tetrahedral, rectangular, and linear shapes of defined dimensions. This method enables the robust construction of complex assemblies, greatly extending the range of DNA-based self-assembly methods.

  15. Mechanisms of recombination and function of DNA in bacteria. Progress report, May 3, 1975--May 5, 1976

    International Nuclear Information System (INIS)

    Guild, W.R.

    1976-01-01

    Results of investigations on phages were obtained with regard to the finding of transfection and characterizing the mode of entry of transfecting DNA; the characterization of a DNAase-resistant gene transfer agent from phage-infected cells which has some of the properties of a generalized transducing phage; and the study of multiplicity reactivation of uv-irradiated phage in a uv-sensitive pneumococcal host. Progress is also reported on a new gene transfer process, cell mutants, fine structure mapping, and stimulated recombination

  16. Single-Molecule Manipulation of Double-Stranded DNA Using Optical Tweezers: Interaction Studies of DNA with RecA and YOYO-1

    NARCIS (Netherlands)

    Bennink, Martin L.; Scharer, Orlando D.; Kanaar, Ronald; Sakata-Sogawa, Kumiko; Schins, J.M.; Kanger, Johannes S.; de Grooth, B.G.; Greve, Jan

    1999-01-01

    By using optical tweezers and a specially designed flow cell with an integrated glass micropipette, we constructed a setup similar to that of Smith et al. (Science 271:795-799, 1996) in which an individual double-stranded DNA (dsDNA) molecule can be captured between two polystyrene beads. The first

  17. Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli.

    Science.gov (United States)

    Geng, Zhirong; Song, Xiaoli; Xing, Zhi; Geng, Jinlong; Zhang, Sichun; Zhang, Xinrong; Wang, Zhilin

    2009-05-01

    The effects of Se(IV) on the structure and function of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT) purified from the cytoplasm of Escherichia coli were studied. The coding region of human AS3MT complementary DNA was amplified from total RNA extracted from HepG2 cell by reverse transcription PCR. Soluble and active human AS3MT was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25 degrees C. Spectra (UV-vis, circular dichroism, and fluorescence) were first used to probe the interaction of Se(IV) and recombinant human AS3MT and the structure-function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% alpha-helix, 23.9% beta-pleated sheet, 17.9% beta-turn, and 29.2% random coil. When Se(IV) was added, the content of the alpha-helix did not change, but that of the beta-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. Se(IV) inhibited the enzymatic methylation of inorganic As(III) in a concentration-dependent manner. The IC(50) value for Se(IV) was 2.38 muM. Double-reciprocal (1/V vs. 1/[inorganic As(III)]) plots showed Se(IV) to be a noncompetitive inhibitor of the methylation of inorganic As(III) by recombinant human AS3MT with a K (i) value of 2.61 muM. We hypothesized that Se(IV) interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When Se(IV) was combined with cysteine(s) in the structural residues, the conformation of recombinant human AS3MT changed and the enzymatic activity decreased. Considering the quenching of tryptophan fluorescence, Cys72 and/or Cys226 are deduced to be primary targets for Se(IV).

  18. Damage-induced ectopic recombination in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Kupiec, M; Steinlauf, R

    1997-06-09

    Mitotic recombination in the yeast Saccharomyces cerevisiae is induced when cells are irradiated with UV or X-rays, reflecting the efficient repair of damage by recombinational repair mechanisms. We have used multiply marked haploid strains that allow the simultaneous detection of several types of ectopic recombination events. We show that inter-chromosomal ectopic conversion of lys2 heteroalleles and, to a lesser extent, direct repeat recombination (DRR) between non-tandem repeats, are increased by DNA-damaging agents; in contrast, ectopic recombination of the naturally occurring Ty element is not induced. We have tested several hypotheses that could explain the preferential lack of induction of Ty recombination by DNA-damaging agents. We have found that the lack of induction cannot be explained by a cell cycle control or by an effect of the mating-type genes. We also found no role for the flanking long terminal repeats (LTRs) of the Ty in preventing the induction. Ectopic conversion, DRR, and forward mutation of artificial repeats show different kinetics of induction at various positions of the cell cycle, reflecting different mechanisms of recombination. We discuss the mechanistic and evolutionary aspects of these results.

  19. Consequences of recombination on traditional phylogenetic analysis

    DEFF Research Database (Denmark)

    Schierup, M H; Hein, J

    2000-01-01

    We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mt......DNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination....... With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may...

  20. Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel target of bisphenol A.

    Science.gov (United States)

    Ito, Yuki; Ito, Takumi; Karasawa, Satoki; Enomoto, Teruya; Nashimoto, Akihiro; Hase, Yasuyoshi; Sakamoto, Satoshi; Mimori, Tsuneyo; Matsumoto, Yoshihisa; Yamaguchi, Yuki; Handa, Hiroshi

    2012-01-01

    Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100-300 μM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

  1. Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

    Science.gov (United States)

    Jancovich, James K; Chapman, Dave; Hansen, Debra T; Robida, Mark D; Loskutov, Andrey; Craciunescu, Felicia; Borovkov, Alex; Kibler, Karen; Goatley, Lynnette; King, Katherine; Netherton, Christopher L; Taylor, Geraldine; Jacobs, Bertram; Sykes, Kathryn; Dixon, Linda K

    2018-04-15

    African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia

  2. Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

    Science.gov (United States)

    Kukiełka, E; Cederbaum, A I

    1995-04-15

    Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

  3. Multifaceted regulation of V(D)J recombination

    Science.gov (United States)

    Wang, Guannan

    V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion to a cell. Multifaceted regulatory mechanisms have been evolved to create great diversity of the antigen receptor repertoire while ensuring genome stability. The RAG-mediated cleavage reaction is stringently regulated at both the pre-cleavage stage and the post-cleavage stage. Specifically, RAG1/2 first forms a pre-cleavage complex assembled at the boarder of RSS and coding flank, which ensures the appropriate DNA targeting. Subsequently, this complex initiates site-specific cleavage, generating two types of double stranded DNA breaks, hairpin-ended coding ends (HP-CEs) and blunt signal ends (SEs). After the cleavage, RAG1/2 proteins bind and retain the recombination ends to form post-cleavage complexes (PCC), which collaborates with the NHEJ machinery for appropriate transfer of recombination ends to NHEJ for proper end resolution. However, little is known about the molecular basis of this collaboration, partly attributed to the lack of sensitive assays to reveal the interaction of PCC with HP-CEs. Here, for the first time, by using two complementary fluorescence-based techniques, fluorescence anisotropy and fluorescence resonance energy transfer (FRET), I managed to monitor the RAG1/2-catalyzed cleavage reaction in real time, from the pre-cleavage to the post-cleavage stages. By

  4. Synthetic lethality between murine DNA repair factors XLF and DNA-PKcs is rescued by inactivation of Ku70

    DEFF Research Database (Denmark)

    Xing, Mengtan; Bjørås, Magnar; Daniel, Jeremy A

    2017-01-01

    DNA double-strand breaks (DSBs) are recognized and repaired by the Classical Non-Homologous End-Joining (C-NHEJ) and Homologous Recombination pathways. C-NHEJ includes the core Ku70 and Ku80 (or Ku86) heterodimer that binds DSBs and thus promotes recruitment of accessory downstream NHEJ factors XLF......, PAXX, DNA-PKcs, Artemis and other core subunits, XRCC4 and DNA Ligase 4 (Lig4). In the absence of core C-NHEJ factors, DNA repair can be performed by Alternative End-Joining, which likely depends on DNA Ligase 1 and DNA Ligase 3. Genetic inactivation of C-NHEJ factors, such as Ku70, Ku80, XLF, PAXX...... with severe apoptosis in the central nervous system. Here, we demonstrate that inactivation of the Ku70 gene rescues the synthetic lethality between XLF and DNA-PKcs, resulting in triple knockout mice that are indistinguishable from Ku70-deficient littermates by size or levels of genomic instability. Moreover...

  5. Cloning and characterization of BKV(MM) DNA and its use for detection of BKV DNA in human urine

    International Nuclear Information System (INIS)

    Harley, E.H.; Olliver, C.L.; Rhodes-Harrison, L.; Mew, R.T.; Lecatsas, G.; Naude, W. du T.

    1982-01-01

    The two fragments produced by restriction of BKV(MM) DNA with the endonucleases Pst I and Eco RI have been cloned separately into the vector pBR322 and amplified in E. coli HB101. Eight recombinant plasmids were characterized by gel electrophoresis of Pst I/Eco RI double digestions or Hind III digestions of the DNA and by hybridization of Southern gel blots to a nick-translated BKV(MM) DNA probe. Four of the recombinant plasmids contained the large Pst I/Eco RI BKV(MM) DNA fragment and four contained the small fragment. Two of these recombinant plasmids were then used to make a probe for the identification of BK DNA in a urine specimen from a patient known to be exreting particles with the morphological features of papovavirus [af

  6. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

    International Nuclear Information System (INIS)

    Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

  7. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  8. Repair on the go: E. coli maintains a high proliferation rate while repairing a chronic DNA double-strand break.

    Directory of Open Access Journals (Sweden)

    Elise Darmon

    Full Text Available DNA damage checkpoints exist to promote cell survival and the faithful inheritance of genetic information. It is thought that one function of such checkpoints is to ensure that cell division does not occur before DNA damage is repaired. However, in unicellular organisms, rapid cell multiplication confers a powerful selective advantage, leading to a dilemma. Is the activation of a DNA damage checkpoint compatible with rapid cell multiplication? By uncoupling the initiation of DNA replication from cell division, the Escherichia coli cell cycle offers a solution to this dilemma. Here, we show that a DNA double-strand break, which occurs once per replication cycle, induces the SOS response. This SOS induction is needed for cell survival due to a requirement for an elevated level of expression of the RecA protein. Cell division is delayed, leading to an increase in average cell length but with no detectable consequence on mutagenesis and little effect on growth rate and viability. The increase in cell length caused by chronic DNA double-strand break repair comprises three components: two types of increase in the unit cell size, one independent of SfiA and SlmA, the other dependent of the presence of SfiA and the absence of SlmA, and a filamentation component that is dependent on the presence of either SfiA or SlmA. These results imply that chronic checkpoint induction in E. coli is compatible with rapid cell multiplication. Therefore, under conditions of chronic low-level DNA damage, the SOS checkpoint operates seamlessly in a cell cycle where the initiation of DNA replication is uncoupled from cell division.

  9. HSV-I and the cellular DNA damage response.

    Science.gov (United States)

    Smith, Samantha; Weller, Sandra K

    2015-04-01

    Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al . that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome.

  10. Telma Reca, an Argentine physician in the state (1930-1948

    Directory of Open Access Journals (Sweden)

    Karina Inés Ramacciotti

    2018-06-01

    Full Text Available This article will reconstruct the biography of the Argentine physician Telma Reca (1904-1979 who managed to get involved in academic fields and the state administration in the 30´s. We will address ourselves to tracking her professional career from her PhD degree in Medicine (1932 until her withdrawal from the División de Maternidad e Infancia del Departamento Nacional de Higiene (Maternity and Childhood Division of the National Hygiene Department in 1948. During this period, she focused her research on the study of the social conditions of juvenile delinquency on the public administration. With a critical eye, far from social exclusion and punitive measures, she managed to promote social integration through health and education policies.

  11. RAD51 interconnects between DNA replication, DNA repair and immunity.

    Science.gov (United States)

    Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

    2017-05-05

    RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. The consequences of sequence erosion in the evolution of recombination hotspots.

    Science.gov (United States)

    Tiemann-Boege, Irene; Schwarz, Theresa; Striedner, Yasmin; Heissl, Angelika

    2017-12-19

    Meiosis is initiated by a double-strand break (DSB) introduced in the DNA by a highly controlled process that is repaired by recombination. In many organisms, recombination occurs at specific and narrow regions of the genome, known as recombination hotspots, which overlap with regions enriched for DSBs. In recent years, it has been demonstrated that conversions and mutations resulting from the repair of DSBs lead to a rapid sequence evolution at recombination hotspots eroding target sites for DSBs. We still do not fully understand the effect of this erosion in the recombination activity, but evidence has shown that the binding of trans -acting factors like PRDM9 is affected. PRDM9 is a meiosis-specific, multi-domain protein that recognizes DNA target motifs by its zinc finger domain and directs DSBs to these target sites. Here we discuss the changes in affinity of PRDM9 to eroded recognition sequences, and explain how these changes in affinity of PRDM9 can affect recombination, leading sometimes to sterility in the context of hybrid crosses. We also present experimental data showing that DNA methylation reduces PRDM9 binding in vitro Finally, we discuss PRDM9-independent hotspots, posing the question how these hotspots evolve and change with sequence erosion.This article is part of the themed issue 'Evolutionary causes and consequences of recombination rate variation in sexual organisms'. © 2017 The Authors.

  13. Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange

    NARCIS (Netherlands)

    H.B. Beverloo (Berna); R.D. Johnson (Roger); M. Jasin (Maria); R. Kanaar (Roland); J.H.J. Hoeijmakers (Jan); M.L.G. Dronkert (Mies)

    2000-01-01

    textabstractCells can achieve error-free repair of DNA double-strand breaks (DSBs) by homologous recombination through gene conversion with or without crossover. In contrast, an alternative homology-dependent DSB repair pathway, single-strand annealing (SSA), results in deletions. In this study, we

  14. Electron-ion recombination rates for merged-beams experiments

    International Nuclear Information System (INIS)

    Pajek, M.

    1994-01-01

    Energy dependence of the electron-ion recombination rates are studied for different recombination processes (radiative recombination, three-body recombination, dissociative recombination) for Maxwellian relative velocity distribution of arbitrary asymmetry. The results are discussed in context of the electron-ion merged beams experiments in cooling ion storage rings. The question of indication of a possible contribution of the three-body recombination to the measured recombination rates versus relative energy is particularly addressed. Its influence on the electron beam temperature derived from the energy dependence of recombination rate is discussed

  15. New vaccine strategies against enterotoxigenic Escherichia coli: II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine

    Directory of Open Access Journals (Sweden)

    M.O. Lásaro

    1999-02-01

    Full Text Available The induction of systemic (IgG and mucosal (IgA antibody responses against the colonization factor I antigen (CFA/I of enterotoxigenic Escherichia coli (ETEC was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

  16. Synergy between the N-terminal and C-terminal domains of Mycobacterium tuberculosis HupB is essential for high-affinity binding, DNA supercoiling and inhibition of RecA-promoted strand exchange.

    Science.gov (United States)

    Sharadamma, N; Khan, Krishnendu; Kumar, Sandeep; Patil, K Neelakanteshwar; Hasnain, Seyed E; Muniyappa, K

    2011-09-01

    The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair. © 2011 The Authors Journal compilation © 2011 FEBS.

  17. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved...... in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved......, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed....

  18. Rad51C deficiency destabilizes XRCC3, impairs recombination and radiosensitizes S/G2-phase cells

    Energy Technology Data Exchange (ETDEWEB)

    Lio, Yi-Ching; Schild, David; Brenneman, Mark A.; Redpath, J. Leslie; Chen, David J.

    2004-05-01

    The highly conserved Rad51 protein plays an essential role in repairing DNA damage through homologous recombination. In vertebrates, five Rad51 paralogs (Rad51B, Rad51C, Rad51D, XRCC2, XRCC3) are expressed in mitotically growing cells, and are thought to play mediating roles in homologous recombination, though their precise functions remain unclear. Here we report the use of RNA interference to deplete expression of Rad51C protein in human HT1080 and HeLa cells. In HT1080 cells, depletion of Rad51C by small interfering RNA caused a significant reduction of frequency in homologous recombination. The level of XRCC3 protein was also sharply reduced in Rad51C-depleted HeLa cells, suggesting that XRCC3 is dependent for its stability upon heterodimerization with Rad51C. In addition, Rad51C-depleted HeLa cells showed hypersensitivity to the DNA cross-linking agent mitomycin C, and moderately increased sensitivity to ionizing radiation. Importantly, the radiosensitivity of Rad51C-deficient HeLa cells was evident in S and G{sub 2}/M phases of the cell cycle but not in G{sub 1} phase. Together, these results provide direct cellular evidence for the importance of human Rad51C in homologous recombinational repair.

  19. DNA template dependent accuracy variation of nucleotide selection in transcription.

    Directory of Open Access Journals (Sweden)

    Harriet Mellenius

    Full Text Available It has been commonly assumed that the effect of erroneous transcription of DNA genes into messenger RNAs on peptide sequence errors are masked by much more frequent errors of mRNA translation to protein. We present a theoretical model of transcriptional accuracy. It uses experimentally estimated standard free energies of double-stranded DNA and RNA/DNA hybrids and predicts a DNA template dependent transcriptional accuracy variation spanning several orders of magnitude. The model also identifies high-error as well a high-accuracy transcription motifs. The source of the large accuracy span is the context dependent variation of the stacking free energy of pairs of correct and incorrect base pairs in the ever moving transcription bubble. Our model predictions have direct experimental support from recent single molecule based identifications of transcriptional errors in the C. elegans transcriptome. Our conclusions challenge the general view that amino acid substitution errors in proteins are mainly caused by translational errors. It suggests instead that transcriptional error hotspots are the dominating source of peptide sequence errors in some DNA template contexts, while mRNA translation is the major cause of protein errors in other contexts.

  20. Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance.

    Science.gov (United States)

    Sedman, Tiina; Gaidutšik, Ilja; Villemson, Karin; Hou, YingJian; Sedman, Juhan

    2014-12-01

    Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.