An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

Science.gov (United States)

Zhao, Wei; Jardine, Paul J.

2015-01-01

ABSTRACT During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor.

Science.gov (United States)

Zhao, Wei; Jardine, Paul J; Grimes, Shelley

2015-12-01

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor

Two high-mobility group box domains act together to underwind and kink DNA

Energy Technology Data Exchange (ETDEWEB)

Sánchez-Giraldo, R.; Acosta-Reyes, F. J. [Universitat Politecnica de Catalunya, 08028 Barcelona (Spain); Malarkey, C. S. [University of Colorado School of Medicine, Aurora, CO 80045 (United States); Saperas, N. [Universitat Politecnica de Catalunya, 08028 Barcelona (Spain); Churchill, M. E. A., E-mail: mair.churchill@ucdenver.edu [University of Colorado School of Medicine, Aurora, CO 80045 (United States); Campos, J. L., E-mail: mair.churchill@ucdenver.edu [Universitat Politecnica de Catalunya, 08028 Barcelona (Spain)

2015-06-30

The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure. High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA.

Two high-mobility group box domains act together to underwind and kink DNA

International Nuclear Information System (INIS)

Sánchez-Giraldo, R.; Acosta-Reyes, F. J.; Malarkey, C. S.; Saperas, N.; Churchill, M. E. A.; Campos, J. L.

2015-01-01

The crystal structure of HMGB1 box A bound to an unmodified AT-rich DNA fragment is reported at a resolution of 2 Å. A new mode of DNA recognition for HMG box proteins is found in which two box A domains bind in an unusual configuration generating a highly kinked DNA structure. High-mobility group protein 1 (HMGB1) is an essential and ubiquitous DNA architectural factor that influences a myriad of cellular processes. HMGB1 contains two DNA-binding domains, box A and box B, which have little sequence specificity but have remarkable abilities to underwind and bend DNA. Although HMGB1 box A is thought to be responsible for the majority of HMGB1–DNA interactions with pre-bent or kinked DNA, little is known about how it recognizes unmodified DNA. Here, the crystal structure of HMGB1 box A bound to an AT-rich DNA fragment is reported at a resolution of 2 Å. Two box A domains of HMGB1 collaborate in an unusual configuration in which the Phe37 residues of both domains stack together and intercalate the same CG base pair, generating highly kinked DNA. This represents a novel mode of DNA recognition for HMGB proteins and reveals a mechanism by which structure-specific HMG boxes kink linear DNA

The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site.

Science.gov (United States)

Claveria-Gimeno, Rafael; Lanuza, Pilar M; Morales-Chueca, Ignacio; Jorge-Torres, Olga C; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

2017-01-31

Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.

The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

Science.gov (United States)

Niraj, Joshi; Caron, Marie-Christine; Drapeau, Karine; Bérubé, Stéphanie; Guitton-Sert, Laure; Coulombe, Yan; Couturier, Anthony M; Masson, Jean-Yves

2017-08-21

Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Isolation and characterisation of the cDNA encoding a glycosylated accessory protein of pea chloroplast DNA polymerase.

OpenAIRE

Gaikwad, A; Tewari, K K; Kumar, D; Chen, W; Mukherjee, S K

1999-01-01

The cDNA encoding p43, a DNA binding protein from pea chloroplasts (ct) that binds to cognate DNA polymerase and stimulates the polymerase activity, has been cloned and characterised. The characteristic sequence motifs of hydroxyproline-rich glyco-proteins (HRGP) are present in the cDNA corres-ponding to the N-terminal domain of the mature p43. The protein was found to be highly O-arabinosylated. Chemically deglycosylated p43 (i.e. p29) retains its binding to both DNA and pea ct-DNA polymeras...

Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

Science.gov (United States)

Reinhardt, H Christian; Yaffe, Michael B

2013-09-01

Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

The role of the Zn(II binding domain in the mechanism of E. coli DNA topoisomerase I

Directory of Open Access Journals (Sweden)

Tse-Dinh Yuk-Ching

2002-05-01

Full Text Available Abstract Background Escherichia coli DNA topoisomerase I binds three Zn(II with three tetracysteine motifs which, together with the 14 kDa C-terminal region, form a 30 kDa DNA binding domain (ZD domain. The 67 kDa N-terminal domain (Top67 has the active site tyrosine for DNA cleavage but cannot relax negatively supercoiled DNA. We analyzed the role of the ZD domain in the enzyme mechanism. Results Addition of purified ZD domain to Top67 partially restored the relaxation activity, demonstrating that covalent linkage between the two domains is not necessary for removal of negative supercoils from DNA. The two domains had similar affinities to ssDNA. However, only Top67 could bind dsDNA with high affinity. DNA cleavage assays showed that the Top67 had the same sequence and structure selectivity for DNA cleavage as the intact enzyme. DNA rejoining also did not require the presence of the ZD domain. Conclusions We propose that during relaxation of negatively supercoiled DNA, Top67 by itself can position the active site tyrosine near the junction of double-stranded and single-stranded DNA for cleavage. However, the interaction of the ZD domain with the passing single-strand of DNA, coupled with enzyme conformational change, is needed for removal of negative supercoils.

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...

The multi-domain protein Np95 connects DNA methylation and histone modification.

Science.gov (United States)

Rottach, Andrea; Frauer, Carina; Pichler, Garwin; Bonapace, Ian Marc; Spada, Fabio; Leonhardt, Heinrich

2010-04-01

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ss. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.

HSI2/VAL1 PHD-like domain promotes H3K27 trimethylation to repress the expression of seed maturation genes and complex transgenes in Arabidopsis seedlings.

Science.gov (United States)

Veerappan, Vijaykumar; Chen, Naichong; Reichert, Angelika I; Allen, Randy D

2014-11-01

The novel mutant allele hsi2-4 was isolated in a genetic screen to identify Arabidopsis mutants with constitutively elevated expression of a glutathione S-transferase F8::luciferase (GSTF8::LUC) reporter gene in Arabidopsis. The hsi2-4 mutant harbors a point mutation that affects the plant homeodomain (PHD)-like domain in HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2 (HSI2)/VIVIPAROUS1/ABI3-LIKE1 (VAL1). In hsi2-4 seedlings, expression of this LUC transgene and certain endogenous seed-maturation genes is constitutively enhanced. The parental reporter line (WT LUC ) that was used for mutagenesis harbors two independent transgene loci, Kan R and Kan S . Both loci express luciferase whereas only the Kan R locus confers resistance to kanamycin. Here we show that both transgene loci harbor multiple tandem insertions at single sites. Luciferase expression from these sites is regulated by the HSI2 PHD-like domain, which is required for the deposition of repressive histone methylation marks (H3K27me3) at both Kan R and Kan S loci. Expression of LUC and Neomycin Phosphotransferase II transgenes is associated with dynamic changes in H3K27me3 levels, and the activation marks H3K4me3 and H3K36me3 but does not appear to involve repressive H3K9me2 marks, DNA methylation or histone deacetylation. However, hsi2-2 and hsi2-4 mutants are partially resistant to growth inhibition associated with exposure to the DNA methylation inhibitor 5-aza-2'-deoxycytidine. HSI2 is also required for the repression of a subset of regulatory and structural seed maturation genes in vegetative tissues and H3K27me3 marks associated with most of these genes are also HSI2-dependent. These data implicate HSI2 PHD-like domain in the regulation of gene expression involving histone modifications and DNA methylation-mediated epigenetic mechanisms.

Detection of Variation in Long-Term Micropropagated Mature Pistachio via DNA-Based Molecular Markers.

Science.gov (United States)

Akdemir, Hülya; Suzerer, Veysel; Tilkat, Engin; Onay, Ahmet; Çiftçi, Yelda Ozden

2016-12-01

Determination of genetic stability of in vitro-grown plantlets is needed for safe and large-scale production of mature trees. In this study, genetic variation of long-term micropropagated mature pistachio developed through direct shoot bud regeneration using apical buds (protocol A) and in vitro-derived leaves (protocol B) was assessed via DNA-based molecular markers. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and amplified fragment length polymorphism (AFLP) were employed, and the obtained PIC values from RAPD (0.226), ISSR (0.220), and AFLP (0.241) showed that micropropagation of pistachio for different periods of time resulted in "reasonable polymorphism" among donor plant and its 18 clones. Mantel's test showed a consistence polymorphism level between marker systems based on similarity matrices. In conclusion, this is the first study on occurrence of genetic variability in long-term micropropagated mature pistachio plantlets. The obtained results clearly indicated that different marker approaches used in this study are reliable for assessing tissue culture-induced variations in long-term cultured pistachio plantlets.

(Br-SCMM) Brazilian Smart City Maturity Model: A Perspective from the Health Domain.

Science.gov (United States)

Afonso, Ricardo Alexandre; dos Santos Brito, Kellyton; Holanda do Nascimento, Clóvis; Campos da Costa, Luciana; Álvaro, Alexandre; Cardoso Garcia, Vinicius

2015-01-01

The term definition "Smart City" still allows various interpretations, and this causes some difficulty in establishing parameters to measure how smart the cities can be. This paper presents a Maturity Model that uses a set of minimum domains and indicators that aim to encourage cities of different sizes to identify their potential and improve processes and public policies.

The multi-domain protein Np95 connects DNA methylation and histone modification

Science.gov (United States)

Rottach, Andrea; Frauer, Carina; Pichler, Garwin; Bonapace, Ian Marc; Spada, Fabio; Leonhardt, Heinrich

2010-01-01

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ß. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways. PMID:20026581

BuD, a helix–loop–helix DNA-binding domain for genome modification

Energy Technology Data Exchange (ETDEWEB)

Stella, Stefano [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Molina, Rafael; López-Méndez, Blanca [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Campos-Olivas, Ramon [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Duchateau, Phillippe [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Montoya, Guillermo, E-mail: guillermo.montoya@cpr.ku.dk [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark)

2014-07-01

Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells.

Science.gov (United States)

Kannappan, Ramaswamy; Mattapally, Saidulu; Wagle, Pooja A; Zhang, Jianyi

2018-05-18

The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human iPS cells has never been studied. p53-TAD was knocked out in iPS cells using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD KO cells were characterized by: accelerated proliferation, decreased population doubling time, and unaltered Bcl2, BBC3, IGF1R, Bax and altered Mdm2, p21, and PIDD transcripts expression. In p53-TAD KO cells p53 regulated DNA repair proteins XPA, DNA polH and DDB2 expression were found to be reduced compared to p53-WT cells. Exposure to low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) measured by RAD50 and MRE11 expression, Checkpoint kinase 2 activation and γH2A.X recruitment at DNA strand breaks in both the cell groups indicating silencing p53-TAD do not affect DDR mechanism upstream of p53. Following removal of Doxo p53-WT hiPS cells underwent DNA repair, corrected their damaged DNA and restored DNA integrity. Conversely, p53-TAD KO hiPS cells did not undergo complete DNA repair and failed to restore DNA integrity. More importantly continuous culture of p53-TAD KO hiPS cells underwent G2/M cell cycle arrest and expressed cellular senescent marker p16 INK4a . Our data clearly shows that silencing transactivation domain of p53 did not affect DDR but affected the DNA repair process implying the crucial role of p53 transactivation domain in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent senescence.

Maturation of Shark Single-Domain (IgNAR) Antibodies: Evidence for Induced-Fit Binding

Energy Technology Data Exchange (ETDEWEB)

Stanfield, R.L.; Dooley, H.; Verdino, P.; Flajnik, M.F.; Wilson, I.A.; /Scripps Res. Inst. /Maryland U.

2007-07-13

Sharks express an unusual heavy-chain isotype called IgNAR, whose variable regions bind antigen as independent soluble domains. To further probe affinity maturation of the IgNAR response, we structurally characterized the germline and somatically matured versions of a type II variable (V) region, both in the presence and absence of its antigen, hen egg-white lysozyme. Despite a disulfide bond linking complementarity determining regions (CDRs) 1 and 3, both germline and somatically matured V regions displayed significant structural changes in these CDRs upon complex formation with antigen. Somatic mutations in the IgNAR V region serve to increase the number of contacts with antigen, as reflected by a tenfold increase in affinity, and one of these mutations appears to stabilize the CDR3 region. In addition, a residue in the HV4 loop plays an important role in antibody-antigen interaction, consistent with the high rate of somatic mutations in this non-CDR loop.

Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase.

Science.gov (United States)

Samai, Poulami; Shuman, Stewart

2011-06-24

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.

Structure-Function Analysis of the OB and Latch Domains of Chlorella Virus DNA Ligase*

Science.gov (United States)

Samai, Poulami; Shuman, Stewart

2011-01-01

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5′-phosphate nucleotide and the 3′-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps. PMID:21527793

Recognition of methylated DNA through methyl-CpG binding domain proteins

DEFF Research Database (Denmark)

Zou, Xueqing; Ma, Wen; Solov'yov, Ilia

2012-01-01

DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although...... the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines...

Overproduction of the poly(ADP-ribose)polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

NARCIS (Netherlands)

M. Molinete; W. Vermeulen (Wim); A. Bürkle; J. Mé nissier-de Murcia; J.H. Küpper; J.H.J. Hoeijmakers (Jan); G. de Murcia

1993-01-01

textabstractThe zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during

MTBP, the partner of Treslin, contains a novel DNA-binding domain that is essential for proper initiation of DNA replication.

Science.gov (United States)

Kumagai, Akiko; Dunphy, William G

2017-11-01

Treslin, which is essential for incorporation of Cdc45 into the replicative helicase, possesses a partner called MTBP (Mdm2-binding protein). We have analyzed Xenopus and human MTBP to assess its role in DNA replication. Depletion of MTBP from Xenopus egg extracts, which also removes Treslin, abolishes DNA replication. These extracts be can rescued with recombinant Treslin-MTBP but not Treslin or MTBP alone. Thus, Treslin-MTBP is collectively necessary for replication. We have identified a C-terminal region of MTBP (the CTM domain) that binds efficiently to both double-stranded DNA and G-quadruplex (G4) DNA. This domain also exhibits homology with budding yeast Sld7. Mutants of MTBP without a functional CTM domain are defective for DNA replication in Xenopus egg extracts. These mutants display an impaired localization to chromatin and the inability to support loading of Cdc45. Human cells harboring such a mutant also display severe S-phase defects. Thus, the CTM domain of MTBP plays a critical role in localizing Treslin-MTBP to the replication apparatus for initiation. © 2017 Kumagai and Dunphy. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly

Science.gov (United States)

D'Lima, Nadia G.

2015-01-01

ABSTRACT Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as “molecular staples” at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging. IMPORTANCE Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

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Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

2017-09-01

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Tyr120Asp mutation alters domain flexibility and dynamics of MeCP2 DNA binding domain leading to impaired DNA interaction: Atomistic characterization of a Rett syndrome causing mutation.

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D'Annessa, Ilda; Gandaglia, Anna; Brivio, Elena; Stefanelli, Gilda; Frasca, Angelisa; Landsberger, Nicoletta; Di Marino, Daniele

2018-05-01

Mutations in the X-linked MECP2 gene represent the main origin of Rett syndrome, causing a profound intellectual disability in females. MeCP2 is an epigenetic transcriptional regulator containing two main functional domains: a methyl-CpG binding domain (MBD) and a transcription repression domain (TRD). Over 600 pathogenic mutations were reported to affect the whole protein; almost half of missense mutations affect the MBD. Understanding the impact of these mutations on the MBD structure and interaction with DNA will foster the comprehension of their pathogenicity and possibly genotype/phenotype correlation studies. Herein, we use molecular dynamics simulations to obtain a detailed view of the dynamics of WT and mutated MBD in the presence and absence of DNA. The pathogenic mutation Y120D is used as paradigm for our studies. Further, since the Y120 residue was previously found to be a phosphorylation site, we characterize the dynamic profile of the MBD also in the presence of Y120 phosphorylation (pY120). We found that addition of a phosphate group to Y120 or mutation in aspartic acid affect domain mobility that samples an alternative conformational space with respect to the WT, leading to impaired ability to interact with DNA. Experimental assays showing a significant reduction in the binding affinity between the mutated MBD and the DNA confirmed our predictions. Copyright © 2018 Elsevier B.V. All rights reserved.

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

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Freemont, P S; Ollis, D L; Steitz, T A; Joyce, C M

1986-09-01

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

N-terminal domains of human DNA polymerase lambda promote primer realignment during translesion DNA synthesis

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Taggart, David J.; Dayeh, Daniel M.; Fredrickson, Saul W.; Suo, Zucai

2014-01-01

The X-family DNA polymerases λ (Polλ) and β (Polβ) possess similar 5′-2-deoxyribose-5-phosphatelyase (dRPase) and polymerase domains. Besides these domains, Polλ also possesses a BRCA1 C-terminal (BRCT) domain and a proline-rich domain at its N terminus. However, it is unclear how these non-enzymatic domains contribute to the unique biological functions of Polλ. Here, we used primer extension assays and a newly developed high-throughput short oligonucleotide sequencing assay (HT-SOSA) to compare the efficiency of lesion bypass and fidelity of human Polβ, Polλ and two N-terminal deletion constructs of Polλ during the bypass of either an abasic site or a 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) lesion. We demonstrate that the BRCT domain of Polλ enhances the efficiency of abasic site bypass by approximately 1.6-fold. In contrast, deletion of the N-terminal domains of Polλ did not affect the efficiency of 8-oxodG bypass relative to nucleotide incorporations opposite undamaged dG. HT-SOSA analysis demonstrated that Polλ and Polβ preferentially generated −1 or −2 frameshift mutations when bypassing an abasic site and the single or double base deletion frequency was highly sequence dependent. Interestingly, the BRCT and proline-rich domains of Polλ cooperatively promoted the generation of −2 frameshift mutations when the abasic site was situated within a sequence context that was susceptible to homology-driven primer realignment. Furthermore, both N-terminal domains of Polλ increased the generation of −1 frameshift mutations during 8-oxodG bypass and influenced the frequency of substitution mutations produced by Polλ opposite the 8-oxodG lesion. Overall, our data support a model wherein the BRCT and proline-rich domains of Polλ act cooperatively to promote primer/template realignment between DNA strands of limited sequence homology. This function of the N-terminal domains may facilitate the role of Polλ as a gap-filling polymerase

Backbone assignment of the little finger domain of a Y-family DNA polymerase.

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Ma, Dejian; Fowler, Jason D; Suo, Zucai

2011-10-01

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms

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Rajendran, Ranjith; Williams, Craig; Lappin, David F.; Millington, Owain; Martins, Margarida

2013-01-01

Aspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy. PMID:23314962

DNA Recognition by the DNA Primase of Bacteriophage T7: A Structure Function Study of the Zinc-Binding Domain

International Nuclear Information System (INIS)

Akabayov, B.; Lee, S.; Akabayov, S.; Rekhi, S.; Zhu, B.; Richardson, C.

2009-01-01

Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.

Molecular dynamics simulations revealed structural differences among WRKY domain-DNA interaction in barley (Hordeum vulgare).

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Pandey, Bharati; Grover, Abhinav; Sharma, Pradeep

2018-02-12

The WRKY transcription factors are a class of DNA-binding proteins involved in diverse plant processes play critical roles in response to abiotic and biotic stresses. Genome-wide divergence analysis of WRKY gene family in Hordeum vulgare provided a framework for molecular evolution and functional roles. So far, the crystal structure of WRKY from barley has not been resolved; moreover, knowledge of the three-dimensional structure of WRKY domain is pre-requisites for exploring the protein-DNA recognition mechanisms. Homology modelling based approach was used to generate structures for WRKY DNA binding domain (DBD) and its variants using AtWRKY1 as a template. Finally, the stability and conformational changes of the generated model in unbound and bound form was examined through atomistic molecular dynamics (MD) simulations for 100 ns time period. In this study, we investigated the comparative binding pattern of WRKY domain and its variants with W-box cis-regulatory element using molecular docking and dynamics (MD) simulations assays. The atomic insight into WRKY domain exhibited significant variation in the intermolecular hydrogen bonding pattern, leading to the structural anomalies in the variant type and differences in the DNA-binding specificities. Based on the MD analysis, residual contribution and interaction contour, wild-type WRKY (HvWRKY46) were found to interact with DNA through highly conserved heptapeptide in the pre- and post-MD simulated complexes, whereas heptapeptide interaction with DNA was missing in variants (I and II) in post-MD complexes. Consequently, through principal component analysis, wild-type WRKY was also found to be more stable by obscuring a reduced conformational space than the variant I (HvWRKY34). Lastly, high binding free energy for wild-type and variant II allowed us to conclude that wild-type WRKY-DNA complex was more stable relative to variants I. The results of our study revealed complete dynamic and structural information

Lune/eye gone, a Pax-like protein, uses a partial paired domain and a homeodomain for DNA recognition.

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Jun, S; Wallen, R V; Goriely, A; Kalionis, B; Desplan, C

1998-11-10

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the PAI and RED domains, has been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. We describe a Pax protein, originally called Lune, that is the product of the Drosophila eye gone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. eyg seems to play a role both in the organogenesis of the salivary gland during embryogenesis and in the development of the eye. A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes.

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

Science.gov (United States)

Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

Three-dimensional structure of N-terminal domain of DnaB helicase and helicase-primase interactions in Helicobacter pylori.

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Tara Kashav

2009-10-01

Full Text Available Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD of H. pylori DnaB (HpDnaB helicase at 2.2 A resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.

Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes

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Brzezinska Agnieszka A

2006-12-01

Full Text Available Abstract Background Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. Results Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes. Conclusion We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.

Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase.

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Wang, Li Kai; Nair, Pravin A; Shuman, Stewart

2008-08-22

NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

Towards the maturity model for feature oriented domain analysis

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Muhammad Javed

2014-09-01

Full Text Available Assessing the quality of a model has always been a challenge for researchers in academia and industry. The quality of a feature model is a prime factor because it is used in the development of products. A degraded feature model leads the development of low quality products. Few efforts have been made on improving the quality of feature models. This paper is an effort to present our ongoing work i.e. development of FODA (Feature Oriented Domain Analysis maturity model which will help to evaluate the quality of a given feature model. In this paper, we provide the quality levels along with their descriptions. The proposed model consists of four levels starting from level 0 to level 3. Design of each level is based on the severity of errors, whereas severity of errors decreases from level 0 to level 3. We elaborate each level with the help of examples. We borrowed all examples from the material published by the research community of Software Product Lines (SPL for the application of our framework.

Identification of the DNA-Binding Domains of Human Replication Protein A That Recognize G-Quadruplex DNA

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Aishwarya Prakash

2011-01-01

Full Text Available Replication protein A (RPA, a key player in DNA metabolism, has 6 single-stranded DNA-(ssDNA- binding domains (DBDs A-F. SELEX experiments with the DBDs-C, -D, and -E retrieve a 20-nt G-quadruplex forming sequence. Binding studies show that RPA-DE binds preferentially to the G-quadruplex DNA, a unique preference not observed with other RPA constructs. Circular dichroism experiments show that RPA-CDE-core can unfold the G-quadruplex while RPA-DE stabilizes it. Binding studies show that RPA-C binds pyrimidine- and purine-rich sequences similarly. This difference between RPA-C and RPA-DE binding was also indicated by the inability of RPA-CDE-core to unfold an oligonucleotide containing a TC-region 5′ to the G-quadruplex. Molecular modeling studies of RPA-DE and telomere-binding proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA-DE and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties.

A structural role for the PHP domain in E. coli DNA polymerase III.

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Barros, Tiago; Guenther, Joel; Kelch, Brian; Anaya, Jordan; Prabhakar, Arjun; O'Donnell, Mike; Kuriyan, John; Lamers, Meindert H

2013-05-14

In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.

Protein Export by the Mycobacterial SecA2 System Is Determined by the Preprotein Mature Domain

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Feltcher, Meghan E.; Gibbons, Henry S.; Ligon, Lauren S.

2013-01-01

At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles “housekeeping” export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm. PMID:23204463

Image-Based Modeling Reveals Dynamic Redistribution of DNA Damageinto Nuclear Sub-Domains

Energy Technology Data Exchange (ETDEWEB)

Costes Sylvain V., Ponomarev Artem, Chen James L.; Nguyen, David; Cucinotta, Francis A.; Barcellos-Hoff, Mary Helen

2007-08-03

Several proteins involved in the response to DNA doublestrand breaks (DSB) f orm microscopically visible nuclear domains, orfoci, after exposure to ionizing radiation. Radiation-induced foci (RIF)are believed to be located where DNA damage occurs. To test thisassumption, we analyzed the spatial distribution of 53BP1, phosphorylatedATM, and gammaH2AX RIF in cells irradiated with high linear energytransfer (LET) radiation and low LET. Since energy is randomly depositedalong high-LET particle paths, RIF along these paths should also berandomly distributed. The probability to induce DSB can be derived fromDNA fragment data measured experimentally by pulsed-field gelelectrophoresis. We used this probability in Monte Carlo simulations topredict DSB locations in synthetic nuclei geometrically described by acomplete set of human chromosomes, taking into account microscope opticsfrom real experiments. As expected, simulations produced DNA-weightedrandom (Poisson) distributions. In contrast, the distributions of RIFobtained as early as 5 min after exposure to high LET (1 GeV/amu Fe) werenon-random. This deviation from the expected DNA-weighted random patterncan be further characterized by "relative DNA image measurements." Thisnovel imaging approach shows that RIF were located preferentially at theinterface between high and low DNA density regions, and were morefrequent than predicted in regions with lower DNA density. The samepreferential nuclear location was also measured for RIF induced by 1 Gyof low-LET radiation. This deviation from random behavior was evidentonly 5 min after irradiation for phosphorylated ATM RIF, while gammaH2AXand 53BP1 RIF showed pronounced deviations up to 30 min after exposure.These data suggest that DNA damage induced foci are restricted to certainregions of the nucleus of human epithelial cells. It is possible that DNAlesions are collected in these nuclear sub-domains for more efficientrepair.

Modelling DNA origami self-assembly at the domain level

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Dannenberg, Frits; Kwiatkowska, Marta [Department of Computer Science, University of Oxford, Wolfson Building, Parks Road, Oxford OX1 3QD (United Kingdom); Dunn, Katherine E. [Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU (United Kingdom); Department of Electronics, University of York, York YO10 5DD (United Kingdom); Bath, Jonathan; Turberfield, Andrew J. [Department of Physics, University of Oxford, Clarendon Laboratory, Parks Road, Oxford OX1 3PU (United Kingdom); Ouldridge, Thomas E. [Department of Physics, University of Oxford, Rudolf Peierls Centre for Theoretical Physics, 1 Keble Road, Oxford OX1 3NP (United Kingdom); Department of Mathematics, Imperial College, 180 Queen’s Gate, London SW7 2AZ (United Kingdom)

2015-10-28

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

Modelling DNA origami self-assembly at the domain level

International Nuclear Information System (INIS)

Dannenberg, Frits; Kwiatkowska, Marta; Dunn, Katherine E.; Bath, Jonathan; Turberfield, Andrew J.; Ouldridge, Thomas E.

2015-01-01

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami

Modelling DNA origami self-assembly at the domain level

Science.gov (United States)

Dannenberg, Frits; Dunn, Katherine E.; Bath, Jonathan; Kwiatkowska, Marta; Turberfield, Andrew J.; Ouldridge, Thomas E.

2015-10-01

We present a modelling framework, and basic model parameterization, for the study of DNA origami folding at the level of DNA domains. Our approach is explicitly kinetic and does not assume a specific folding pathway. The binding of each staple is associated with a free-energy change that depends on staple sequence, the possibility of coaxial stacking with neighbouring domains, and the entropic cost of constraining the scaffold by inserting staple crossovers. A rigorous thermodynamic model is difficult to implement as a result of the complex, multiply connected geometry of the scaffold: we present a solution to this problem for planar origami. Coaxial stacking of helices and entropic terms, particularly when loop closure exponents are taken to be larger than those for ideal chains, introduce interactions between staples. These cooperative interactions lead to the prediction of sharp assembly transitions with notable hysteresis that are consistent with experimental observations. We show that the model reproduces the experimentally observed consequences of reducing staple concentration, accelerated cooling, and absent staples. We also present a simpler methodology that gives consistent results and can be used to study a wider range of systems including non-planar origami.

NMR structure of the N-terminal domain of the replication initiator protein DnaA

Energy Technology Data Exchange (ETDEWEB)

Wemmer, David E.; Lowery, Thomas J.; Pelton, Jeffrey G.; Chandonia, John-Marc; Kim, Rosalind; Yokota, Hisao; Wemmer, David E.

2007-08-07

DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 Angstrom based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.

Different domains of P21Cip1/waf1 regulate DNA replication and DNA repair-associated processes after UV

International Nuclear Information System (INIS)

Soria, Gaston; Speroni, Juliana; Podhajcer, Osvaldo L.; Gottifredi, Vanesa; Prives, Carol

2007-01-01

Full text: Many genotoxic insults result in p21 up-regulation and p21-dependent cell cycle arrest but UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated to DNA repair. While the role of p21 in Nucleotide Excision Repair (NER) remains controversial, two recent reports explore its effect on Translesion DNA Synthesis (TLS), a process that avoids replication blockage during S phase. The first report shows that p21 degradation is required for efficient PCNA ubiquitination, a post transcriptional modification that is relevant for TLS. The second report demonstrates that p21 (-/-) cells have increased TLS-associated mutagenic rates. Herein we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK binding domain of p21 is required for cell cycle arrest in unstressed cells; neither the CDK- nor the PCNA-binding domains of p21 are able to block early and late steps of NER. Intriguingly, through its PCNA binding domain, p21 inhibited recruitment of the TLS-polymerase, polη to PCNA foci after UV. Moreover, this obstruction correlates with accumulation of γH2AX and increased apoptosis. Taking together, our data emphasizes the link between p21 turnover and efficient TLS. This might also suggest a potential effect of p21 on other activities of polζ, a DNA polymerase with central roles in other biological scenarios such as genetic conversion, homologous recombination and modulation of the cellular response to genotoxic agents [es

Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain

International Nuclear Information System (INIS)

Eisen, L.P.; Reichman, M.E.; Thompson, E.B.; Gametchu, B.; Harrison, R.W.; Eisen, H.J.

1985-01-01

The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA

Antibacterial small molecules targeting the conserved TOPRIM domain of DNA gyrase.

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Scott S Walker

Full Text Available To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient Escherichia coli and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of E. coli DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of E. coli and Pseudomonas aeruginosa DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.

Detailed kinetic analysis of the interaction between the FOXO4–DNA-binding domain and DNA

Czech Academy of Sciences Publication Activity Database

Vácha, P.; Zusková, Iva; Bumba, Ladislav; Večeř, J.; Obšilová, Veronika; Obšil, T.

2013-01-01

Roč. 184, DEC 31 (2013), s. 68-78 ISSN 0301-4622 R&D Projects: GA ČR(CZ) GAP207/11/0717 Institutional support: RVO:67985823 ; RVO:61388971 Keywords : binding kinetics * DNA-binding domain * FOXO4 forkhead transcription factor Subject RIV: BO - Biophysics; CE - Biochemistry (MBU-M) Impact factor: 2.319, year: 2013

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies

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Pant, Kiran; Anderson, Brian; Perdana, Hendrik; Malinowski, Matthew A.; Win, Aye T.; Williams, Mark C.

2018-01-01

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes. PMID:29634784

DNA content of rodent brains during maturation and aging, and autoradiography of postnatal DNA synthesis in monkey brain

International Nuclear Information System (INIS)

Howard, E.

1973-01-01

[ 3 H]Thymidine is taken up by cells synthesizing DNA prepatory to cell division and remains incorporated in the DNA molecules as a lasting radioactive cell marker unless diluted out by repeated cell divisions. With the mouse and rat, histological studies after [ 3 H]thymidine injections have demonstrated that the cells of the external granular layer of the cerebellum proliferate abundantly during the first 2 weeks of postnatal life. Development of the primate brain is a gradual process extending over a much longer time than is required in the rodent. Despite the relative histological maturity of the monkey cerebellum at birth, the cells of the external granular layer are still actively synthesizing DNA at this time. Two monkeys were given [ 3 H]thymidine at birth and killed within 4 hours. Intense radioactivity was present in the cells of the external granular layer. Cells near the Prukinje perikarya were rather frequently labelled in this monkey, as described by Miale and Sidman in the mouse. In the molecular layer and in the body of the granular layer, relatively few cells were labelled. The labelling was present throughout the cerebellum, although the number of cells labelled varied from one microscopic field to another

Variations in DNA methylation, acetylated histone H4, and methylated histone H3 during Pinus radiata needle maturation in relation to the loss of in vitro organogenic capability.

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Valledor, Luis; Meijón, Mónica; Hasbún, Rodrigo; Jesús Cañal, Maria; Rodríguez, Roberto

2010-03-15

Needle differentiation is a very complex process associated with the formation of a mature photosynthetic organ. From meristem differentiation to leaf maturation, gene control must play an important role switching required genes on and off to define tissue functions, with the epigenetic code being one of the main regulation mechanisms. In this work, we examined the connections between the variation in the levels of some epigenetic players (DNA methylation, acetylated histone H4 and histone H3 methylation at Lys 4 and Lys 9) at work during needle maturation. Our results indicate that needle maturation, which is associated with a decrease in organogenic capability, is related to an increase in heterochromatin-related epigenetic markers (high DNA methylation and low acetylated histone H4 levels, and the presence of histone H3 methylated at lys 9). Immunohistochemical analyses also showed that the DNA methylation of palisade parenchyma cell layers during the transition from immature to mature scions is associated with the loss of the capacity to induce adventitious organs. Copyright 2009 Elsevier GmbH. All rights reserved.

Purification, crystallization and preliminary X-ray crystallographic studies of the Mycobacterium tuberculosis DNA gyrase ATPase domain

International Nuclear Information System (INIS)

Roué, Mélanie; Agrawal, Alka; Volker, Craig; Mossakowska, Danuta; Mayer, Claudine; Bax, Benjamin D.

2013-01-01

The ATPase domain of M. tuberculosis DNA gyrase was crystallized using hanging-drop vapour diffusion. The crystals belonged to space groups P1 and P2 1 . Diffraction data were collected to resolutions of 2.9 and 3.3 Å, respectively. Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47 C1 and Mtb-GyrB47 C2 , have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P2 1 and diffracted to resolutions of 2.9 and 3.3 Å, respectively

Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the POL domain of bacterial DNA ligase D.

Science.gov (United States)

Zhu, Hui; Bhattarai, Hitesh; Yan, Han-Guang; Shuman, Stewart; Glickman, Michael S

2012-12-21

Mycobacteria exploit nonhomologous end-joining (NHEJ) to repair DNA double-strand breaks. The core NHEJ machinery comprises the homodimeric DNA end-binding protein Ku and DNA ligase D (LigD), a modular enzyme composed of a C-terminal ATP-dependent ligase domain (LIG), a central 3'-phosphoesterase domain (PE), and an N-terminal polymerase domain (POL). LigD POL is proficient at adding templated and nontemplated deoxynucleotides and ribonucleotides to DNA ends in vitro and is the catalyst in vivo of unfaithful NHEJ events involving nontemplated single-nucleotide additions to blunt DSB ends. Here, we identify two mycobacterial proteins, PolD1 and PolD2, as stand-alone homologues of the LigD POL domain. Biochemical characterization of PolD1 and PolD2 shows that they resemble LigD POL in their monomeric quaternary structures, their ability to add templated and nontemplated nucleotides to primer-templates and blunt ends, and their preference for rNTPs versus dNTPs. Deletion of polD1, polD2, or both from a Mycobacterium smegmatis strain carrying an inactivating mutation in LigD POL failed to reveal a role for PolD1 or PolD2 in templated nucleotide additions during NHEJ of 5'-overhang DSBs or in clastogen resistance. Whereas our results document the existence and characteristics of new stand-alone members of the LigD POL family of RNA/DNA polymerases, they imply that other polymerases can perform fill-in synthesis during mycobacterial NHEJ.

Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'-5' exonuclease activity.

Science.gov (United States)

Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2009-04-01

The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'-5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg(2+)/Mn(2+)-dependent DNA/RNA polymerase, Mn(2+)-dependent 3'-5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn(2+)-dependent 3'-5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'-5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'-5' exonuclease activity.

Structure-guided Mutational Analysis of the Nucleotidyltransferase Domain of Escherichia coli DNA Ligase (LigA).

Science.gov (United States)

Wang, Li Kai; Zhu, Hui; Shuman, Stewart

2009-03-27

NAD(+)-dependent DNA ligases (LigA) are ubiquitous in bacteria, where they are essential for growth and present attractive targets for antimicrobial drug discovery. LigA has a distinctive modular structure in which a nucleotidyltransferase catalytic domain is flanked by an upstream NMN-binding module and by downstream OB-fold, zinc finger, helix-hairpin-helix, and BRCT domains. Here we conducted a structure-function analysis of the nucleotidyltransferase domain of Escherichia coli LigA, guided by the crystal structure of the LigA-DNA-adenylate intermediate. We tested the effects of 29 alanine and conservative mutations at 15 amino acids on ligase activity in vitro and in vivo. We thereby identified essential functional groups that coordinate the reactive phosphates (Arg(136)), contact the AMP adenine (Lys(290)), engage the phosphodiester backbone flanking the nick (Arg(218), Arg(308), Arg(97) plus Arg(101)), or stabilize the active domain fold (Arg(171)). Finer analysis of the mutational effects revealed step-specific functions for Arg(136), which is essential for the reaction of LigA with NAD(+) to form the covalent ligase-AMP intermediate (step 1) and for the transfer of AMP to the nick 5'-PO(4) to form the DNA-adenylate intermediate (step 2) but is dispensable for phosphodiester formation at a preadenylylated nick (step 3).

D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp.

Science.gov (United States)

Dagar, Sumit S; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K

2011-09-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

The flexible loop L1 of the H3K4 demethylase JARID1B ARID domain has a crucial role in DNA-binding activity

International Nuclear Information System (INIS)

Yao, Wenming; Peng, Yu; Lin, Donghai

2010-01-01

JARID1B, a member of the JmjC demethylase family, has a crucial role in H3K4me3 demethylation. The ARID domain is a potential DNA-binding domain of JARID1B. Previous studies indicate that a GC-rich DNA motif is the specific target of the ARID domain. However, the details of the interaction between the ARID domain and duplex DNA require further study. Here, we utilized NMR spectroscopy to assign the backbone amino acids and mapped the DNA-binding sites of the human JARID1B ARID domain. Perturbations to 1 H- 15 N correlation spectra revealed that the flexible loop L1 of ARID was the main DNA-binding interface. EMSA and intrinsic fluorescence experiments demonstrated that mutations on loop L1 strongly reduced the DNA-binding activity of JARID1B ARID. Furthermore, transfection of mutant forms resulted in a distinct loss of intrinsic H3K4 demethylase activity, implying that the flexible loop L1 made a major contribution to sustaining the DNA-binding ability of JARID1B ARID domain.

The impact of the human DNA topoisomerase II C-terminal domain on activity.

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Emma L Meczes

2008-03-01

Full Text Available Type II DNA topoisomerases (topos are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, alpha and beta, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity.We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIalpha and beta and topoIIalpha+beta-tail and topoIIbeta+alpha-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIalpha-CTD increasing activity, and the topoIIbeta-CTD decreasing activity.In vivo complementation data show that the topoIIalpha C-terminal domain is needed for growth, but the topoIIbeta isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIbeta has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIalpha or beta C-terminal domain can affect strand passage activity. Data indicates that the topoIIbeta-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs.

Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

Science.gov (United States)

de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

2014-06-01

The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

Science.gov (United States)

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Purification, crystallization and preliminary X-ray diffraction experiments on the breakage-reunion domain of the DNA gyrase from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Piton, Jérémie; Matrat, Stéphanie; Petrella, Stéphanie; Jarlier, Vincent; Aubry, Alexandra; Mayer, Claudine

2009-01-01

The breakage-reunion domain of M. tuberculosis DNA gyrase was crystallized using the hanging-drop vapour-diffusion method. One of the four crystal forms obtained belonged to space group C2 and diffraction data were collected to a resolution of 2.7 Å. Mycobacterium tuberculosis DNA gyrase, a nanomachine that is involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target for fluoroquinolone action. The breakage-reunion domain of the A subunit plays an essential role in DNA binding during the catalytic cycle. Two constructs of 53 and 57 kDa (termed GA53BK and GA57BK) corresponding to this domain have been overproduced, purified and crystallized. Diffraction data were collected from four crystal forms. The resolution limits ranged from 4.6 to 2.7 Å depending on the crystal form. The best diffracting crystals belonged to space group C2, with a biological dimer in the asymmetric unit. This is the first report of the crystallization and preliminary X-ray diffraction analysis of the breakage-reunion domain of DNA gyrase from a species containing one unique type II topoisomerase

Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

International Nuclear Information System (INIS)

Korzhnev, Dmitry M.; Neculai, Dante; Dhe-Paganon, Sirano; Arrowsmith, Cheryl H.; Bezsonova, Irina

2016-01-01

HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

Solution NMR structure of the HLTF HIRAN domain: a conserved module in SWI2/SNF2 DNA damage tolerance proteins

Energy Technology Data Exchange (ETDEWEB)

Korzhnev, Dmitry M. [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States); Neculai, Dante [Zhejiang University, School of Medicine (China); Dhe-Paganon, Sirano [Dana-Farber Cancer Institute, Department of Cancer Biology (United States); Arrowsmith, Cheryl H. [University of Toronto, Structural Genomics Consortium (Canada); Bezsonova, Irina, E-mail: bezsonova@uchc.edu [University of Connecticut Health, Department of Molecular Biology and Biophysics (United States)

2016-11-15

HLTF is a SWI2/SNF2-family ATP-dependent chromatin remodeling enzyme that acts in the error-free branch of DNA damage tolerance (DDT), a cellular mechanism that enables replication of damaged DNA while leaving damage repair for a later time. Human HLTF and a closely related protein SHPRH, as well as their yeast homologue Rad5, are multi-functional enzymes that share E3 ubiquitin-ligase activity required for activation of the error-free DDT. HLTF and Rad5 also function as ATP-dependent dsDNA translocases and possess replication fork reversal activities. Thus, they can convert Y-shaped replication forks into X-shaped Holliday junction structures that allow error-free replication over DNA lesions. The fork reversal activity of HLTF is dependent on 3′-ssDNA-end binding activity of its N-terminal HIRAN domain. Here we present the solution NMR structure of the human HLTF HIRAN domain, an OB-like fold module found in organisms from bacteria (as a stand-alone domain) to plants, fungi and metazoan (in combination with SWI2/SNF2 helicase-like domain). The obtained structure of free HLTF HIRAN is similar to recently reported structures of its DNA bound form, while the NMR analysis also reveals that the DNA binding site of the free domain exhibits conformational heterogeneity. Sequence comparison of N-terminal regions of HLTF, SHPRH and Rad5 aided by knowledge of the HLTF HIRAN structure suggests that the SHPRH N-terminus also includes an uncharacterized structured module, exhibiting weak sequence similarity with HIRAN regions of HLTF and Rad5, and potentially playing a similar functional role.

DNA loop domain organization in nucleoids from cells of different types.

Science.gov (United States)

Afanasieva, Katerina; Chopei, Marianna; Lozovik, Alexandra; Semenova, Anastasia; Lukash, Lyubov; Sivolob, Andrei

2017-01-29

The loop domain organization of chromatin plays an important role in transcription regulation and thus may be assumed to vary in cells of different types. We investigated the kinetics of DNA loop migration during single cell gel electrophoresis (the comet assay) for nucleoids obtained from human lymphocytes, lymphoblasts and glioblastoma T98G cells. The results confirm our previous observation that there are three parts of DNA in nucleoids: DNA on the nucleoid surface, loops up to ∼150 kb inside the nucleoid, and larger loops that cannot migrate. However, the relative amounts of the three parts were found to be very different for different cell types. The distributions of the loop length up to 150 kb were shown to be exponential, with the distribution parameter, the loop density, to be dependent on the cell type. Copyright © 2016 Elsevier Inc. All rights reserved.

The BRCT domain is a phospho-protein binding domain.

Science.gov (United States)

Yu, Xiaochun; Chini, Claudia Christiano Silva; He, Miao; Mer, Georges; Chen, Junjie

2003-10-24

The carboxyl-terminal domain (BRCT) of the Breast Cancer Gene 1 (BRCA1) protein is an evolutionarily conserved module that exists in a large number of proteins from prokaryotes to eukaryotes. Although most BRCT domain-containing proteins participate in DNA-damage checkpoint or DNA-repair pathways, or both, the function of the BRCT domain is not fully understood. We show that the BRCA1 BRCT domain directly interacts with phosphorylated BRCA1-Associated Carboxyl-terminal Helicase (BACH1). This specific interaction between BRCA1 and phosphorylated BACH1 is cell cycle regulated and is required for DNA damage-induced checkpoint control during the transition from G2 to M phase of the cell cycle. Further, we show that two other BRCT domains interact with their respective physiological partners in a phosphorylation-dependent manner. Thirteen additional BRCT domains also preferentially bind phospho-peptides rather than nonphosphorylated control peptides. These data imply that the BRCT domain is a phospho-protein binding domain involved in cell cycle control.

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

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Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino (Rutgers); (LBNL); (Connecticut); (TJU); (MSU)

2017-01-30

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid’) built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging’ is a DNA-dependent symmetrization of portal protein.

Portal protein functions akin to a DNA-sensor that couples genome-packaging to icosahedral capsid maturation

Science.gov (United States)

Lokareddy, Ravi K.; Sankhala, Rajeshwer S.; Roy, Ankoor; Afonine, Pavel V.; Motwani, Tina; Teschke, Carolyn M.; Parent, Kristin N.; Cingolani, Gino

2017-01-01

Tailed bacteriophages and herpesviruses assemble infectious particles via an empty precursor capsid (or ‘procapsid') built by multiple copies of coat and scaffolding protein and by one dodecameric portal protein. Genome packaging triggers rearrangement of the coat protein and release of scaffolding protein, resulting in dramatic procapsid lattice expansion. Here, we provide structural evidence that the portal protein of the bacteriophage P22 exists in two distinct dodecameric conformations: an asymmetric assembly in the procapsid (PC-portal) that is competent for high affinity binding to the large terminase packaging protein, and a symmetric ring in the mature virion (MV-portal) that has negligible affinity for the packaging motor. Modelling studies indicate the structure of PC-portal is incompatible with DNA coaxially spooled around the portal vertex, suggesting that newly packaged DNA triggers the switch from PC- to MV-conformation. Thus, we propose the signal for termination of ‘Headful Packaging' is a DNA-dependent symmetrization of portal protein. PMID:28134243

The C-terminal domain of the Bloom syndrome DNA helicase is essential for genomic stability

Directory of Open Access Journals (Sweden)

Noonan James P

2001-07-01

Full Text Available Abstract Background Bloom syndrome is a rare cancer-prone disorder in which the cells of affected persons have a high frequency of somatic mutation and genomic instability. Bloom syndrome cells have a distinctive high frequency of sister chromatid exchange and quadriradial formation. BLM, the protein altered in BS, is a member of the RecQ DNA helicase family, whose members share an average of 40% identity in the helicase domain and have divergent N-terminal and C-terminal flanking regions of variable lengths. The BLM DNA helicase has been shown to localize to the ND10 (nuclear domain 10 or PML (promyelocytic leukemia nuclear bodies, where it associates with TOPIIIα, and to the nucleolus. Results This report demonstrates that the N-terminal domain of BLM is responsible for localization of the protein to the nuclear bodies, while the C-terminal domain directs the protein to the nucleolus. Deletions of the N-terminal domain of BLM have little effect on sister chromatid exchange frequency and chromosome stability as compared to helicase and C-terminal mutations which can increase SCE frequency and chromosome abnormalities. Conclusion The helicase activity and the C-terminal domain of BLM are critical for maintaining genomic stability as measured by the sister chromatid exchange assay. The localization of BLM into the nucleolus by the C-terminal domain appears to be more important to genomic stability than localization in the nuclear bodies.

Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo.

Science.gov (United States)

Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M; Melendy, Thomas; Archambault, Jacques

2016-01-06

The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed for the structural

Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

Science.gov (United States)

Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

2016-01-01

ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

Crystal structure and DNA-binding property of the ATPase domain of bacterial mismatch repair endonuclease MutL from Aquifex aeolicus.

Science.gov (United States)

Fukui, Kenji; Iino, Hitoshi; Baba, Seiki; Kumasaka, Takashi; Kuramitsu, Seiki; Yano, Takato

2017-09-01

DNA mismatch repair (MMR) system corrects mismatched bases that are generated mainly by DNA replication errors. The repair system excises the error-containing single-stranded region and enables the re-synthesis of the strand. In the early reactions of MMR, MutL endonuclease incises the newly-synthesized/error-containing strand of the duplex to initiate the downstream excision reaction. MutL endonuclease consists of the N-terminal ATPase and C-terminal endonuclease domains. In this study, we report the crystal structure of the ATPase domain of MutL endonuclease from Aquifex aeolicus. The overall structure of the domain was similar to those of human MutL homologs and Escherichia coli MutL, although E. coli MutL has no endonuclease activity. The ATPase domain was comprised of two subdomains: the N-terminal ATP-binding subdomain and the C-terminal α-β sandwich subdomain. Site-directed mutagenesis experiment identified DNA-interacting eight basic amino acid residues, which were distributed across both the two subdomains and formed a DNA-binding cleft. Docking simulation between the structures of the ATPase and endonuclease domains generated a reliable model structure for the full-length A. aeolicus MutL, which satisfies our previous result of small-angle X-ray scattering analysis. On the basis of the model structure and further experimental results, we concluded that the two separate DNA-binding sites in the full-length A. aeolicus MutL simultaneously bind a dsDNA molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

Purification, crystallization and preliminary X-ray crystallographic analysis of the ETS domain of human Ergp55 in complex with the cfos promoter DNA sequence

International Nuclear Information System (INIS)

Gangwar, Shanti P.; Meena, Sita R.; Saxena, Ajay K.

2012-01-01

The ETS domain of human Ergp55 was purified and crystallized in native, complexes with E74, and cfos promoter DNA sequences. The X-ray intensity data set was collected on ETS–cfos promoter DNA complex crystal at 3.1 Å resolution to analyze the structure by molecular replacement technique. The Ergp55 protein belongs to the Ets family of transciption factors. The Ets transcription factors are involved in various developmental processes and the regulation of cancer metabolism. They contain a highly similar DNA-binding domain known as the ETS domain and have diverse functions in oncogenesis and physiology. The Ets transcription factors differ in their DNA-binding preference at the ETS site and the mechanisms by which they target genes are not clearly understood. To understand its DNA-binding mechanism, the ETS domain of Ergp55 was expressed and purified. The ETS domain was crystallized in the native form and in complex forms with DNA sequences from the E74 and cfos promoters. An X-ray diffraction data set was collected from an ETS–cfos DNA complex crystal at a wavelength of 0.9725 Å on the BM14 synchrotron beamline at the ESRF, France. The ETS–cfos DNA complex crystal belonged to space group C222 1 , with four molecules in the asymmetric unit. For structure analysis, initial phases for the ETS–cfos DNA complex were obtained by the molecular-replacement technique with Phaser in the CCP4 suite using the coordinates of Fli-1 protein and cfos DNA as search models. Structure analysis of the ETS–cfos DNA complex may possibly explain the DNA-binding specificity and its mechanism of interaction with the ETS domain of Ergp55

D1/D2 Domain of Large-Subunit Ribosomal DNA for Differentiation of Orpinomyces spp.▿

Science.gov (United States)

Dagar, Sumit S.; Kumar, Sanjay; Mudgil, Priti; Singh, Rameshwar; Puniya, Anil K.

2011-01-01

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation. PMID:21784906

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

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Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko, E-mail: yamagata@gpo.kumamoto-u.ac.jp [Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973 (Japan)

2008-03-01

The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V{sub M} value of 3.56 Å{sup 3} Da{sup −1}. Diffraction data were collected to a resolution of 3.0 Å.

Preparation, crystallization and preliminary X-ray diffraction analysis of the DNA-binding domain of the Ets transcription factor in complex with target DNA

International Nuclear Information System (INIS)

Suwa, Yoshiaki; Nakamura, Teruya; Toma, Sachiko; Ikemizu, Shinji; Kai, Hirofumi; Yamagata, Yuriko

2008-01-01

The complex between the Ets domain of Ets2 and its target DNA has been crystallized. The crystals diffracted to 3.0 Å resolution. The Ets2 transcription factor is a member of the Ets transcription-factor family. Ets2 plays a role in the malignancy of cancer and in Down’s syndrome by regulating the transcription of various genes. The DNA-binding domain of Ets2 (Ets domain; ETSD), which contains residues that are highly conserved among Ets transcription-factor family members, was expressed as a GST-fusion protein. The aggregation of ETSD produced after thrombin cleavage could be prevented by treatment with NDSB-195 (nondetergent sulfobetaine 195). ETSD was crystallized in complex with DNA containing the Ets2 target sequence (GGAA) by the hanging-drop vapour-diffusion method. The best crystals were grown using 25% PEG 3350, 80 mM magnesium acetate, 50 mM sodium cacodylate pH 5.0/5.5 as the reservoir at 293 K. The crystals belonged to space group C2, with unit-cell parameters a = 85.89, b = 95.52, c = 71.89 Å, β = 101.7° and a V M value of 3.56 Å 3 Da −1 . Diffraction data were collected to a resolution of 3.0 Å

Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

International Nuclear Information System (INIS)

Spicer, E.K.; Horton, R.; Bloem, L.

1987-01-01

Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. λ Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC)

The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

Science.gov (United States)

Landry, Aaron P; Ding, Huangen

2014-01-01

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

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Aaron P. Landry

2014-01-01

Full Text Available Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0. The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss and double-stranded (ds DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

Mutation and crystallization of the first KH domain of human polycytosine-binding protein 1 (PCBP1) in complex with DNA

International Nuclear Information System (INIS)

Yoga, Yano M. K.; Traore, Daouda A. K.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

2011-01-01

The successful preparation of a mutant KH domain representing the first KH domain of PCBP1 and its crystallization in complex with a C-rich DNA are reported. This structure is anticipated to provide high-resolution information that will allow better understanding of the basis of cytosine specificity by PCBPs. Polycytosine-binding proteins (PCBPs) are triple KH-domain proteins that play an important role in the regulation of translation of eukaryotic mRNA. They are also utilized by viral RNA and have been shown to interact with ssDNA. Underlying their function is the specific recognition of C-rich nucleotides by their KH domains. However, the structural basis of this recognition is only partially understood. Here, the preparation of a His-tagged KH domain is described, representing the first domain of PCBP1 that incorporates a C54S mutation as well as the addition of a C-terminal tryptophan. This construct has facilitated the preparation of highly diffracting crystals in complex with C-rich DNA (sequence ACCCCA). Crystals of the KH1–DNA complex were grown using the hanging-drop vapour-diffusion method in 0.1 M phosphate–citrate pH 4.2, 40%(v/v) PEG 300. X-ray diffraction data were collected to 1.77 Å resolution and the diffraction was consistent with space group P2 1 , with unit-cell parameters a = 38.59, b = 111.88, c = 43.42 Å, α = γ = 90.0, β = 93.37°. The structure of the KH1–DNA complex will further our insight into the basis of cytosine specificity by PCBPs

Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors

International Nuclear Information System (INIS)

Pasquinelli, C.; Garreau, F.; Bougueleret, L.; Cariani, E.; Thiers, V.; Croissant, O.; Hadchouel, M.; Tiollais, P.; Brechot, C.; Grzeschik, K.H.

1988-01-01

Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. The authors have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possible related to HBV integration

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

Czech Academy of Sciences Publication Activity Database

Gillard, N.; Goffinont, S.; Buré, C.; Davídková, Marie; Maurizot, J. C.; Cadene, M.; Spotheim-Maurizot, M.

2007-01-01

Roč. 403, part 3 (2007), s. 463-472 ISSN 0264-6021 R&D Projects: GA MŠk 1P05OC085 Institutional research plan: CEZ:AV0Z10480505 Keywords : ionizing radiation * oxidative damage * DNA binding domain * lac repressor Subject RIV: CE - Biochemistry Impact factor: 4.009, year: 2007

Essential and distinct roles of the F-box and helicase domains of Fbh1 in DNA damage repair

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Shinagawa Hideo

2008-03-01

Full Text Available Abstract Background DNA double-strand breaks (DSBs are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage. Results To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant. Conclusion These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

Maturity Models

DEFF Research Database (Denmark)

Lasrado, Lester Allan; Vatrapu, Ravi

2016-01-01

Recent advancements in set theory and readily available software have enabled social science researchers to bridge the variable-centered quantitative and case-based qualitative methodological paradigms in order to analyze multi-dimensional associations beyond the linearity assumptions, aggregate...... effects, unicausal reduction, and case specificity. Based on the developments in set theoretical thinking in social sciences and employing methods like Qualitative Comparative Analysis (QCA), Necessary Condition Analysis (NCA), and set visualization techniques, in this position paper, we propose...... and demonstrate a new approach to maturity models in the domain of Information Systems. This position paper describes the set-theoretical approach to maturity models, presents current results and outlines future research work....

Maturity Models Development in IS Research

DEFF Research Database (Denmark)

Lasrado, Lester Allan; Vatrapu, Ravi; Andersen, Kim Normann

2015-01-01

Maturity models are widespread in IS research and in particular, IT practitioner communities. However, theoretically sound, methodologically rigorous and empirically validated maturity models are quite rare. This literature review paper focuses on the challenges faced during the development...... literature reveals that researchers have primarily focused on developing new maturity models pertaining to domain-specific problems and/or new enterprise technologies. We find rampant re-use of the design structure of widely adopted models such as Nolan’s Stage of Growth Model, Crosby’s Grid, and Capability...... Maturity Model (CMM). Only recently have there been some research efforts to standardize maturity model development. We also identify three dominant views of maturity models and provide guidelines for various approaches of constructing maturity models with a standard vocabulary. We finally propose using...

DNA double strand break repair is enhanced by P53 following induction by DNA damage and is dependent on the C-terminal domain of P53

International Nuclear Information System (INIS)

Wei Tang; Powell, Simon N.

1996-01-01

Purpose: The tumor suppressor gene p53 can mediate cell cycle arrest or apoptosis in response to DNA damage. Accumulating evidence suggests that it may also directly or indirectly influence the DNA repair machinery. In the present study, we investigated whether p53, induced by DNA damage, could enhance the rejoining of double-strand DNA breaks. Materials and Methods: DNA double-strand breaks (dsb) were made by restriction enzyme digestion of a plasmid, between a promoter and a 'reporter' gene: luciferase (LUC) or chloramphenicol acetyl-transferase (CAT). Linear or circular plasmid DNA (LUC or CAT) was co-transfected with circular β-Gal plasmid (to normalize for uptake) into mouse embryonic fibroblasts genetically matched to be (+/+) or (-/-) for p53. Their ability to rejoin linearized plasmid was measured by the luciferase or CAT activity detected in rescued plasmids. The activity detected in cells transfected with linear plasmid was scored relative to the activity detected in cells transfected with circular plasmid. Results: Ionizing radiation (IR, 2 Gy) enhanced the dsb repair activity in wild type p53 cells; however, p53 null cells lose this effect, indicating that the enhancement of dsb repair was p53-dependent. REF cells with dominant-negative mutant p53 showed a similar induction compared with the parental REF cells with wild-type p53. This ala-143 mutant p53 prevents cell cycle arrest and transactivation of p21 WAF1/cip1) following IR, indicating that the p53-dependent enhancement of DNA repair is distinct from transactivation. Immortalized murine embryonic fibroblasts, 10(1)VasK1 cells, which express p53 cDNA encoding a temperature-sensitive mutant in the DNA sequence specific binding domain (ala135 to val135) with an alternatively spliced C-terminal domain (ASp53: amino-acids 360-381) and, 10(1)Val5 cells, which express the normal spliced p53 (NSp53) with the same temperature-sensitive mutant were compared. It was found that 10(1)VasK1 cells showed no DNA

Leveraging People-Related Maturity Issues for Achieving Higher Maturity and Capability Levels

Science.gov (United States)

Buglione, Luigi

During the past 20 years Maturity Models (MM) become a buzzword in the ICT world. Since the initial Crosby's idea in 1979, plenty of models have been created in the Software & Systems Engineering domains, addressing various perspectives. By analyzing the content of the Process Reference Models (PRM) in many of them, it can be noticed that people-related issues have little weight in the appraisals of the capabilities of organizations while in practice they are considered as significant contributors in traditional process and organizational performance appraisals, as stressed instead in well-known Performance Management models such as MBQA, EFQM and BSC. This paper proposes some ways for leveraging people-related maturity issues merging HR practices from several types of maturity models into the organizational Business Process Model (BPM) in order to achieve higher organizational maturity and capability levels.

Crystal structure of the gamma-2 herpesvirus LANA DNA binding domain identifies charged surface residues which impact viral latency.

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Bruno Correia

Full Text Available Latency-associated nuclear antigen (LANA mediates γ2-herpesvirus genome persistence and regulates transcription. We describe the crystal structure of the murine gammaherpesvirus-68 LANA C-terminal domain at 2.2 Å resolution. The structure reveals an alpha-beta fold that assembles as a dimer, reminiscent of Epstein-Barr virus EBNA1. A predicted DNA binding surface is present and opposite this interface is a positive electrostatic patch. Targeted DNA recognition substitutions eliminated DNA binding, while certain charged patch mutations reduced bromodomain protein, BRD4, binding. Virus containing LANA abolished for DNA binding was incapable of viable latent infection in mice. Virus with mutations at the charged patch periphery exhibited substantial deficiency in expansion of latent infection, while central region substitutions had little effect. This deficiency was independent of BRD4. These results elucidate the LANA DNA binding domain structure and reveal a unique charged region that exerts a critical role in viral latent infection, likely acting through a host cell protein(s.

Solution structure of a DNA mimicking motif of an RNA aptamer against transcription factor AML1 Runt domain.

Science.gov (United States)

Nomura, Yusuke; Tanaka, Yoichiro; Fukunaga, Jun-ichi; Fujiwara, Kazuya; Chiba, Manabu; Iibuchi, Hiroaki; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Kozu, Tomoko; Sakamoto, Taiichi

2013-12-01

AML1/RUNX1 is an essential transcription factor involved in the differentiation of hematopoietic cells. AML1 binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. In a previous study, we obtained RNA aptamers against the AML1 Runt domain by systematic evolution of ligands by exponential enrichment and revealed that RNA aptamers exhibit higher affinity for the Runt domain than that for RDE and possess the 5'-GCGMGNN-3' and 5'-N'N'CCAC-3' conserved motif (M: A or C; N and N' form Watson-Crick base pairs) that is important for Runt domain binding. In this study, to understand the structural basis of recognition of the Runt domain by the aptamer motif, the solution structure of a 22-mer RNA was determined using nuclear magnetic resonance. The motif contains the AH(+)-C mismatch and base triple and adopts an unusual backbone structure. Structural analysis of the aptamer motif indicated that the aptamer binds to the Runt domain by mimicking the RDE sequence and structure. Our data should enhance the understanding of the structural basis of DNA mimicry by RNA molecules.

Crystal Structure of the Dimeric Oct6 (Pou3fl) POU Domain Bound to Palindromic MORE DNA

Energy Technology Data Exchange (ETDEWEB)

R Jauch; S Choo; C Ng; P Kolatkar

2011-12-31

POU domains (named after their identification in Pit1, Oct1 unc86) are found in around 15 transcription factors encoded in mammalian genomes many of which feature prominently as key regulators at development bifurcations. For example, the POU III class Octamer binding protein 6 (Oct6) is expressed in embryonic stem cells and during neural development and drives the differentia5tion of myelinated cells in the central and peripheral nervous system. Defects in oct6 expression levels are linked to neurological disorders such as schizophrenia. POU proteins contain a bi-partite DNA binding domain that assembles on various DNA motifs with differentially configured subdomains. Intriguingly, alternative configurations of POU domains on different DNA sites were shown to affect the subsequent recruitment of transcriptional coactivators. Namely, binding of Oct1 to a Palindromic Oct-factor Recognition Element (PORE) was shown to facilitate the recruitment of the OBF1 coactivator whereas More of PORE (MORE) bound Oct1 does not. Moreover, Pit1 was shown to recruit the corepressor N-CoR only when bound to a variant MORE motif with a 2 bp half-site spacing. Therefore, POU proteins are seen as a paradigm for DNA induced allosteric effects on transcription factors modulating their regulatory potential. However, a big unresolved conundrum for the POU class and for most if not all other transcription factor classes is how highly similar proteins regulate different sets of genes causing fundamentally different biological responses. Ultimately, there must be subtle features enabling those factors to engage in contrasting molecular interactions in the cell. Thus, the dissection of the molecular details of the transcription-DNA recognition in general, and the formation of multimeric regulatory complexes, in particular, is highly desirable. To contribute to these efforts they solved the 2.05 {angstrom} crystal structure of Oct6 bound as a symmetrical homodimer to palindromic MORE DNA.

The N-terminal domain of the repressor of Staphylococcus aureus phage Φ11 possesses an unusual dimerization ability and DNA binding affinity.

Directory of Open Access Journals (Sweden)

Anindya Biswas

Full Text Available Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors.

RING finger and WD repeat domain 3 (RFWD3) associates with replication protein A (RPA) and facilitates RPA-mediated DNA damage response.

Science.gov (United States)

Liu, Shangfeng; Chu, Jessica; Yucer, Nur; Leng, Mei; Wang, Shih-Ya; Chen, Benjamin P C; Hittelman, Walter N; Wang, Yi

2011-06-24

DNA damage response is crucial for maintaining genomic integrity and preventing cancer by coordinating the activation of checkpoints and the repair of damaged DNA. Central to DNA damage response are the two checkpoint kinases ATM and ATR that phosphorylate a wide range of substrates. RING finger and WD repeat domain 3 (RFWD3) was initially identified as a substrate of ATM/ATR from a proteomic screen. Subsequent studies showed that RFWD3 is an E3 ubiquitin ligase that ubiquitinates p53 in vitro and positively regulates p53 levels in response to DNA damage. We report here that RFWD3 associates with replication protein A (RPA), a single-stranded DNA-binding protein that plays essential roles in DNA replication, recombination, and repair. Binding of RPA to single-stranded DNA (ssDNA), which is generated by DNA damage and repair, is essential for the recruitment of DNA repair factors to damaged sites and the activation of checkpoint signaling. We show that RFWD3 is physically associated with RPA and rapidly localizes to sites of DNA damage in a RPA-dependent manner. In vitro experiments suggest that the C terminus of RFWD3, which encompass the coiled-coil domain and the WD40 domain, is necessary for binding to RPA. Furthermore, DNA damage-induced phosphorylation of RPA and RFWD3 is dependent upon each other. Consequently, loss of RFWD3 results in the persistent foci of DNA damage marker γH2AX and the repair protein Rad51 in damaged cells. These findings suggest that RFWD3 is recruited to sites of DNA damage and facilitates RPA-mediated DNA damage signaling and repair.

Comprehensive Interrogation of Natural TALE DNA Binding Modules and Transcriptional Repressor Domains

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Cong, Le; Zhou, Ruhong; Kuo, Yu-chi; Cunniff, Margaret; Zhang, Feng

2012-01-01

Transcription activator-like effectors (TALE) are sequence-specific DNA binding proteins that harbor modular, repetitive DNA binding domains. TALEs have enabled the creation of customizable designer transcriptional factors and sequence-specific nucleases for genome engineering. Here we report two improvements of the TALE toolbox for achieving efficient activation and repression of endogenous gene expression in mammalian cells. We show that the naturally occurring repeat variable diresidue (RVD) Asn-His (NH) has high biological activity and specificity for guanine, a highly prevalent base in mammalian genomes. We also report an effective TALE transcriptional repressor architecture for targeted inhibition of transcription in mammalian cells. These findings will improve the precision and effectiveness of genome engineering that can be achieved using TALEs. PMID:22828628

The DNA binding and activation domains of Gal4p are sufficient for conveying its regulatory signals.

OpenAIRE

Ding, W V; Johnston, S A

1997-01-01

The transcriptional activation function of the Saccharomyces cerevisiae activator Gal4p is known to rely on a DNA binding activity at its amino terminus and an activation domain at its carboxy terminus. Although both domains are required for activation, truncated forms of Gal4p containing only these domains activate poorly in vivo. Also, mutations in an internal conserved region of Gal4p inactivate the protein, suggesting that this internal region has some function critical to the activity of...

ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB

International Nuclear Information System (INIS)

Koo, Hyeon-Sook; Liu, L.F.; Claassen, L.; Grossman, L.

1991-01-01

The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils. In the presence of E. coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template. Positive supercoils were not produced when E. coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E. coli and eukaryotic DNA topoisomerases I were added simultaneously. These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAM complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained. The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 C-terminal amino acids and is defective in preferential binding to UV-damaged DNA. UvrAB was found to preferentially supercoil the UV-damaged DNA template, whereas the mutant protein supercoiled UV-damaged and undamaged DNA with equal efficiency. The authors results therefore suggest that the DNA helix-tracking activity of UvrAB may be involved in searching and/or prepriming the damaged DNA for UvrC incision. A possible role of supercoiled domains in the incision process is discussed

An elastic network model of HK97 capsid maturation.

Science.gov (United States)

Kim, Moon K; Jernigan, Robert L; Chirikjian, Gregory S

2003-08-01

The structure of the capsid of bacteriophage HK97 has been solved at various stages of maturity by crystallography and cryo-electron microscopy, and has been reported previously in the literature. Typically the capsid assembles through polymerization and maturation processes. Maturation is composed of proteolytic cleavages to the precursor capsid (called Prohead II), expansion triggered by DNA packaging (in which the largest conformational changes of the capsid appear), and covalent cross-links of neighboring subunits to create the mature capsid called Head II. We apply a coarse-grained elastic network interpolation (ENI) to generate a feasible pathway for conformational change from Prohead II to Head II. The icosahedral symmetry of the capsid structure offers a significant computational advantage because it is not necessary to consider the whole capsid structure but only an asymmetric unit consisting of one hexamer plus an additional subunit from an adjacent pentamer. We also analyze normal modes of the capsid structure using an elastic network model which is also subject to symmetry constraints. Using our model, we can visualize the smooth evolution of capsid expansion and revisit in more detail several interesting geometric changes recognized in early experimental works such as rigid body motion of two compact domains (A and P) with two refolding extensions (N-arm and E-loop) and track the approach of the two particular residues associated with isopeptide bonds that make hexagonal cross-links in Head II. The feasibility of the predicted pathway is also supported by the results of our normal mode analysis.

The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

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Miles, Jennifer A; Frost, Mark G; Carroll, Eilis; Rowe, Michelle L; Howard, Mark J; Sidhu, Ateesh; Chaugule, Viduth K; Alpi, Arno F; Walden, Helen

2015-08-21

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL. FANCL comprises 3 domains, a RING domain that interacts with E2 conjugating enzymes, a central domain required for substrate interaction, and an N-terminal E2-like fold (ELF) domain. The ELF domain is found in all FANCL homologues, yet the function of the domain remains unknown. We report here that the ELF domain of FANCL is required to mediate a non-covalent interaction between FANCL and ubiquitin. The interaction involves the canonical Ile44 patch on ubiquitin, and a functionally conserved patch on FANCL. We show that the interaction is not necessary for the recognition of the core complex, it does not enhance the interaction between FANCL and Ube2T, and is not required for FANCD2 monoubiquitination in vitro. However, we demonstrate that the ELF domain is required to promote efficient DNA damage-induced FANCD2 monoubiquitination in vertebrate cells, suggesting an important function of ubiquitin binding by FANCL in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Imperfect DNA mirror repeats in the gag gene of HIV-1 (HXB2 identify key functional domains and coincide with protein structural elements in each of the mature proteins

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Lang Dorothy M

2007-10-01

Full Text Available Abstract Background A DNA mirror repeat is a sequence segment delimited on the basis of its containing a center of symmetry on a single strand, e.g. 5'-GCATGGTACG-3'. It is most frequently described in association with a functionally significant site in a genomic sequence, and its occurrence is regarded as noteworthy, if not unusual. However, imperfect mirror repeats (IMRs having ≥ 50% symmetry are common in the protein coding DNA of monomeric proteins and their distribution has been found to coincide with protein structural elements – helices, β sheets and turns. In this study, the distribution of IMRs is evaluated in a polyprotein – to determine whether IMRs may be related to the position or order of protein cleavage or other hierarchal aspects of protein function. The gag gene of HIV-1 [GenBank:K03455] was selected for the study because its protein motifs and structural components are well documented. Results There is a highly specific relationship between IMRs and structural and functional aspects of the Gag polyprotein. The five longest IMRs in the polyprotein translate a key functional segment in each of the five cleavage products. Throughout the protein, IMRs coincide with functionally significant segments of the protein. A detailed annotation of the protein, which combines structural, functional and IMR data illustrates these associations. There is a significant statistical correlation between the ends of IMRs and the ends of PSEs in each of the mature proteins. Weakly symmetric IMRs (≥ 33% are related to cleavage positions and processes. Conclusion The frequency and distribution of IMRs in HIV-1 Gag indicates that DNA symmetry is a fundamental property of protein coding DNA and that different levels of symmetry are associated with different functional aspects of the gene and its protein. The interaction between IMRs and protein structure and function is precise and interwoven over the entire length of the polyprotein. The

Blue light-induced LOV domain dimerization enhances the affinity of Aureochrome 1a for its target DNA sequence

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Heintz, Udo; Schlichting, Ilme

2016-01-01

The design of synthetic optogenetic tools that allow precise spatiotemporal control of biological processes previously inaccessible to optogenetic control has developed rapidly over the last years. Rational design of such tools requires detailed knowledge of allosteric light signaling in natural photoreceptors. To understand allosteric communication between sensor and effector domains, characterization of all relevant signaling states is required. Here, we describe the mechanism of light-dependent DNA binding of the light-oxygen-voltage (LOV) transcription factor Aureochrome 1a from Phaeodactylum tricornutum (PtAu1a) and present crystal structures of a dark state LOV monomer and a fully light-adapted LOV dimer. In combination with hydrogen/deuterium-exchange, solution scattering data and DNA-binding experiments, our studies reveal a light-sensitive interaction between the LOV and basic region leucine zipper DNA-binding domain that together with LOV dimerization results in modulation of the DNA affinity of PtAu1a. We discuss the implications of these results for the design of synthetic LOV-based photosensors with application in optogenetics. DOI: http://dx.doi.org/10.7554/eLife.11860.001 PMID:26754770

In Situ Dark Adaptation Enhances the Efficiency of DNA Extraction from Mature Pin Oak (Quercus palustris Leaves, Facilitating the Identification of Partial Sequences of the 18S rRNA and Isoprene Synthase (IspS Genes

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Csengele E. Barta

2017-10-01

Full Text Available Mature oak (Quercus spp. leaves, although abundantly available during the plants’ developmental cycle, are rarely exploited as viable sources of genomic DNA. These leaves are rich in metabolites difficult to remove during standard DNA purification, interfering with downstream molecular genetics applications. The current work assessed whether in situ dark adaptation, to deplete sugar reserves and inhibit secondary metabolite synthesis could compensate for the difficulties encountered when isolating DNA from mature leaves rich in secondary metabolites. We optimized a rapid, commercial kit based method to extract genomic DNA from dark- and light-adapted leaves. We demonstrated that in situ dark adaptation increases the yield and quality of genomic DNA obtained from mature oak leaves, yielding templates of sufficiently high quality for direct downstream applications, such as PCR amplification and gene identification. The quality of templates isolated from dark-adapted pin oak leaves particularly improved the amplification of larger fragments in our experiments. From DNA extracts prepared with our optimized method, we identified for the first time partial segments of the genes encoding 18S rRNA and isoprene synthase (IspS from pin oak (Quercus palustris, whose full genome has not yet been sequenced.

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

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Zhu, Hui; Shuman, Stewart

2005-04-01

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains

International Nuclear Information System (INIS)

Vallee, B.L.; Auld, D.S.; Coleman, J.E.

1991-01-01

The authors recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families of proteins. Those of the DNA-binding domains of the fungal transcription factor GAL4 and the rat glucocorticoid receptor are the first to have been determined. Both proteins contain two zinc binding sites, and in both, cysteine residues are the sole zinc ligands. In GAL4, two zinc atoms are bound to six cysteine residues which form a zinc cluster akin to that of metallothionein; the distance between the two zinc atoms of GAL4 is ∼3.5 angstrom. In the glucocorticoid receptor, each zinc atom is bound to four cysteine residues; the interatomic zinc-zinc distance is ∼13 angstrom, and in this instance, a zinc twist is represented by a helical DNA recognition site located between the two zinc atoms. Zinc clusters and zinc twists are here recognized as two distinctive motifs in DNA-binding proteins containing multiple zinc atoms. For native zinc fingers, structural data do not exist as yet; consequently, the interatomic distances between zinc atoms are not known. As further structural data become available, the structural and functional significance of these different motifs in their binding to DNA and other proteins participating in the transmission of the genetic message will become apparent

The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome

Energy Technology Data Exchange (ETDEWEB)

Kuo, Alex J; Song, Jikui; Cheung, Peggie; Ishibe-Murakami, Satoko; Yamazoe, Sayumi; Chen, James K; Patel, Dinshaw J; Gozani, Or [Stanford; (MSKCC); (Stanford-MED)

2012-07-11

The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1 - a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing - contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

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Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

2015-01-01

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the central zinc-binding domain of the human Mcm10 DNA-replication factor

International Nuclear Information System (INIS)

Jung, Nam Young; Bae, Won Jin; Chang, Jeong Ho; Kim, Young Chang; Cho, Yunje

2008-01-01

Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. The initiation of eukaryotic DNA replication requires the tightly controlled assembly of a set of replication factors. Mcm10 is a highly conserved nuclear protein that plays a key role in the initiation and elongation processes of DNA replication by providing a physical link between the Mcm2–7 complex and DNA polymerases. The central domain, which contains the CCCH zinc-binding motif, is most conserved within Mcm10 and binds to DNA and several proteins, including proliferative cell nuclear antigen. In this study, the central domain of human Mcm10 was crystallized using the hanging-drop vapour-diffusion method in the presence of PEG 3350. An X-ray diffraction data set was collected to a resolution of 2.6 Å on a synchrotron beamline. The crystals formed belonged to space group R3, with unit-cell parameters a = b = 99.5, c = 133.0 Å. According to Matthews coefficient calculations, the crystals were predicted to contain six MCM10 central domain molecules in the asymmetric unit

Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

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Narasimhan, Kamesh; Hilbig, Antonia; Udayasuryan, Barath; Jayabal, Sriram; Kolatkar, Prasanna R; Jauch, Ralf

2014-10-01

Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.

Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element

Science.gov (United States)

Narasimhan, Kamesh; Hilbig, Antonia; Udayasuryan, Barath; Jayabal, Sriram; Kolatkar, Prasanna R.; Jauch, Ralf

2014-01-01

Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6–DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Å resolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit. PMID:25286939

Effect of pH on the Structure and DNA Binding of the FOXP2 Forkhead Domain.

Science.gov (United States)

Blane, Ashleigh; Fanucchi, Sylvia

2015-06-30

Forkhead box P2 (FOXP2) is a transcription factor expressed in cardiovascular, intestinal, and neural tissues during embryonic development and is implicated in language development. FOXP2 like other FOX proteins contains a DNA binding domain known as the forkhead domain (FHD). The FHD interacts with DNA by inserting helix 3 into the major groove. One of these DNA-protein interactions is a direct hydrogen bond that is formed with His554. FOXP2 is localized in the nuclear compartment that has a pH of 7.5. Histidine contains an imidazole side chain in which the amino group typically has a pKa of ~6.5. It seems possible that pH fluctuations around 6.5 may result in changes in the protonation state of His554 and thus the ability of the FOXP2 FHD to bind DNA. To investigate the effect of pH on the FHD, both the structure and the binding affinity were studied in the pH range of 5-9. This was done in the presence and absence of DNA. The structure was assessed using size exclusion chromatography, far-UV circular dichroism, and intrinsic and extrinsic fluorescence. The results indicated that while pH did not affect the secondary structure in the presence or absence of DNA, the tertiary structure was pH sensitive and the protein was less compact at low pH. Furthermore, the presence of DNA caused the protein to become more compact at low pH and also had the potential to increase the dimerization propensity. Fluorescence anisotropy was used to investigate the effect of pH on the FOXP2 FHD DNA binding affinity. It was found that pH had a direct effect on binding affinity. This was attributed to the altered hydrogen bonding patterns upon protonation or deprotonation of His554. These results could implicate pH as a means of regulating transcription by the FOXP2 FHD, which may also have repercussions for the behavior of this protein in cancer cells.

Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: Identification of peptides in the DNA binding domain

International Nuclear Information System (INIS)

Farrar, Y.J.K.; Evans, R.K.; Beach, C.M.; Coleman, M.S.

1991-01-01

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32 P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32 P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp 221 -Lys 231 (peptide B8) and Cys 234 -Lys 249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an α-helical array of 39 angstrom which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase β that has been implicated in the binding of DNA template

Melatonin in maturation media fails to improve oocyte maturation, embryo development rates and DNA damage of bovine embryos Melatonina no meio de maturação não melhorou as taxas de maturação dos ovócitos, de desenvolvimento embrionário e a fragmentação do DNA dos embriões bovinos

Directory of Open Access Journals (Sweden)

Luciana Takada

2010-08-01

Full Text Available Melatonin (MEL acts as a powerful scavenger of free radicals and direct gonadal responses to melatonin have been reported in the literature. Few studies, however, have evaluated the effect of MEL during in vitro maturation (IVM on bovine embryos. This study tested the addition of MEL to maturation medium (MM with no gonadotropins on nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos. Cumulus-oocyte complexes were aspirated from abattoir ovaries and cultured in MM (TCM-199 medium supplemented with 10% fetal calf serum - FCS at 39ºC and 5% CO2 in air. After 24 hours of culture in MM with 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH; 10-9 M MEL or 10-9 M MEL, 0.5 µg mL-1 FSH and 5.0 µg mL-1 LH, the oocytes were stained with Hoechst 33342 to evaluate nuclear maturation rate. After in vitro fertilization and embryo culture, development rates were evaluated and the blastocysts were assessed for DNA damage by Comet assay. There was no effect of melatonin added to the MM, alone or in combination with gonadotropins, on nuclear maturation, cleavage and blastocyst rates. These rates ranged between 88% to 90%, 85% to 88% and 42% to 46%, respectively. The extent of DNA damage in embryos was also not affected by MEL supplementation during IVM. The addition of 10-9 M MEL to the MM failed to improve nuclear maturation and embryo development rates and the incidence of DNA damage in resulting embryos, but was able to properly substitute for gonadotropins during IVM.Melatonin (MEL atua como um potente redutor de radicais livres. Efeito direto da MEL na função gonadal também foi observado. Existem poucos estudos relacionados ao efeito da MEL durante a maturação no desenvolvimento embrionário in vitro. Avaliou-se a adição de MEL no meio de maturação (sem gonadotrofinas nas taxas de maturação nuclear e de desenvolvimento embrionário e na incidência de fragmentação do DNA nos embriões produzidos in vitro

Redox-sensitive structural change in the A-domain of HMGB1 and its implication for the binding to cisplatin modified DNA

International Nuclear Information System (INIS)

Wang, Jing; Tochio, Naoya; Takeuchi, Aya; Uewaki, Jun-ichi; Kobayashi, Naohiro; Tate, Shin-ichi

2013-01-01

Highlights: •The structure of the oxidized A-domain of human HMGB1 was solved. •Phe38 ring was flipped in the oxidized structure from that in the reduced form. •The flipped ring disables the intercalation into the cisplatinated lesions. •The functionally relevant redox-dependent structural change was described. -- Abstract: HMGB1 (high-mobility group B1) is a ubiquitously expressed bifunctional protein that acts as a nuclear protein in cells and also as an inflammatory mediator in the extracellular space. HMGB1 changes its functions according to the redox states in both intra- and extra-cellular environments. Two cysteines, Cys23 and Cys45, in the A-domain of HMGB1 form a disulfide bond under oxidative conditions. The A-domain with the disulfide bond shows reduced affinity to cisplatin modified DNA. We have solved the oxidized A-domain structure by NMR. In the structure, Phe38 has a flipped ring orientation from that found in the reduced form; the phenyl ring in the reduced form intercalates into the platinated lesion in DNA. The phenyl ring orientation in the oxidized form is stabilized through intramolecular hydrophobic contacts. The reorientation of the Phe38 ring by the disulfide bond in the A-domain may explain the reduced HMGB1 binding affinity towards cisplatinated DNA

ICoNOs MM: The IT-enabled Collaborative Networked Organizations Maturity Model

NARCIS (Netherlands)

Santana Tapia, R.G.

2009-01-01

The focus of this paper is to introduce a comprehensive model for assessing and improving maturity of business-IT alignment (B-ITa) in collaborative networked organizations (CNOs): the ICoNOs MM. This two dimensional maturity model (MM) addresses five levels of maturity as well as four domains to

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

Science.gov (United States)

Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

2017-10-01

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

Mature Microsatellites: Mechanisms Underlying Dinucleotide Microsatellite Mutational Biases in Human Cells

OpenAIRE

Baptiste, Beverly A.; Ananda, Guruprasad; Strubczewski, Noelle; Lutzkanin, Andrew; Khoo, Su Jen; Srikanth, Abhinaya; Kim, Nari; Makova, Kateryna D.; Krasilnikova, Maria M.; Eckert, Kristin A.

2013-01-01

Dinucleotide microsatellites are dynamic DNA sequences that affect genome stability. Here, we focused on mature microsatellites, defined as pure repeats of lengths above the threshold and unlikely to mutate below it in a single mutational event. We investigated the prevalence and mutational behavior of these sequences by using human genome sequence data, human cells in culture, and purified DNA polymerases. Mature dinucleotides (?10 units) are present within exonic sequences of >350 genes, re...

Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

International Nuclear Information System (INIS)

Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie; Cotmore, Susan F.; Tattersall, Peter; Zhao, Haiyan; Tang, Liang

2015-01-01

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication

Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

Energy Technology Data Exchange (ETDEWEB)

Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan; Lynn, Annie [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Cotmore, Susan F. [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Tattersall, Peter [Departments of Laboratory Medicine, Yale University Medical School, New Haven, CT 06510 (United States); Departments of Genetics, Yale University Medical School, New Haven, CT 06510 (United States); Zhao, Haiyan, E-mail: zhaohy@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States); Tang, Liang, E-mail: tangl@ku.edu [Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045 (United States)

2015-02-15

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase active site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.

Fingerprinting of near-homogeneous DNA ligase I and II from human cells. Similarity of their AMP-binding domains.

Science.gov (United States)

Yang, S W; Becker, F F; Chan, J Y

1990-10-25

DNA ligases play obligatory roles during replication, repair, and recombination. Multiple forms of DNA ligase have been reported in mammalian cells including DNA ligase I, the high molecular mass species which functions during replication, and DNA ligase II, the low molecular mass species which is associated with repair. In addition, alterations in DNA ligase activities have been reported in acute lymphocytic leukemia cells, Bloom's syndrome cells, and cells undergoing differentiation and development. To better distinguish the biochemical and molecular properties of the various DNA ligases from human cells, we have developed a method of purifying multiple species of DNA ligase from HeLa cells by chromatography through DEAE-Bio-Gel, CM-Bio-Gel, hydroxylapatite, Sephacryl S-300, Mono P, and DNA-cellulose. DNA-cellulose chromatography of the partially purified enzymes resolved multiple species of DNA ligase after labeling the enzyme with [alpha-32P]ATP to form the ligase-[32P]AMP adduct. The early eluting enzyme activity (0.25 M NaCl) contained a major 67-kDa-labeled protein, while the late eluting activity (0.48 M NaCl) contained two major labeled proteins of 90 and 78 kDa. Neutralization experiments with antiligase I antibodies indicated that the early and late eluting activity peaks were DNA ligase II and I, respectively. The three major ligase-[32P]AMP polypeptides (90, 78, and 67 kDa) were subsequently purified to near homogeneity by elution from preparative sodium dodecyl sulfate-polyacrylamide gels. All three polypeptides retained DNA ligase activities after gel elution and renaturation. To further reveal the relationship between these enzymes, partial digestion by V8-protease was performed. All three purified polypeptides gave rise to a common 22-kDa-labeled fragment for their AMP-binding domains, indicating that the catalytic sites of ligase I and II are quite similar, if not identical. Similar findings were obtained from the two-dimensional gel

WHERE MULTIFUNCTIONAL DNA REPAIR PROTEINS MEET: MAPPING THE INTERACTION DOMAINS BETWEEN XPG AND WRN

Energy Technology Data Exchange (ETDEWEB)

Rangaraj, K.; Cooper, P.K.; Trego, K.S.

2009-01-01

The rapid recognition and repair of DNA damage is essential for the maintenance of genomic integrity and cellular survival. Multiple complex and interconnected DNA damage responses exist within cells to preserve the human genome, and these repair pathways are carried out by a specifi c interplay of protein-protein interactions. Thus a failure in the coordination of these processes, perhaps brought about by a breakdown in any one multifunctional repair protein, can lead to genomic instability, developmental and immunological abnormalities, cancer and premature aging. This study demonstrates a novel interaction between two such repair proteins, Xeroderma pigmentosum group G protein (XPG) and Werner syndrome helicase (WRN), that are both highly pleiotropic and associated with inherited genetic disorders when mutated. XPG is a structure-specifi c endonuclease required for the repair of UV-damaged DNA by nucleotide excision repair (NER), and mutations in XPG result in the diseases Xeroderma pigmentosum (XP) and Cockayne syndrome (CS). A loss of XPG incision activity results in XP, whereas a loss of non-enzymatic function(s) of XPG causes CS. WRN is a multifunctional protein involved in double-strand break repair (DSBR), and consists of 3’–5’ DNA-dependent helicase, 3’–5’ exonuclease, and single-strand DNA annealing activities. Nonfunctional WRN protein leads to Werner syndrome, a premature aging disorder with increased cancer incidence. Far Western analysis was used to map the interacting domains between XPG and WRN by denaturing gel electrophoresis, which separated purifi ed full length and recombinant XPG and WRN deletion constructs, based primarily upon the length of each polypeptide. Specifi c interacting domains were visualized when probed with the secondary protein of interest which was then detected by traditional Western analysis using the antibody of the secondary protein. The interaction between XPG and WRN was mapped to the C-terminal region of

The DNA translocase RAD5A acts independently of the other main DNA repair pathways, and requires both its ATPase and RING domain for activity in Arabidopsis thaliana.

Science.gov (United States)

Klemm, Tobias; Mannuß, Anja; Kobbe, Daniela; Knoll, Alexander; Trapp, Oliver; Dorn, Annika; Puchta, Holger

2017-08-01

Multiple pathways exist to repair DNA damage induced by methylating and crosslinking agents in Arabidopsis thaliana. The SWI2/SNF2 translocase RAD5A, the functional homolog of budding yeast Rad5 that is required for the error-free branch of post-replicative repair, plays a surprisingly prominent role in the repair of both kinds of lesions in Arabidopsis. Here we show that both the ATPase domain and the ubiquitination function of the RING domain of the Arabidopsis protein are essential for the cellular response to different forms of DNA damage. To define the exact role of RAD5A within the complex network of DNA repair pathways, we crossed the rad5a mutant line with mutants of different known repair factors of Arabidopsis. We had previously shown that RAD5A acts independently of two main pathways of replication-associated DNA repair defined by the helicase RECQ4A and the endonuclease MUS81. The enhanced sensitivity of all double mutants tested in this study indicates that the repair of damaged DNA by RAD5A also occurs independently of nucleotide excision repair (AtRAD1), single-strand break repair (AtPARP1), as well as microhomology-mediated double-strand break repair (AtTEB). Moreover, RAD5A can partially complement for a deficient AtATM-mediated DNA damage response in plants, as the double mutant shows phenotypic growth defects. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Cutting edge: double-stranded DNA breaks in the IgV region gene were detected at lower frequency in affinity-maturation impeded GANP-/- mice.

Science.gov (United States)

Kawatani, Yousuke; Igarashi, Hideya; Matsui, Takeshi; Kuwahara, Kazuhiko; Fujimura, Satoru; Okamoto, Nobukazu; Takagi, Katsumasa; Sakaguchi, Nobuo

2005-11-01

Double-stranded DNA breaks (DSBs) at the IgV region (IgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken gamma-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP-/- B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANPTg) B cells showed an increased level. These results suggested that the level of IgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation.

The Cold Shock Domain of YB-1 Segregates RNA from DNA by Non-Bonded Interactions.

Directory of Open Access Journals (Sweden)

Vladislav Kljashtorny

Full Text Available The human YB-1 protein plays multiple cellular roles, of which many are dictated by its binding to RNA and DNA through its Cold Shock Domain (CSD. Using molecular dynamics simulation approaches validated by experimental assays, the YB1 CSD was found to interact with nucleic acids in a sequence-dependent manner and with a higher affinity for RNA than DNA. The binding properties of the YB1 CSD were close to those observed for the related bacterial Cold Shock Proteins (CSP, albeit some differences in sequence specificity. The results provide insights in the molecular mechanisms whereby YB-1 interacts with nucleic acids.

Affinity maturation in an HIV broadly neutralizing B-cell lineage through reorientation of variable domains.

Science.gov (United States)

Fera, Daniela; Schmidt, Aaron G; Haynes, Barton F; Gao, Feng; Liao, Hua-Xin; Kepler, Thomas B; Harrison, Stephen C

2014-07-15

Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus-antibody "arms race." Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus-antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule.

Predicting and analyzing DNA-binding domains using a systematic approach to identifying a set of informative physicochemical and biochemical properties

Science.gov (United States)

2011-01-01

Background Existing methods of predicting DNA-binding proteins used valuable features of physicochemical properties to design support vector machine (SVM) based classifiers. Generally, selection of physicochemical properties and determination of their corresponding feature vectors rely mainly on known properties of binding mechanism and experience of designers. However, there exists a troublesome problem for designers that some different physicochemical properties have similar vectors of representing 20 amino acids and some closely related physicochemical properties have dissimilar vectors. Results This study proposes a systematic approach (named Auto-IDPCPs) to automatically identify a set of physicochemical and biochemical properties in the AAindex database to design SVM-based classifiers for predicting and analyzing DNA-binding domains/proteins. Auto-IDPCPs consists of 1) clustering 531 amino acid indices in AAindex into 20 clusters using a fuzzy c-means algorithm, 2) utilizing an efficient genetic algorithm based optimization method IBCGA to select an informative feature set of size m to represent sequences, and 3) analyzing the selected features to identify related physicochemical properties which may affect the binding mechanism of DNA-binding domains/proteins. The proposed Auto-IDPCPs identified m=22 features of properties belonging to five clusters for predicting DNA-binding domains with a five-fold cross-validation accuracy of 87.12%, which is promising compared with the accuracy of 86.62% of the existing method PSSM-400. For predicting DNA-binding sequences, the accuracy of 75.50% was obtained using m=28 features, where PSSM-400 has an accuracy of 74.22%. Auto-IDPCPs and PSSM-400 have accuracies of 80.73% and 82.81%, respectively, applied to an independent test data set of DNA-binding domains. Some typical physicochemical properties discovered are hydrophobicity, secondary structure, charge, solvent accessibility, polarity, flexibility, normalized Van Der

Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

International Nuclear Information System (INIS)

Zhang, Ruoxi; Fang, Liurong; Wang, Dang; Cai, Kaimei; Zhang, Huan; Xie, Lilan; Li, Yi; Chen, Huanchun; Xiao, Shaobo

2015-01-01

To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9). Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses. - Highlights: • Porcine bocavirus (PBoV) NP1 interferes with the IFN α/β signaling pathway. • PBoV NP1 does not prevent STAT1/STAT2 phosphorylation and nuclear translocation. • PBoV NP1 inhibits the DNA-binding activity of ISGF3. • PBoV NP1 interacts with the DNA-binding domain of IRF9.

Porcine bocavirus NP1 negatively regulates interferon signaling pathway by targeting the DNA-binding domain of IRF9

Energy Technology Data Exchange (ETDEWEB)

Zhang, Ruoxi [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Fang, Liurong, E-mail: fanglr@mail.hzau.edu.cn [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Wang, Dang; Cai, Kaimei; Zhang, Huan [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China); Xie, Lilan; Li, Yi [College of Life Science and Technology, Wuhan Institute of Bioengineering, Wuhan 430415 (China); Chen, Huanchun; Xiao, Shaobo [State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070 (China); The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070 (China)

2015-11-15

To subvert host antiviral immune responses, many viruses have evolved countermeasures to inhibit IFN signaling pathway. Porcine bocavirus (PBoV), a newly identified porcine parvovirus, has received attention because it shows clinically high co-infection prevalence with other pathogens in post-weaning multisystemic wasting syndrome (PWMS) and diarrheic piglets. In this study, we screened the structural and non-structural proteins encoded by PBoV and found that the non-structural protein NP1 significantly suppressed IFN-stimulated response element (ISRE) activity and subsequent IFN-stimulated gene (ISG) expression. However, NP1 affected neither the activation and translocation of STAT1/STAT2, nor the formation of the heterotrimeric transcription factor complex ISGF3 (STAT1/STAT2/IRF9). Detailed analysis demonstrated that PBoV NP1 blocked the ISGF3 DNA-binding activity by combining with the DNA-binding domain (DBD) of IRF9. In summary, these results indicate that PBoV NP1 interferes with type I IFN signaling pathway by blocking DNA binding of ISGF3 to attenuate innate immune responses. - Highlights: • Porcine bocavirus (PBoV) NP1 interferes with the IFN α/β signaling pathway. • PBoV NP1 does not prevent STAT1/STAT2 phosphorylation and nuclear translocation. • PBoV NP1 inhibits the DNA-binding activity of ISGF3. • PBoV NP1 interacts with the DNA-binding domain of IRF9.

Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide γ-phosphate derivative

Science.gov (United States)

Hansen, Connie J.; Wu, Lydia; Fox, Jeffrey D.; Arezi, Bahram; Hogrefe, Holly H.

2011-01-01

Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (−1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure. PMID:21062827

Engineered split in Pfu DNA polymerase fingers domain improves incorporation of nucleotide gamma-phosphate derivative.

Science.gov (United States)

Hansen, Connie J; Wu, Lydia; Fox, Jeffrey D; Arezi, Bahram; Hogrefe, Holly H

2011-03-01

Using compartmentalized self-replication (CSR), we evolved a version of Pyrococcus furiosus (Pfu) DNA polymerase that tolerates modification of the γ-phosphate of an incoming nucleotide. A Q484R mutation in α-helix P of the fingers domain, coupled with an unintended translational termination-reinitiation (split) near the finger tip, dramatically improve incorporation of a bulky γ-phosphate-O-linker-dabcyl substituent. Whether synthesized by coupled translation from a bicistronic (-1 frameshift) clone, or reconstituted from separately expressed and purified fragments, split Pfu mutant behaves identically to wild-type DNA polymerase with respect to chromatographic behavior, steady-state kinetic parameters (for dCTP), and PCR performance. Although naturally-occurring splits have been identified previously in the finger tip region of T4 gp43 variants, this is the first time a split (in combination with a point mutation) has been shown to broaden substrate utilization. Moreover, this latest example of a split hyperthermophilic archaeal DNA polymerase further illustrates the modular nature of the Family B DNA polymerase structure.

Dynamics of water around the complex structures formed between the KH domains of far upstream element binding protein and single-stranded DNA molecules

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, Kaushik; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

2015-07-28

Single-stranded DNA (ss-DNA) binding proteins specifically bind to the single-stranded regions of the DNA and protect it from premature annealing, thereby stabilizing the DNA structure. We have carried out atomistic molecular dynamics simulations of the aqueous solutions of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein complexed with two short ss-DNA segments. Attempts have been made to explore the influence of the formation of such complex structures on the microscopic dynamics and hydrogen bond properties of the interfacial water molecules. It is found that the water molecules involved in bridging the ss-DNA segments and the protein domains form a highly constrained thin layer with extremely retarded mobility. These water molecules play important roles in freezing the conformational oscillations of the ss-DNA oligomers and thereby forming rigid complex structures. Further, it is demonstrated that the effect of complexation on the slow long-time relaxations of hydrogen bonds at the interface is correlated with hindered motions of the surrounding water molecules. Importantly, it is observed that the highly restricted motions of the water molecules bridging the protein and the DNA components in the complexed forms originate from more frequent hydrogen bond reformations.

Cooperative interactions between paired domain and homeodomain.

Science.gov (United States)

Jun, S; Desplan, C

1996-09-01

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

Effect of point substitutions within the minimal DNA-binding domain of xeroderma pigmentosum group A protein on interaction with DNA intermediates of nucleotide excision repair.

Science.gov (United States)

Maltseva, E A; Krasikova, Y S; Naegeli, H; Lavrik, O I; Rechkunova, N I

2014-06-01

Xeroderma pigmentosum factor A (XPA) is one of the key proteins in the nucleotide excision repair (NER) process. The effects of point substitutions in the DNA-binding domain of XPA (positively charged lysine residues replaced by negatively charged glutamate residues: XPA K204E, K179E, K141E, and tandem mutant K141E/K179E) on the interaction of the protein with DNA structures modeling intermediates of the damage recognition and pre-incision stages in NER were analyzed. All these mutations decreased the affinity of the protein to DNA, the effect depending on the substitution and the DNA structure. The mutant as well as wild-type proteins bind with highest efficiency partly open damaged DNA duplex, and the affinity of the mutants to this DNA is reduced in the order: K204E > K179E > K141E = K141/179E. For all the mutants, decrease in DNA binding efficiency was more pronounced in the case of full duplex and single-stranded DNA than with bubble-DNA structure, the difference between protein affinities to different DNA structures increasing as DNA binding activity of the mutant decreased. No effect of the studied XPA mutations on the location of the protein on the partially open DNA duplex was observed using photoinduced crosslinking with 5-I-dUMP in different positions of the damaged DNA strand. These results combined with earlier published data suggest no direct correlation between DNA binding and activity in NER for these XPA mutants.

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.

Laser-scanning astrocyte mapping reveals increased glutamate-responsive domain size and disrupted maturation of glutamate uptake following neonatal cortical freeze-lesion

Directory of Open Access Journals (Sweden)

Mortiz eArmbruster

2014-09-01

Full Text Available Astrocytic uptake of glutamate shapes extracellular neurotransmitter dynamics, receptor activation, and synaptogenesis. During development, glutamate transport becomes more robust. How neonatal brain insult affects the functional maturation of glutamate transport remains unanswered. Neonatal brain insult can lead to developmental delays, cognitive losses, and epilepsy; the disruption of glutamate transport is known to cause changes in synaptogenesis, receptor activation, and seizure. Using the neonatal freeze-lesion (FL model, we have investigated how insult affects the maturation of astrocytic glutamate transport. As lesioning occurs on the day of birth, a time when astrocytes are still functionally immature, this model is ideal for identifying changes in astrocyte maturation following insult. Reactive astrocytosis, astrocyte proliferation, and in vitro hyperexcitability are known to occur in this model. To probe astrocyte glutamate transport with better spatial precision we have developed a novel technique, Laser Scanning Astrocyte Mapping (LSAM, which combines glutamate transport current (TC recording from astrocytes with laser scanning glutamate photolysis. LSAM allows us to identify the area from which a single astrocyte can transport glutamate and to quantify spatial heterogeneity in the rate of glutamate clearance kinetics within that domain. Using LSAM, we report that cortical astrocytes have an increased glutamate-responsive area following FL and that TCs have faster decay times in distal, as compared to proximal processes. Furthermore, the developmental shift from GLAST- to GLT-1-dominated clearance is disrupted following FL. These findings introduce a novel method to probe astrocyte glutamate uptake and show that neonatal cortical FL disrupts the functional maturation of cortical astrocytes.

The crystal structure of the Sox4 HMG domain-DNA complex suggests a mechanism for positional interdependence in DNA recognition.

Science.gov (United States)

Jauch, Ralf; Ng, Calista K L; Narasimhan, Kamesh; Kolatkar, Prasanna R

2012-04-01

It has recently been proposed that the sequence preferences of DNA-binding TFs (transcription factors) can be well described by models that include the positional interdependence of the nucleotides of the target sites. Such binding models allow for multiple motifs to be invoked, such as principal and secondary motifs differing at two or more nucleotide positions. However, the structural mechanisms underlying the accommodation of such variant motifs by TFs remain elusive. In the present study we examine the crystal structure of the HMG (high-mobility group) domain of Sox4 [Sry (sex-determining region on the Y chromosome)-related HMG box 4] bound to DNA. By comparing this structure with previously solved structures of Sox17 and Sox2, we observed subtle conformational differences at the DNA-binding interface. Furthermore, using quantitative electrophoretic mobility-shift assays we validated the positional interdependence of two nucleotides and the presence of a secondary Sox motif in the affinity landscape of Sox4. These results suggest that a concerted rearrangement of two interface amino acids enables Sox4 to accommodate primary and secondary motifs. The structural adaptations lead to altered dinucleotide preferences that mutually reinforce each other. These analyses underline the complexity of the DNA recognition by TFs and provide an experimental validation for the conceptual framework of positional interdependence and secondary binding motifs.

Four levels of hierarchical organization, including noncovalent chainmail, brace the mature tumor herpesvirus capsid against pressurization.

Science.gov (United States)

Zhou, Z Hong; Hui, Wong Hoi; Shah, Sanket; Jih, Jonathan; O'Connor, Christine M; Sherman, Michael B; Kedes, Dean H; Schein, Stan

2014-10-07

Like many double-stranded DNA viruses, tumor gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus withstand high internal pressure. Bacteriophage HK97 uses covalent chainmail for this purpose, but how this is achieved noncovalently in the much larger gammaherpesvirus capsid is unknown. Our cryoelectron microscopy structure of a gammaherpesvirus capsid reveals a hierarchy of four levels of organization: (1) Within a hexon capsomer, each monomer of the major capsid protein (MCP), 1,378 amino acids and six domains, interacts with its neighboring MCPs at four sites. (2) Neighboring capsomers are linked in pairs by MCP dimerization domains and in groups of three by heterotrimeric triplex proteins. (3) Small (∼280 amino acids) HK97-like domains in MCP monomers alternate with triplex heterotrimers to form a belt that encircles each capsomer. (4) One hundred sixty-two belts concatenate to form noncovalent chainmail. The triplex heterotrimer orchestrates all four levels and likely drives maturation to an angular capsid that can withstand pressurization. Copyright © 2014 Elsevier Ltd. All rights reserved.

Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow

Directory of Open Access Journals (Sweden)

A. M. Luciano

2009-12-01

Full Text Available DNA methyltransferase-1 (Dnmt1 is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation.We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM. RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8-16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals.

GROWING UP IS HARD TO DO: AN EMPIRICAL EVALUATION OF MATURATION AND DESISTANCE

Science.gov (United States)

Rocque, Michael; Posick, Chad; White, Helene R.

2016-01-01

Purpose With an increase in longitudinal datasets and analyses, scholars have made theoretical advances toward understanding desistance, using biological, social, and psychological factors. In an effort to integrate the theoretical views on desistance, some scholars have argued that each of these views represents a piece of adult maturation. Yet to date, research has not empirically examined an integrated perspective. The purpose of this study is to conduct an exploratory examination of various “domains” of maturation to determine whether they explain desistance from crime separately and as a whole. Methods Using the Rutgers Health and Human Development Project, a longitudinal study spanning ages 12–31, we develop exploratory measures of maturation in five domains: 1) adult social roles, 2) identity/cognitive, 3) psychosocial, 4) civic, and 5) neurocognitive. We then utilize growth curve models to examine the relationship between these domains and crime over time. Results Although each of the domains is associated with crime at the bivariate level, only three (i.e., psychosocial, identity/cognitive transformation, and adult social role) remain significant in the growth curve models (2 in within-individual analyses). In addition, a combined measure of maturation is related to crime, indicating that greater maturation through emerging adulthood has a negative effect on criminal behavior and is, therefore, a factor influencing desistance. Conclusions Maturation emerges as a promising approach to integrating the multiple theoretical views that characterize the literature on desistance from crime. Further research should develop additional domains and determine the best approach for measurement. PMID:28580234

In silico maturation of binding-specificity of DNA aptamers against Proteus mirabilis.

Science.gov (United States)

Savory, Nasa; Lednor, Danielle; Tsukakoshi, Kaori; Abe, Koichi; Yoshida, Wataru; Ferri, Stefano; Jones, Brian V; Ikebukuro, Kazunori

2013-10-01

Proteus mirabilis is a prominent cause of catheter-associated urinary tract infections (CAUTIs) among patients undergoing long-term bladder catheterization. There are currently no effective means of preventing P. mirabilis infections, and strategies for prophylaxis and rapid early diagnosis are urgently required. Aptamers offer significant potential for development of countermeasures against P. mirabilis CAUTI and are an ideal class of molecules for the development of diagnostics and therapeutics. Here we demonstrate the application of Cell-SELEX to identify DNA aptamers that show high affinity for P. mirabilis. While the aptamers identified displayed high affinity for P. mirabilis cells in dot blotting assays, they also bound to other uropathogenic bacteria. To improve aptamer specificity for P. mirabilis, an in silico maturation (ISM) approach was employed. Two cycles of ISM allowed the identification of an aptamer showing 36% higher specificity, evaluated as a ratio of binding signal for P. mirabilis to that for Escherichia coli (also a cause of CAUTI and the most common urinary tract pathogen). Aptamers that specifically recognize P. mirabilis would have diagnostic and therapeutic values and constitute useful tools for studying membrane-associated proteins in this organism. Copyright © 2013 Wiley Periodicals, Inc.

The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element.

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Fukunaga, Junichi; Nomura, Yusuke; Tanaka, Yoichiro; Amano, Ryo; Tanaka, Taku; Nakamura, Yoshikazu; Kawai, Gota; Sakamoto, Taiichi; Kozu, Tomoko

2013-07-01

AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5'-NNCCAC-3' and 5'-GCGMGN'N'-3' (M:A or C; N and N' form Watson-Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences.

Human pro. cap alpha. 1)(I) collagen: cDNA sequence for the C-propeptide domain

Energy Technology Data Exchange (ETDEWEB)

Maekelae, J K; Raassina, M; Virta, A; Vuorio, E

1988-01-11

The authors have previously constructed a cDNA clone pHCAL1, covering most of the C-terminal propeptide domain of human pro..cap alpha..1(I) collagen mRNA,by inserting a 678 bp EcoRI-XhoI fragment of cDNA into pBR322. Since the XhoI/SalI ligation prevented removal of the insert, they used the same strategy to obtain a similar clone in pUC8. RNA was isolated from fetal calvarial bones. The cDNA was digested with EcoRI and XhoI and fractionated on a 1 % agarose gel. Fragments of 650-700 bp were cloned in pUC8 at the polylinker site, which now permits easy removal of the insert. The new clone was named pHCAL1U since the RNA was isolated from another individual. The approach outlined is useful for studies on individual variation which is important to recognize when searching for disease-related mutations in type I collagen.

Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA.

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Mouw, M; Pintel, D J

1998-11-10

GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well. Copyright 1998 Academic Press.

The demise of chloroplast DNA in Arabidopsis.

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Rowan, Beth A; Oldenburg, Delene J; Bendich, Arnold J

2004-09-01

Although it might be expected that chloroplast DNA (cpDNA) would be stably maintained in mature leaves, we report the surprising observation that cpDNA levels decline during plastid development in Arabidopsis thaliana (Col.) until most of the leaves contain little or no DNA long before the onset of senescence. We measured the cpDNA content in developing cotyledons, rosette leaves, and cauline leaves. The amount of cpDNA per chloroplast decreases as the chloroplasts develop, reaching undetectable levels in mature leaves. In young cauline leaves, most individual molecules of cpDNA are found in complex, branched forms. In expanded cauline leaves, cpDNA is present in smaller branched forms only at the base of the leaf and is virtually absent in the distal part of the leaf. We conclude that photosynthetic activity may persist long after the demise of the cpDNA. Copyright 2004 Springer-Verlag

The Enzyme-Like Domain of Arabidopsis Nuclear β-Amylases Is Critical for DNA Sequence Recognition and Transcriptional Activation.

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Soyk, Sebastian; Simková, Klára; Zürcher, Evelyne; Luginbühl, Leonie; Brand, Luise H; Vaughan, Cara K; Wanke, Dierk; Zeeman, Samuel C

2014-04-01

Plant BZR1-BAM transcription factors contain a β-amylase (BAM)-like domain, characteristic of proteins involved in starch breakdown. The enzyme-derived domains appear to be noncatalytic, but they determine the function of the two Arabidopsis thaliana BZR1-BAM isoforms (BAM7 and BAM8) during transcriptional initiation. Removal or swapping of the BAM domains demonstrates that the BAM7 BAM domain restricts DNA binding and transcriptional activation, while the BAM8 BAM domain allows both activities. Furthermore, we demonstrate that BAM7 and BAM8 interact on the protein level and cooperate during transcriptional regulation. Site-directed mutagenesis of residues in the BAM domain of BAM8 shows that its function as a transcriptional activator is independent of catalysis but requires an intact substrate binding site, suggesting it may bind a ligand. Microarray experiments with plants overexpressing truncated versions lacking the BAM domain indicate that the pseudo-enzymatic domain increases selectivity for the preferred cis-regulatory element BBRE (BZR1-BAM Responsive Element). Side specificity toward the G-box may allow crosstalk to other signaling networks. This work highlights the importance of the enzyme-derived domain of BZR1-BAMs, supporting their potential role as metabolic sensors. © 2014 American Society of Plant Biologists. All rights reserved.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

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Haruta, Mayumi [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Nishiyama, Atsuya; Johmura, Yoshikazu [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan); Le Tallec, Benoît; Debatisse, Michelle [Institut Curie, Centre de Recherche, 26 rue d’Ulm, CNRS UMR 3244, 75248 ParisCedex 05 (France); Nakanishi, Makoto, E-mail: mkt-naka@med.nagoya-cu.ac.jp [Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601 (Japan)

2016-01-22

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

International Nuclear Information System (INIS)

Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

2016-01-01

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.

GCR1, a transcriptional activator in Saccharomyces cerevisiae, complexes with RAP1 and can function without its DNA binding domain.

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Tornow, J; Zeng, X; Gao, W; Santangelo, G M

1993-01-01

In Saccharomyces cerevisiae, efficient expression of glycolytic and translational component genes requires two DNA binding proteins, RAP1 (which binds to UASRPG) and GCR1 (which binds to the CT box). We generated deletions in GCR1 to test the validity of several different models for GCR1 function. We report here that the C-terminal half of GCR1, which includes the domain required for DNA binding to the CT box in vitro, can be removed without affecting GCR1-dependent transcription of either the glycolytic gene ADH1 or the translational component genes TEF1 and TEF2. We have also identified an activation domain within a segment of the GCR1 protein (the N-terminal third) that is essential for in vivo function. RAP1 and GCR1 can be co-immunoprecipitated from whole cell extracts, suggesting that they form a complex in vivo. The data are most consistent with a model in which GCR1 is attracted to DNA through contact with RAP1. Images PMID:8508768

Purification, crystallization and preliminary X-ray diffraction analysis of the HMG domain of Sox17 in complex with DNA

International Nuclear Information System (INIS)

Ng, Calista Keow Leng; Palasingam, Paaventhan; Venkatachalam, Rajakannan; Baburajendran, Nithya; Cheng, Jason; Jauch, Ralf; Kolatkar, Prasanna R.

2008-01-01

Crystals of the Sox17 HMG domain bound to LAMA1 enhancer DNA-element crystals that diffracted to 2.75 Å resolution were obtained. Sox17 is a member of the SRY-related high-mobility group (HMG) of transcription factors that have been shown to direct endodermal differentiation in early mammalian development. The LAMA1 gene encoding the α-chain of laminin-1 has been reported to be directly bound and regulated by Sox17. This paper describes the details of initial crystallization attempts with the HMG domain of mouse Sox17 (mSox17-HMG) with a 16-mer DNA element derived from the LAMA1 enhancer and optimization strategies to obtain a better diffracting crystal. The best diffracting crystal was obtained in a condition containing 0.1 M Tris–HCl pH 7.4, 0.2 M MgCl 2 , 30% PEG 3350 using the hanging-drop vapour-diffusion method. A highly redundant in-house data set was collected to 2.75 Å resolution with 99% completeness. The presence of the mSox17-HMG–DNA complex within the crystals was confirmed and Matthews analysis indicated the presence of one complex per asymmetric unit

Crystallization and preliminary X-ray analysis of a complex formed between the antibiotic simocyclinone D8 and the DNA breakage–reunion domain of Escherichia coli DNA gyrase

International Nuclear Information System (INIS)

Edwards, Marcus J.; Flatman, Ruth H.; Mitchenall, Lesley A.; Stevenson, Clare E. M.; Maxwell, Anthony; Lawson, David M.

2009-01-01

Crystals of a complex formed between the 59 kDa N-terminal fragment of the E. coli DNA gyrase A subunit and the antibiotic simocyclinone D8 were obtained and X-ray data were recorded to a resolution of 2.75 Å. Crystals of a complex formed between the 59 kDa N-terminal fragment of the Escherichia coli DNA gyrase A subunit (also known as the breakage–reunion domain) and the antibiotic simocyclinone D8 were grown by vapour diffusion. The complex crystallized with I-centred orthorhombic symmetry and X-ray data were recorded to a resolution of 2.75 Å from a single crystal at the synchrotron. DNA gyrase is an essential bacterial enzyme and thus represents an attractive target for drug development

Antibody affinity maturation

DEFF Research Database (Denmark)

Skjødt, Mette Louise

Yeast surface display is an effective tool for antibody affinity maturation because yeast can be used as an all-in-one workhorse to assemble, display and screen diversified antibody libraries. By employing the natural ability of yeast Saccharomyces cerevisiae to efficiently recombine multiple DNA...... laboratory conditions. A particular emphasis was put on using molecular techniques in conjunction with microenvironmental measurements (O2, pH, irradiance), a combination that is rarely found but provides a much more detailed understanding of “cause and effect” in complex natural systems...

DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription*

Science.gov (United States)

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H.; Rothblum, Katrina; Schneider, David A.; Rothblum, Lawrence I.

2013-01-01

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I. PMID:23393135

DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

Science.gov (United States)

Stepanchick, Ann; Zhi, Huijun; Cavanaugh, Alice H; Rothblum, Katrina; Schneider, David A; Rothblum, Lawrence I

2013-03-29

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

Thermodynamic characterization of binding Oxytricha nova single strand telomere DNA with the alpha protein N-terminal domain.

Science.gov (United States)

Buczek, Pawel; Horvath, Martin P

2006-06-23

The Oxytricha nova telemere binding protein alpha subunit binds single strand DNA and participates in a nucleoprotein complex that protects the very ends of chromosomes. To understand how the N-terminal, DNA binding domain of alpha interacts with DNA we measured the stoichiometry, enthalpy (DeltaH), entropy (DeltaS), and dissociation constant (K(D-DNA)) for binding telomere DNA fragments at different temperatures and salt concentrations using native gel electrophoresis and isothermal titration calorimetry (ITC). About 85% of the total free energy of binding corresponded with non-electrostatic interactions for all DNAs. Telomere DNA fragments d(T(2)G(4)), d(T(4)G(4)), d(G(3)T(4)G(4)), and d(G(4)T(4)G(4)) each formed monovalent protein complexes. In the case of d(T(4)G(4)T(4)G(4)), which has two tandemly repeated d(TTTTTGGGG) telomere motifs, two binding sites were observed. The high-affinity "A site" has a dissociation constant, K(D-DNA(A)) = 13(+/-4) nM, while the low-affinity "B site" is characterized by K(D-DNA(B)) = 5600(+/-600) nM at 25 degrees C. Nucleotide substitution variants verified that the A site corresponds principally with the 3'-terminal portion of d(T(4)G(4)T(4)G(4)). The relative contributions of entropy (DeltaS) and enthalpy (DeltaH) for binding reactions were DNA length-dependent as was heat capacity (DeltaCp). These trends with respect to DNA length likely reflect structural transitions in the DNA molecule that are coupled with DNA-protein association. Results presented here are important for understanding early intermediates and subsequent stages in the assembly of the full telomere nucleoprotein complex and how binding events can prepare the telomere DNA for extension by telomerase, a critical event in telomere biology.

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.

The solution structure of the forkhead box-O DNA binding domain of Brugia malayi DAF-16a.

Science.gov (United States)

Casper, Sarah K; Schoeller, Scott J; Zgoba, Danielle M; Phillips, Andrew J; Morien, Thomas J; Chaffee, Gary R; Sackett, Peter C; Peterson, Francis C; Crossgrove, Kirsten; Veldkamp, Christopher T

2014-12-01

Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. Here the solution structure of the forkhead DNA binding domain of Brugia malayi DAF-16a, a putative ortholog of Caenorhabditis elegans DAF-16, is reported. It is believed to be the first structure of a forkhead or winged helix domain from an invertebrate. C. elegans DAF-16 is involved in the insulin/IGF-I signaling pathway and helps control metabolism, longevity, and development. Conservation of sequence and structure with human FOXO proteins suggests that B. malayi DAF-16a is a member of the FOXO family of forkhead proteins. © 2014 Wiley Periodicals, Inc.

DNA-binding proteins essential for protein-primed bacteriophage ø29 DNA replication

Directory of Open Access Journals (Sweden)

Margarita Salas

2016-08-01

Full Text Available Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5’ ends of the DNA. This protein, called terminal protein (TP, is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3’-5’ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding

Editing of misaligned 3'-termini by an intrinsic 3'-5' exonuclease activity residing in the PHP domain of a family X DNA polymerase.

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Baños, Benito; Lázaro, José M; Villar, Laurentino; Salas, Margarita; de Vega, Miguel

2008-10-01

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolX(Bs)), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolX(Bs) possesses an intrinsic 3'-5' exonuclease activity specialized in resecting unannealed 3'-termini in a gapped DNA substrate. Biochemical analysis of a PolX(Bs) deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3'-5' exonuclease activity of PolX(Bs) resides in its PHP domain. Furthermore, site-directed mutagenesis of PolX(Bs) His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3'-termini resection by the 3'-5' exonuclease activity of PolX(Bs) in the DNA repair context are discussed.

Characterization of the roles of the catalytic domains of Mycobacterium tuberculosis ligase D in Ku-dependent error-prone DNA end joining.

Science.gov (United States)

Wright, Douglas; DeBeaux, Austin; Shi, Runhua; Doherty, Aidan J; Harrison, Lynn

2010-09-01

We previously established an Escherichia coli strain capable of re-circularizing linear plasmid DNA by expressing the Mycobacterium tuberculosis Ku (Mt-Ku) and Mycobacterium tuberculosis ligase D (Mt-LigD) proteins from the E.coli chromosome. Repair was predominately mutagenic due to deletions at the termini. We hypothesized that these deletions could be due to a nuclease activity of Mt-LigD that was previously detected in vitro. Mt-LigD has three domains: an N-terminal polymerase domain (PolDom), a central domain with 3'-phosphoesterase and nuclease activity and a C-terminal ligase domain (LigDom). We generated bacterial strains expressing Mt-Ku and mutant versions of Mt-LigD. Plasmid re-circularization experiments in bacteria showed that the PolDom alone had no re-circularization activity. However, an increase in the total and accurate repair was found when the central domain was deleted. This provides further evidence that this central domain does have nuclease activity that can generate deletions during repair. Deletion of only the PolDom of Mt-LigD resulted in a complete loss of accurate repair and a significant reduction in total repair. This is in agreement with published in vitro work indicating that the PolDom is the major Mt-Ku-binding site. Interestingly, the LigDom alone was able to re-circularize plasmid DNA but only in an Mt-Ku-dependent manner, suggesting a potential second site for Ku-LigD interaction. This work has increased our understanding of the mutagenic repair by Mt-Ku and Mt-LigD and has extended the in vitro biochemical experiments by examining the importance of the Mt-LigD domains during repair in bacteria.

Role of ataxia-telangiectasia mutated (ATM) in porcine oocyte in vitro maturation.

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Lin, Zi-Li; Kim, Nam-Hyung

2015-06-01

Ataxia-telangiectasia mutated (ATM) is critical for the DNA damage response, cell cycle checkpoints, and apoptosis. Significant effort has focused on elucidating the relationship between ATM and other nuclear signal transducers; however, little is known about the connection between ATM and oocyte meiotic maturation. We investigated the function of ATM in porcine oocytes. ATM was expressed at all stages of oocyte maturation and localized predominantly in the nucleus. Furthermore, the ATM-specific inhibitor KU-55933 blocked porcine oocyte maturation, reducing the percentages of oocytes that underwent germinal vesicle breakdown (GVBD) and first polar body extrusion. KU-55933 also decreased the expression of DNA damage-related genes (breast cancer 1, budding uninhibited by benzimidazoles 1, and P53) and reduced the mRNA and protein levels of AKT and other cell cycle-regulated genes that are predominantly expressed during G2/M phase, including bone morphogenetic protein 15, growth differentiation factor 9, cell division cycle protein 2, cyclinB1, and AKT. KU-55933 treatment decreased the developmental potential of blastocysts following parthenogenetic activation and increased the level of apoptosis. Together, these data suggested that ATM influenced the meiotic and cytoplasmic maturation of porcine oocytes, potentially by decreasing their sensitivity to DNA strand breaks, stimulating the AKT pathway, and/or altering the expression of other maternal genes. © 2015 International Federation for Cell Biology.

Involvement of the yeast DNA polymerase delta in DNA repair in vivo

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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)

Biophysical characterization of the basic cluster in the transcription repression domain of human MeCP2 with AT-rich DNA.

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Mushtaq, Ameeq Ul; Lee, Yejin; Hwang, Eunha; Bang, Jeong Kyu; Hong, Eunmi; Byun, Youngjoo; Song, Ji-Joon; Jeon, Young Ho

2018-01-01

MeCP2 is a chromatin associated protein which is highly expressed in brain and relevant with Rett syndrome (RTT). There are AT-hook motifs in MeCP2 which can bind with AT-rich DNA, suggesting a role in chromatin binding. Here, we report the identification and characterization of another AT-rich DNA binding motif (residues 295 to 313) from the C-terminal transcription repression domain of MeCP2 by nuclear magnetic resonance (NMR) and isothermal calorimetry (ITC). This motif shows a micromolar affinity to AT-rich DNA, and it binds to the minor groove of DNA like AT-hook motifs. Together with the previous studies, our results provide an insight into a critical role of this motif in chromatin structure and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Methylation changes associated with early maturation stages in the Atlantic salmon

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Pérez-Figueroa Andrés

2011-10-01

Full Text Available Abstract Background Early maturation in the Atlantic salmon is an interesting subject for numerous research lines. Prior to sea migration, parr can reach sexual maturation and successfully fertilize adult female eggs during the reproductive season. These individuals are known as precocious parr, mature parr or "sneakers". Reasons for early maturation are unknown and this transitory stage is usually considered to be a threshold trait. Here, we compare methylation patterns between mature and immature salmon parr from two different rivers in order to infer if such methylation differences may be related to their maturation condition. First we analyzed genetic differences between rivers by means of AFLPs. Then, we compared the DNA methylation differences between mature and immature parrs, using a Methylation-Sensitive Amplified Polymorphism (MSAP, which is a modification of the AFLPs method by making use of the differential sensitivity of a pair of restriction enzymes isoschizomeres to cytosine methylation. The tissues essayed included brain, liver and gonads. Results AFLPs statistical analysis showed that there was no significant differentiation between rivers or a significant differentiation between maturation states in each river. MSAP statistical analysis showed that among the three tissues sampled, the gonads had the highest number of significant single-locus variation among populations with 74 loci followed by brain with 70 and finally liver with only 12. Principal components analysis (PCA of the MSAP profiles revealed different profiles among different tissues (liver, brain and testis clearly separating maturation states in the testis tissue when compared to the liver. Conclusions Our results reveal that genetically-similar mature and immature salmon parr present high levels of DNA methylation variation in two of the three analyzed tissues. We hypothesize that early maturation may be mostly mediated by epigenetic processes rather than by

ITS2 barcoding DNA region combined with high resolution melting (HRM) analysis of Hyoscyami Semen, the mature seed of Hyoscyamus niger.

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Xiong, Chao; Hu, Zhi-Gang; Tu, Yuan; Liu, He-Gang; Wang, Ping; Zhao, Ming-Ming; SHIi, Yu-Hua; Wu, Lan; Sun, Wei; Chen, Shi-Lin

2016-12-01

Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 domain bound to a palindromic SBE DNA element

International Nuclear Information System (INIS)

Baburajendran, Nithya; Palasingam, Paaventhan; Ng, Calista Keow Leng; Jauch, Ralf; Kolatkar, Prasanna R.

2009-01-01

Crystals of palindromic SBE DNA-bound Smad1 MH1 domain diffracting to 2.7 Å resolution have been obtained. The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 Å resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit

Eccentric localization of catalase to protect chromosomes from oxidative damages during meiotic maturation in mouse oocytes.

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Park, Yong Seok; You, Seung Yeop; Cho, Sungrae; Jeon, Hyuk-Joon; Lee, Sukchan; Cho, Dong-Hyung; Kim, Jae-Sung; Oh, Jeong Su

2016-09-01

The maintenance of genomic integrity and stability is essential for the survival of every organism. Unfortunately, DNA is vulnerable to attack by a variety of damaging agents. Oxidative stress is a major cause of DNA damage because reactive oxygen species (ROS) are produced as by-products of normal cellular metabolism. Cells have developed eloquent antioxidant defense systems to protect themselves from oxidative damage along with aerobic metabolism. Here, we show that catalase (CAT) is present in mouse oocytes to protect the genome from oxidative damage during meiotic maturation. CAT was expressed in the nucleus to form unique vesicular structures. However, after nuclear envelope breakdown, CAT was redistributed in the cytoplasm with particular focus at the chromosomes. Inhibition of CAT activity increased endogenous ROS levels, but did not perturb meiotic maturation. In addition, CAT inhibition produced chromosomal defects, including chromosome misalignment and DNA damage. Therefore, our data suggest that CAT is required not only to scavenge ROS, but also to protect DNA from oxidative damage during meiotic maturation in mouse oocytes.

The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

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Nakane, Shuhei; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2012-11-01

Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER. Copyright © 2012 Elsevier B.V. All rights reserved.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

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Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

2016-01-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

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Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

2016-12-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation.

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Elvira-Matelot, Emilie; Hachet, Mélanie; Shamandi, Nahid; Comella, Pascale; Sáez-Vásquez, Julio; Zytnicki, Matthias; Vaucheret, Hervé

2016-02-01

RNaseIII enzymes catalyze the cleavage of double-stranded RNA (dsRNA) and have diverse functions in RNA maturation. Arabidopsis thaliana RNASE THREE LIKE2 (RTL2), which carries one RNaseIII and two dsRNA binding (DRB) domains, is a unique Arabidopsis RNaseIII enzyme resembling the budding yeast small interfering RNA (siRNA)-producing Dcr1 enzyme. Here, we show that RTL2 modulates the production of a subset of small RNAs and that this activity depends on both its RNaseIII and DRB domains. However, the mode of action of RTL2 differs from that of Dcr1. Whereas Dcr1 directly cleaves dsRNAs into 23-nucleotide siRNAs, RTL2 likely cleaves dsRNAs into longer molecules, which are subsequently processed into small RNAs by the DICER-LIKE enzymes. Depending on the dsRNA considered, RTL2-mediated maturation either improves (RTL2-dependent loci) or reduces (RTL2-sensitive loci) the production of small RNAs. Because the vast majority of RTL2-regulated loci correspond to transposons and intergenic regions producing 24-nucleotide siRNAs that guide DNA methylation, RTL2 depletion modifies DNA methylation in these regions. Nevertheless, 13% of RTL2-regulated loci correspond to protein-coding genes. We show that changes in 24-nucleotide siRNA levels also affect DNA methylation levels at such loci and inversely correlate with mRNA steady state levels, thus implicating RTL2 in the regulation of protein-coding gene expression. © 2016 American Society of Plant Biologists. All rights reserved.

The Effect of Growth Hormone on Lipid Accumulation or Maturation in Adipocytes

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Yuchao Zhang

2016-11-01

Full Text Available Background: Adipogenesis of adipocytes includes two stages: initiation and maturation. Growth hormone (GH secretion is decreased in obese subjects and GH levels are inversely correlated with abdominal fat mass. The effects of growth hormone (GH on lipids accumulation or maturation of adipocytes remains elusive. Methods: In the present study, effect of GH on lipid accumulation in vitro and in vivo was examined. cDNA microarray, quantitative real time-PCR (qPCR and western blotting was used to analyze the expression of genes related to adipocyte lipid accumulation or degradation in pre- or mature 3T3-F442A adipocytes treated with GH and in epididymal adipose tissue of C57BL/6 mice administrated with GH. Level of adiponectin in supernatants of cultured F442A adipocytes was determined by enzyme-linked immune-sorbent assay. Results: We found that in 3T3-F442A especially 6 days post initiation of adipogenesis, GH intervention resulted in decreased expression of adipocyte maturation regulators (C/EBPα, PPARγ and prominent genes related to lipid synthesis such as FAS and FABP, while the expression of UCP1 was markedly enhanced. cDNA microarray analysis and qPCR showed that the expression of SOCS2 and Adipor2 was increased under GH-treatment in mature 3T3-F442A adipocytes. GH treatment increased the mRNA expression of adiponectin and UCP1 in mature adipocytes. The above results were confirmed by in vivo study. Conclusions: GH potentially negatively modulates the maturation and accumulation of lipid in adipocytes.

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange*

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Fenyk, Stepan; Dixon, Christopher H.; Gittens, William H.; Townsend, Philip D.; Sharples, Gary J.; Pålsson, Lars-Olof; Takken, Frank L. W.; Cann, Martin J.

2016-01-01

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. PMID:26601946

The Tomato Nucleotide-binding Leucine-rich Repeat Immune Receptor I-2 Couples DNA-binding to Nucleotide-binding Domain Nucleotide Exchange.

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Fenyk, Stepan; Dixon, Christopher H; Gittens, William H; Townsend, Philip D; Sharples, Gary J; Pålsson, Lars-Olof; Takken, Frank L W; Cann, Martin J

2016-01-15

Plant nucleotide-binding leucine-rich repeat (NLR) proteins enable plants to recognize and respond to pathogen attack. Previously, we demonstrated that the Rx1 NLR of potato is able to bind and bend DNA in vitro. DNA binding in situ requires its genuine activation following pathogen perception. However, it is unknown whether other NLR proteins are also able to bind DNA. Nor is it known how DNA binding relates to the ATPase activity intrinsic to NLR switch function required to immune activation. Here we investigate these issues using a recombinant protein corresponding to the N-terminal coiled-coil and nucleotide-binding domain regions of the I-2 NLR of tomato. Wild type I-2 protein bound nucleic acids with a preference of ssDNA ≈ dsDNA > ssRNA, which is distinct from Rx1. I-2 induced bending and melting of DNA. Notably, ATP enhanced DNA binding relative to ADP in the wild type protein, the null P-loop mutant K207R, and the autoactive mutant S233F. DNA binding was found to activate the intrinsic ATPase activity of I-2. Because DNA binding by I-2 was decreased in the presence of ADP when compared with ATP, a cyclic mechanism emerges; activated ATP-associated I-2 binds to DNA, which enhances ATP hydrolysis, releasing ADP-bound I-2 from the DNA. Thus DNA binding is a general property of at least a subset of NLR proteins, and NLR activation is directly linked to its activity at DNA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fungal mediator tail subunits contain classical transcriptional activation domains.

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Liu, Zhongle; Myers, Lawrence C

2015-04-01

Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Mapping the structural and dynamical features of multiple p53 DNA binding domains: insights into loop 1 intrinsic dynamics.

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Suryani Lukman

Full Text Available The transcription factor p53 regulates cellular integrity in response to stress. p53 is mutated in more than half of cancerous cells, with a majority of the mutations localized to the DNA binding domain (DBD. In order to map the structural and dynamical features of the DBD, we carried out multiple copy molecular dynamics simulations (totaling 0.8 μs. Simulations show the loop 1 to be the most dynamic element among the DNA-contacting loops (loops 1-3. Loop 1 occupies two major conformational states: extended and recessed; the former but not the latter displays correlations in atomic fluctuations with those of loop 2 (~24 Å apart. Since loop 1 binds to the major groove whereas loop 2 binds to the minor groove of DNA, our results begin to provide some insight into the possible mechanism underpinning the cooperative nature of DBD binding to DNA. We propose (1 a novel mechanism underlying the dynamics of loop 1 and the possible tread-milling of p53 on DNA and (2 possible mutations on loop 1 residues to restore the transcriptional activity of an oncogenic mutation at a distant site.

Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

International Nuclear Information System (INIS)

Carr, Stephen B.; Makris, George; Phillips, Simon E. V.; Thomas, Christopher D.

2006-01-01

The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2 1 , diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2 1 2 1 2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism

Site-directed mutational analysis of structural interactions of low molecule compounds binding to the N-terminal 8 kDa domain of DNA polymerase β

International Nuclear Information System (INIS)

Murakami, Shizuka; Kamisuki, Shinji; Takata, Kei-ichi; Kasai, Nobuyuki; Kimura, Seisuke; Mizushina, Yoshiyuki; Ohta, Keisuke; Sugawara, Fumio; Sakaguchi, Kengo

2006-01-01

We previously reported the mode of inhibition of DNA polymerase β (pol. β) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. β interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. 1 H- 15 N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not only were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA

High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

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Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

2015-01-01

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

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Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

2015-02-13

SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of DNA Packaging by Large Double-Stranded DNA Viruses

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Rao, Venigalla B.; Feiss, Michael

2016-01-01

Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead’s portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL’s N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage φ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics. PMID:26958920

The Application of Work Domain Analysis to Defining Australia’s Air Combat Capability

Science.gov (United States)

2014-03-01

Capability. References Brady, A., Naikar, N., & Treadwell, A. (2013). Organisational storytelling with work domain analysis: Case study of air power...UNCLASSIFIED 20 Brady, A., Naikar, N., & Treadwell, A. (2013). Organisational storytelling with work domain analysis: Case study of air power doctrine and...maturity model? A framework of general design principles for maturity model and its demonstration in business process management. Proceedings of the

Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori

Energy Technology Data Exchange (ETDEWEB)

Gardiennet, Carole [Université de Lorraine, CNRS, CRM2, UMR 7036 (France); Wiegand, Thomas [ETH Zurich, Physical Chemistry (Switzerland); Bazin, Alexandre [Université de Lyon 1, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS (France); Cadalbert, Riccardo [ETH Zurich, Physical Chemistry (Switzerland); Kunert, Britta; Lacabanne, Denis [Université de Lyon 1, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS (France); Gutsche, Irina [Université Grenoble Alpes, Institut de Biologie Structurale (IBS), CNRS, IBS, CEA, IBS (France); Terradot, Laurent, E-mail: l.terradot@ibcp.fr [Université de Lyon 1, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); Böckmann, Anja, E-mail: a.bockmann@ibcp.fr [Université de Lyon 1, Molecular Microbiology and Structural Biochemistry, Labex Ecofect, UMR 5086 CNRS (France)

2016-03-15

We here investigate the interactions between the DnaB helicase and the C-terminal domain of the corresponding DnaG primase of Helicobacter pylori using solid-state NMR. The difficult crystallization of this 387 kDa complex, where the two proteins interact in a six to three ratio, is circumvented by simple co-sedimentation of the two proteins directly into the MAS-NMR rotor. While the amount of information that can be extracted from such a large protein is still limited, we can assign a number of amino-acid residues experiencing significant chemical-shift perturbations upon helicase-primase complex formation. The location of these residues is used as a guide to model the interaction interface between the two proteins in the complex. Chemical-shift perturbations also reveal changes at the interaction interfaces of the hexameric HpDnaB assembly on HpDnaG binding. A structural model of the complex that explains the experimental findings is obtained.

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

OpenAIRE

van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

2015-01-01

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand brea...

A Methodological Demonstration of Set-theoretical Approach to Social Media Maturity Models Using Necessary Condition Analysis

DEFF Research Database (Denmark)

Lasrado, Lester Allan; Vatrapu, Ravi; Andersen, Kim Normann

2016-01-01

Despite being widely accepted and applied across research domains, maturity models have been criticized for lacking academic rigor, especially methodologically rigorous and empirically grounded or tested maturity models are quite rare. Attempting to close this gap, we adopt a set-theoretic approach...... and evaluate some of arguments presented by previous conceptual focused social media maturity models....... by applying the Necessary Condition Analysis (NCA) technique to derive maturity stages and stage boundaries conditions. The ontology is to view stages (boundaries) in maturity models as a collection of necessary condition. Using social media maturity data, we demonstrate the strength of our approach...

Genomic DNA methylation-demethylation during aging and reinvigoration of Pinus radiata.

Science.gov (United States)

Fraga, Mario F; Rodríguez, Roberto; Cañal, Maria Jesús

2002-08-01

In animals, DNA methylation is related to gene silencing during ontogenic development. Little is known about DNA methylation in plants, although occasional changes in the DNA methylation state of specific gene promoters have been reported in angiosperms during some developmental processes. We found large differences in the extent of DNA methylation between meristematic areas of juvenile and mature Pinus radiata D. Don. trees, whereas differences in the extent of DNA methylation between differentiated tissues of juvenile and mature trees were small. In meristematic areas, there was a gradual decrease in extent of DNA methylation as the degree of reinvigoration increased. The observed changes in extent of DNA methylation during aging and reinvigoration indicate that reinvigoration could be a consequence of epigenetic modifications opposite in direction to those that occur during aging.

The Responsible Research and Innovation (RRI) Maturity Model

DEFF Research Database (Denmark)

Stahl, Bernd; Obach, Michael; Yaghmaei, Emad

2017-01-01

Responsible research and innovation (RRI) is an approach to research and innovation governance aiming to ensure that research purpose, process and outcomes are acceptable, sustainable and even desirable. In order to achieve this ambitious aim, RRI must be relevant to research and innovation...... in industry. In this paper, we discuss a way of understanding and representing RRI that resonates with private companies and lends itself to practical implementation and action. We propose the development of an RRI maturity model in the tradition of other well-established maturity models, linked...... with a corporate research and development (R&D) process. The foundations of this model lie in the discourse surrounding RRI and selected maturity models from other domains as well as the results of extensive empirical investigation. The model was tested in three industry environments and insights from these case...

Thermodynamics of complex structures formed between single-stranded DNA oligomers and the KH domains of the far upstream element binding protein

Energy Technology Data Exchange (ETDEWEB)

Chakraborty, Kaushik; Sinha, Sudipta Kumar; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)

2016-05-28

The noncovalent interaction between protein and DNA is responsible for regulating the genetic activities in living organisms. The most critical issue in this problem is to understand the underlying driving force for the formation and stability of the complex. To address this issue, we have performed atomistic molecular dynamics simulations of two DNA binding K homology (KH) domains (KH3 and KH4) of the far upstream element binding protein (FBP) complexed with two single-stranded DNA (ss-DNA) oligomers in aqueous media. Attempts have been made to calculate the individual components of the net entropy change for the complexation process by adopting suitable statistical mechanical approaches. Our calculations reveal that translational, rotational, and configurational entropy changes of the protein and the DNA components have unfavourable contributions for this protein-DNA association process and such entropy lost is compensated by the entropy gained due to the release of hydration layer water molecules. The free energy change corresponding to the association process has also been calculated using the Free Energy Perturbation (FEP) method. The free energy gain associated with the KH4–DNA complex formation has been found to be noticeably higher than that involving the formation of the KH3–DNA complex.

Maturation of osteoblast-like SaoS2 induced by carbon nanotubes

International Nuclear Information System (INIS)

Li Xiaoming; Uo, Motohiro; Akasaka, Tsukasa; Abe, Shigeaki; Watari, Fumio; Gao Hong; Sato, Yoshinori; Feng Qingling; Cui Fuzhai

2009-01-01

Osteogenic maturation of the osteoblast is crucial for bone formation. In this study, multi-walled carbon nanotubes (MWCNTs) and graphite (GP) were pressed as compacts. The greater ability of carbon nanotubes to adsorb proteins, compared with graphite, was shown. Human osteoblast-like SaoS2 cells were cultured and the cell response to the two kinds of compacts was compared in vitro. Meanwhile, we used cell culture on the culture plate as a control. Assays for osteonectin, osteopontin and osteocalcin gene expression, total protein (TP) amount, alkaline phosphatase activity (ALP) and DNA of cells cultured on the samples were done. During the conventional culture, significantly higher osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA on carbon nanotubes were found. To confirm the hypothesis that the larger amount of specific proteins adsorbed on the carbon nanotubes was crucial for this, the compacts were pre-soaked in culture medium having additional recombinant human bone morphogenetic protein-2 (rhBMP-2) before cell culture. Compared with GP, osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA of the cells tested increased more on the MWCNTs after the compacts were pre-soaked in the culture medium with rhBMP-2. The results indicated that the carbon nanotubes might induce osteogenic maturation of the osteoblast by adsorbing more specific proteins.

Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery.

Directory of Open Access Journals (Sweden)

Felipe Merino

2015-06-01

Full Text Available Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

Relationship of Structure and Function of DNA-Binding Domain in Vitamin D Receptor

Directory of Open Access Journals (Sweden)

Lin-Yan Wan

2015-07-01

Full Text Available While the structure of the DNA-binding domain (DBD of the vitamin D receptor (VDR has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE, at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR, while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE. For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers–VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110 of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

Mycobacterium smegmatis Ku binds DNA without free ends.

Science.gov (United States)

Kushwaha, Ambuj K; Grove, Anne

2013-12-01

Ku is central to the non-homologous end-joining pathway of double-strand-break repair in all three major domains of life, with eukaryotic homologues being associated with more diversified roles compared with prokaryotic and archaeal homologues. Ku has a conserved central 'ring-shaped' core domain. While prokaryotic homologues lack the N- and C-terminal domains that impart functional diversity to eukaryotic Ku, analyses of Ku from certain prokaryotes such as Pseudomonas aeruginosa and Mycobacterium smegmatis have revealed the presence of distinct C-terminal extensions that modulate DNA-binding properties. We report in the present paper that the lysine-rich C-terminal extension of M. smegmatis Ku contacts the core protein domain as evidenced by an increase in DNA-binding affinity and a decrease in thermal stability and intrinsic tryptophan fluorescence upon its deletion. Ku deleted for this C-terminus requires free DNA ends for binding, but translocates to internal DNA sites. In contrast, full-length Ku can directly bind DNA without free ends, suggesting that this property is conferred by its C-terminus. Such binding to internal DNA sites may facilitate recruitment to sites of DNA damage. The results of the present study also suggest that extensions beyond the shared core domain may have independently evolved to expand Ku function.

Application of Ancient DNA Methods to the Study of the Transatlantic Slave Trade

DEFF Research Database (Denmark)

Sandoval Velasco, Marcela

As one of a limited number of biomolecules recording evolutionary events, DNA provides an unparalleled means of investigating genetic processes. Over three decades, ancient DNA research has matured in many ways, growing alongside technological and methodological advancements. However, due to DNA...... means of investigating genetic processes. Over three decades, ancient DNA research has matured in many ways, growing alongside technological and methodological advancements. However, due to DNA preservation, degradation and contamination, ancient DNA research presents significant limitations...... preservation, degradation and contamination, ancient DNA research presents significant limitations and challenges. Until recently, it was thought that DNA did not survive more than few hundred thousand years, and that it was impossible to retrieve whole genome data from ancient samples preserved under...

Designable DNA-binding domains enable construction of logic circuits in mammalian cells.

Science.gov (United States)

Gaber, Rok; Lebar, Tina; Majerle, Andreja; Šter, Branko; Dobnikar, Andrej; Benčina, Mojca; Jerala, Roman

2014-03-01

Electronic computer circuits consisting of a large number of connected logic gates of the same type, such as NOR, can be easily fabricated and can implement any logic function. In contrast, designed genetic circuits must employ orthogonal information mediators owing to free diffusion within the cell. Combinatorial diversity and orthogonality can be provided by designable DNA- binding domains. Here, we employed the transcription activator-like repressors to optimize the construction of orthogonal functionally complete NOR gates to construct logic circuits. We used transient transfection to implement all 16 two-input logic functions from combinations of the same type of NOR gates within mammalian cells. Additionally, we present a genetic logic circuit where one input is used to select between an AND and OR function to process the data input using the same circuit. This demonstrates the potential of designable modular transcription factors for the construction of complex biological information-processing devices.

MARs Wars: heterogeneity and clustering of DNA-binding domains in the nuclear matrix

Directory of Open Access Journals (Sweden)

Ioudinkova E. S.

2009-12-01

Full Text Available Aim. CO326 is a chicken nuclear scaffold/matrix attachment region (MAR associated with the nuclear matrix in several types of chicken cells. It contains a binding site for a sequence-specific DNA-binding protein, F326. We have studied its interaction with the nuclear matrix. Methods. We have used an in vitro MAR assay with isolated matrices from chicken HD3 cells. Results. We have found that an oligonucleotide binding site for the F326 inhibits binding of the CO326 to the nuclear matrix. At the same time, the binding of heterologous MARs is enhanced. Conclusions. Taken together, these data suggest that there exist several classes of MARs and MAR-binding domains and that the MAR-binding proteins may be clustered in the nuclear matrix.

The escherichia coli chromosome replication initiator protein, DnaA

DEFF Research Database (Denmark)

Nyborg, Malene

The experimental work presented in this thesis involve mutational analysis of the DNA binding domain of the DnaA protein and analysis of the A184V substitution in the ATP area of domain III and other amino acid substitutions found in the DnaA5 and DnaA4G proteins....

An assessment system for the system safety engineering capability maturity model in the case of spent fuel reprocessing

International Nuclear Information System (INIS)

Yang Xiaohua; Liu Zhenghai; Liu Zhiming; Wan Yaping; Bai Xiaofeng

2012-01-01

We can improve the processing, the evaluation of capability and promote the user's trust by using system security engineering capability maturity model (SSE-CMM). SSE-CMM is the common method for organizing and implementing safety engineering, and it is a mature method for system safety engineering. Combining capability maturity model (CMM) with total quality management and statistic theory, SSE-CMM turns systems security engineering into a well-defined, mature, measurable, advanced engineering discipline. Lack of domain knowledge, the size of data, the diversity of evidences, the cumbersomeness of processes, and the complexity of matching evidences with problems are the main issues that SSE-CMM assessment has to face. To improve effectively the efficiency of assessment of spent fuel reprocessing system security engineering capability maturity model (SFR-SSE-CMM), in this paper we de- signed an intelligent assessment software based on domain ontology and that uses methods such as ontology, evidence theory, semantic web, intelligent information retrieval and intelligent auto-matching techniques. This software includes four subsystems, which are domain ontology creation and management system, evidence auto collection system, and a problem and evidence matching system. The architecture of the software is divided into five layers: a data layer, an oncology layer, a knowledge layer, a service layer arid a presentation layer. (authors)

Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

Energy Technology Data Exchange (ETDEWEB)

Carr, Stephen B., E-mail: bmbsbc@bmb.leeds.ac.uk [Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom); Makris, George [Omega Mediation Hellas Ltd, Clinical and Pharma Consulting, 11525 N. Psychiko, Athens (Greece); Phillips, Simon E. V.; Thomas, Christopher D. [Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom)

2006-11-01

The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2{sub 1}, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

Science.gov (United States)

van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

2015-01-01

The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

Comparison of structure, function and regulation of plant cold shock domain proteins to bacterial and animal cold shock domain proteins.

Science.gov (United States)

Chaikam, Vijay; Karlson, Dale T

2010-01-01

The cold shock domain (CSD) is among the most ancient and well conserved nucleic acid binding domains from bacteria to higher animals and plants. The CSD facilitates binding to RNA, ssDNA and dsDNA and most functions attributed to cold shock domain proteins are mediated by this nucleic acid binding activity. In prokaryotes, cold shock domain proteins only contain a single CSD and are termed cold shock proteins (Csps). In animal model systems, various auxiliary domains are present in addition to the CSD and are commonly named Y-box proteins. Similar to animal CSPs, plant CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. Cold shock domain proteins have been shown to play important roles in development and stress adaptation in wide variety of organisms. In this review, the structure, function and regulation of plant CSPs are compared and contrasted to the characteristics of bacterial and animal CSPs. [BMB reports 2010; 43(1): 1-8].

Comparative transcriptome analyses of a late-maturing mandarin mutant and its original cultivar reveals gene expression profiling associated with citrus fruit maturation

Directory of Open Access Journals (Sweden)

Lu Wang

2017-05-01

Full Text Available Characteristics of late maturity in fruit are good agronomic traits for extending the harvest period and marketing time. However, underlying molecular basis of the late-maturing mechanism in fruit is largely unknown. In this study, RNA sequencing (RNA-Seq technology was used to identify differentially expressed genes (DEGs related to late-maturing characteristics from a late-maturing mutant ‘Huawan Wuzishatangju’ (HWWZSTJ (Citrus reticulata Blanco and its original line ‘Wuzishatangju’ (WZSTJ. A total of approximately 17.0 Gb and 84.2 M paried-end reads were obtained. DEGs were significantly enriched in the pathway of photosynthesis, phenylpropanoid biosynthesis, carotenoid biosynthesis, chlorophyll and abscisic acid (ABA metabolism. Thirteen candidate transcripts related to chlorophyll metabolism, carotenoid biosynthesis and ABA metabolism were analyzed using real-time quantitative PCR (qPCR at all fruit maturing stages of HWWZSTJ and WZSTJ. Chlorophyllase (CLH and divinyl reductase (DVR from chlorophyll metabolism, phytoene synthase (PSY and capsanthin/capsorubin synthase (CCS from carotenoid biosynthesis, and abscisic acid 8′-hydroxylase (AB1 and 9-cis-epoxycarotenoid dioxygenase (NCED1 from ABA metabolism were cloned and analyzed. The expression pattern of NCED1 indicated its role in the late-maturing characteristics of HWWZSTJ. There were 270 consecutive bases missing in HWWZSTJ in comparison with full-length sequences of NCED1 cDNA from WZSTJ. Those results suggested that NCED1 might play an important role in the late maturity of HWWZSTJ. This study provides new information on complex process that results in the late maturity of Citrus fruit at the transcriptional level.

Eukaryotic DNA Replication Fork.

Science.gov (United States)

Burgers, Peter M J; Kunkel, Thomas A

2017-06-20

This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and δ carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

Editing of misaligned 3′-termini by an intrinsic 3′–5′ exonuclease activity residing in the PHP domain of a family X DNA polymerase

Science.gov (United States)

Baños, Benito; Lázaro, José M.; Villar, Laurentino; de Vega, Miguel

2008-01-01

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolXBs), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolXBs possesses an intrinsic 3′–5′ exonuclease activity specialized in resecting unannealed 3′-termini in a gapped DNA substrate. Biochemical analysis of a PolXBs deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3′–5′ exonuclease activity of PolXBs resides in its PHP domain. Furthermore, site-directed mutagenesis of PolXBs His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3′-termini resection by the 3′–5′ exonuclease activity of PolXBs in the DNA repair context are discussed. PMID:18776221

Generation of antibodies against disintegrin and cysteine-rich domains by DNA immunization: An approach to neutralize snake venom-induced haemorrhage

Directory of Open Access Journals (Sweden)

Sidgi Syed Anwer Abdo Hasson

2017-03-01

Conclusions: Antibodies generated against the E. ocellatus venom prothrombin activator-like metalloprotease and disintegrin-cysteine-rich domains modulated and inhibited the catalytic activity both in vitro and in vivo of venom metalloproteinase disintegrin cysteine rich molecules. Thus, generating of venom specific-toxin antibodies by DNA immunization offer a more rational treatment of snake envenoming than conventional antivenom.

Targeted DNA demethylation of the Arabidopsis genome using the human TET1 catalytic domain

Science.gov (United States)

Gallego-Bartolomé, Javier; Gardiner, Jason; Liu, Wanlu; Papikian, Ashot; Ghoshal, Basudev; Kuo, Hsuan Yu; Zhao, Jenny Miao-Chi; Jacobsen, Steven E.

2018-01-01

DNA methylation is an important epigenetic modification involved in gene regulation and transposable element silencing. Changes in DNA methylation can be heritable and, thus, can lead to the formation of stable epialleles. A well-characterized example of a stable epiallele in plants is fwa, which consists of the loss of DNA cytosine methylation (5mC) in the promoter of the FLOWERING WAGENINGEN (FWA) gene, causing up-regulation of FWA and a heritable late-flowering phenotype. Here we demonstrate that a fusion between the catalytic domain of the human demethylase TEN-ELEVEN TRANSLOCATION1 (TET1cd) and an artificial zinc finger (ZF) designed to target the FWA promoter can cause highly efficient targeted demethylation, FWA up-regulation, and a heritable late-flowering phenotype. Additional ZF–TET1cd fusions designed to target methylated regions of the CACTA1 transposon also caused targeted demethylation and changes in expression. Finally, we have developed a CRISPR/dCas9-based targeted demethylation system using the TET1cd and a modified SunTag system. Similar to the ZF–TET1cd fusions, the SunTag–TET1cd system is able to target demethylation and activate gene expression when directed to the FWA or CACTA1 loci. Our study provides tools for targeted removal of 5mC at specific loci in the genome with high specificity and minimal off-target effects. These tools provide the opportunity to develop new epialleles for traits of interest, and to reactivate expression of previously silenced genes, transgenes, or transposons. PMID:29444862

Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.

Science.gov (United States)

Shizu, Ryota; Min, Jungki; Sobhany, Mack; Pedersen, Lars C; Mutoh, Shingo; Negishi, Masahiko

2018-01-05

The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

A novel heavy domain antibody library with functionally optimized complementarity determining regions.

Directory of Open Access Journals (Sweden)

Ole Aalund Mandrup

Full Text Available Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.

Birth of scale-free molecular networks and the number of distinct DNA and protein domains per genome.

Science.gov (United States)

Rzhetsky, A; Gomez, S M

2001-10-01

Current growth in the field of genomics has provided a number of exciting approaches to the modeling of evolutionary mechanisms within the genome. Separately, dynamical and statistical analyses of networks such as the World Wide Web and the social interactions existing between humans have shown that these networks can exhibit common fractal properties-including the property of being scale-free. This work attempts to bridge these two fields and demonstrate that the fractal properties of molecular networks are linked to the fractal properties of their underlying genomes. We suggest a stochastic model capable of describing the evolutionary growth of metabolic or signal-transduction networks. This model generates networks that share important statistical properties (so-called scale-free behavior) with real molecular networks. In particular, the frequency of vertices connected to exactly k other vertices follows a power-law distribution. The shape of this distribution remains invariant to changes in network scale: a small subgraph has the same distribution as the complete graph from which it is derived. Furthermore, the model correctly predicts that the frequencies of distinct DNA and protein domains also follow a power-law distribution. Finally, the model leads to a simple equation linking the total number of different DNA and protein domains in a genome with both the total number of genes and the overall network topology. MatLab (MathWorks, Inc.) programs described in this manuscript are available on request from the authors. ar345@columbia.edu.

Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression

DEFF Research Database (Denmark)

Beloin, C.; Valle, J.; Latour-Lambert, P.

2004-01-01

The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K-12 biofilm. We show that, when biofilm is compared...... that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological...

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

International Nuclear Information System (INIS)

Yun, Ji-Hye; Lee, Won Kyung; Kim, Heeyoun; Kim, Eunhee; Cheong, Chaejoon; Cho, Myeon Haeng; Lee, Weontae

2014-01-01

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2 1–64 ) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2 1–64 and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences

Molecular dynamics simulations of DNA-free and DNA-bound TAL effectors.

Directory of Open Access Journals (Sweden)

Hua Wan

Full Text Available TAL (transcriptional activator-like effectors (TALEs are DNA-binding proteins, containing a modular central domain that recognizes specific DNA sequences. Recently, the crystallographic studies of TALEs revealed the structure of DNA-recognition domain. In this article, molecular dynamics (MD simulations are employed to study two crystal structures of an 11.5-repeat TALE, in the presence and absence of DNA, respectively. The simulated results indicate that the specific binding of RVDs (repeat-variable diresidues with DNA leads to the markedly reduced fluctuations of tandem repeats, especially at the two ends. In the DNA-bound TALE system, the base-specific interaction is formed mainly by the residue at position 13 within a TAL repeat. Tandem repeats with weak RVDs are unfavorable for the TALE-DNA binding. These observations are consistent with experimental studies. By using principal component analysis (PCA, the dominant motions are open-close movements between the two ends of the superhelical structure in both DNA-free and DNA-bound TALE systems. The open-close movements are found to be critical for the recognition and binding of TALE-DNA based on the analysis of free energy landscape (FEL. The conformational analysis of DNA indicates that the 5' end of DNA target sequence has more remarkable structural deformability than the other sites. Meanwhile, the conformational change of DNA is likely associated with the specific interaction of TALE-DNA. We further suggest that the arrangement of N-terminal repeats with strong RVDs may help in the design of efficient TALEs. This study provides some new insights into the understanding of the TALE-DNA recognition mechanism.

The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.

Directory of Open Access Journals (Sweden)

Audrey Costes

2010-12-01

Full Text Available We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSB(Cter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSB(Cter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.

Genomic DNA extraction from sapwood of Pinus roxburghii for ...

African Journals Online (AJOL)

A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, where collection of needle tissues is extremely difficult has been standardized. The extracted DNA was comparable to that obtained from the needle tissue in terms of yield and purity. The yield of extracted DNA ranged ...

E-learning process maturity level: a conceptual framework

Science.gov (United States)

Rahmah, A.; Santoso, H. B.; Hasibuan, Z. A.

2018-03-01

ICT advancement is a sure thing with the impact influencing many domains, including learning in both formal and informal situations. It leads to a new mindset that we should not only utilize the given ICT to support the learning process, but also improve it gradually involving a lot of factors. These phenomenon is called e-learning process evolution. Accordingly, this study attempts to explore maturity level concept to provide the improvement direction gradually and progression monitoring for the individual e-learning process. Extensive literature review, observation, and forming constructs are conducted to develop a conceptual framework for e-learning process maturity level. The conceptual framework consists of learner, e-learning process, continuous improvement, evolution of e-learning process, technology, and learning objectives. Whilst, evolution of e-learning process depicted as current versus expected conditions of e-learning process maturity level. The study concludes that from the e-learning process maturity level conceptual framework, it may guide the evolution roadmap for e-learning process, accelerate the evolution, and decrease the negative impact of ICT. The conceptual framework will be verified and tested in the future study.

A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

International Nuclear Information System (INIS)

Ohsaki, Eriko; Ueda, Keiji

2017-01-01

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. - Highlights: •KSHV replication in latency depends on LANA localization to the nuclear matrix. •LANA DBD was fused with NuMA, a nuclear matrix protein, at the N- and C-terminus. •NuMA-DBD was in the nuclear matrix and supported the ori-P dependent replication. •LANA in the nuclear matrix should be important for the KSHV replication in latency.

A chimeric protein composed of NuMA fused to the DNA binding domain of LANA is sufficient for the ori-P-dependent DNA replication

Energy Technology Data Exchange (ETDEWEB)

Ohsaki, Eriko; Ueda, Keiji, E-mail: kueda@virus.med.osaka-u.ac.jp

2017-01-15

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome is stably maintained in KSHV-infected PEL cell lines during cell division. We previously showed that accumulation of LANA in the nuclear matrix fraction could be important for the latent DNA replication, and that the functional significance of LANA should be its recruitment of ori-P to the nuclear matrix. Here, we investigated whether the forced localization of the LANA-DNA binding domain (DBD) to the nuclear matrix facilitated ori-P-containing plasmid replication. We demonstrated that chimeric proteins constructed by fusion of LANA DBD with the nuclear mitotic apparatus protein (NuMA), which is one of the components of the nuclear matrix, could bind with ori-P and enhance replication of an ori-P-containing plasmid, compared with that in the presence of DBD alone. These results further suggested that the ori-P recruitment to the nuclear matrix through the binding with DBD is important for latent viral DNA replication. - Highlights: •KSHV replication in latency depends on LANA localization to the nuclear matrix. •LANA DBD was fused with NuMA, a nuclear matrix protein, at the N- and C-terminus. •NuMA-DBD was in the nuclear matrix and supported the ori-P dependent replication. •LANA in the nuclear matrix should be important for the KSHV replication in latency.

Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

Science.gov (United States)

Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q.; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M.

2014-01-01

Summary Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi, and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1 and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a new pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

Science.gov (United States)

Kim, Yucheol; De Zoysa, Mahanama; Lee, Youngdeuk; Whang, Ilson; Lee, Jehee

2010-11-01

A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone. Copyright 2010 Elsevier Ltd. All rights reserved.

Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

Science.gov (United States)

Paliwal, Anupam; Temkin, Alexis M; Kerkel, Kristi; Yale, Alexander; Yotova, Iveta; Drost, Natalia; Lax, Simon; Nhan-Chang, Chia-Ling; Powell, Charles; Borczuk, Alain; Aviv, Abraham; Wapner, Ronald; Chen, Xiaowei; Nagy, Peter L; Schork, Nicholas; Do, Catherine; Torkamani, Ali; Tycko, Benjamin

2013-08-01

Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.

Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

Directory of Open Access Journals (Sweden)

Anupam Paliwal

2013-08-01

Full Text Available Allele-specific DNA methylation (ASM is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons, one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs, each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS peaks near CTCF binding sites with ASM.

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

KAUST Repository

Zaher, Manal S.; Rashid, Fahad; Song, Bo; Joudeh, Luay I; Sobhy, Mohamed Abdelmaboud; Tehseen, Muhammad; Hingorani, Manju M; Hamdan, Samir

2018-01-01

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Missed cleavage opportunities by FEN1 lead to Okazaki fragment maturation via the long-flap pathway

KAUST Repository

Zaher, Manal S.

2018-01-27

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5\\'-single-stranded flap is cleaved by structure-specific 5\\'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Polyphenolics free DNA isolation and optimization of PCR-RAPD for ...

African Journals Online (AJOL)

, and good quality high molecular weight DNA samples. DNA was isolated from the mature and fresh young tender leaves obtained from various Italian wild populations of fennel. We performed a modified cetyl trimethyl ammonium bromide ...

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

Energy Technology Data Exchange (ETDEWEB)

Wu, Hong; Zeng, Hong; Lam, Robert; Tempel, Wolfram; Amaya, Maria F.; Xu, Chao; Dombrovski, Ludmila; Qiu, Wei; Wang, Yanming; Min, Jinrong (Toronto); (Penn)

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.

Solution structure of telomere binding domain of AtTRB2 derived from Arabidopsis thaliana

Energy Technology Data Exchange (ETDEWEB)

Yun, Ji-Hye [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Won Kyung [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Heeyoun [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Eunhee; Cheong, Chaejoon [Magnetic Resonance Team, Korea Basic Science Institute (KBSI), Ochang, Chungbuk 363-883 (Korea, Republic of); Cho, Myeon Haeng [Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Lee, Weontae, E-mail: wlee@spin.yonsei.ac.kr [Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

2014-09-26

Highlights: • We have determined solution structure of Myb domain of AtTRB2. • The Myb domain of AtTRB2 is located in the N-terminal region. • The Myb domain of AtTRB2 binds to plant telomeric DNA without fourth helix. • Helix 2 and 3 of the Myb domain of AtTRB2 are involved in DNA recognition. • AtTRB2 is a novel protein distinguished from other known plant TBP. - Abstract: Telomere homeostasis is regulated by telomere-associated proteins, and the Myb domain is well conserved for telomere binding. AtTRB2 is a member of the SMH (Single-Myb-Histone)-like family in Arabidopsis thaliana, having an N-terminal Myb domain, which is responsible for DNA binding. The Myb domain of AtTRB2 contains three α-helices and loops for DNA binding, which is unusual given that other plant telomere-binding proteins have an additional fourth helix that is essential for DNA binding. To understand the structural role for telomeric DNA binding of AtTRB2, we determined the solution structure of the Myb domain of AtTRB2 (AtTRB2{sub 1–64}) using nuclear magnetic resonance (NMR) spectroscopy. In addition, the inter-molecular interaction between AtTRB2{sub 1–64} and telomeric DNA has been characterized by the electrophoretic mobility shift assay (EMSA) and NMR titration analyses for both plant (TTTAGGG)n and human (TTAGGG)n telomere sequences. Data revealed that Trp28, Arg29, and Val47 residues located in Helix 2 and Helix 3 are crucial for DNA binding, which are well conserved among other plant telomere binding proteins. We concluded that although AtTRB2 is devoid of the additional fourth helix in the Myb-extension domain, it is able to bind to plant telomeric repeat sequences as well as human telomeric repeat sequences.

Cellular response to DNA damage. Link between p53 and DNA-PK

International Nuclear Information System (INIS)

Salles-Passador, I.; Fotedar, R.; Fotedar, A.

1999-01-01

Cells which lack DNA-activated protein kinase (DNA-PK) are very susceptible to ionizing radiation and display an inability to repair double-strand DNA breaks. DNA-PK is a member of a protein kinase family that includes ATR and ATM which have strong homology in their carboxy-terminal kinase domain with Pl-3 kinase. ATM has been proposed to act upstream of p53 in cellular response to ionizing radiation. DNA-PK may similarly interact with p53 in cellular growth control and in mediation of the response to ionizing radiation. (author)

The DnaJ-like zinc finger domain protein PSA2 affects light acclimation and chloroplast development in Arabidopsis thaliana

Directory of Open Access Journals (Sweden)

Yan-Wen eWang

2016-03-01

Full Text Available The biosynthesis of chlorophylls and carotenoids and the assembly of thylakoid membranes are critical for the photoautotrophic growth of plants. Different factors are involved in these two processes. In recent years, members of the DnaJ-like zinc finger domain proteins have been found to take part in the biogenesis and/or the maintenance of plastids. One member of this family of proteins, PSA2, was recently found to localize to the thylakoid lumen and regulate the accumulation of photosystem I. In this study, we report that the silencing of PSA2 in Arabidopsis thaliana resulted in variegated leaves and retarded growth. Although both chlorophylls and total carotenoids decreased in the psa2 mutant, violaxanthin and zeaxanthin accumulated in the mutant seedlings grown under growth condition. Lower levels of non-photochemical quenching and electron transport rate were also found in the psa2 mutant seedlings under growth condition compared with those of the wild-type plants, indicating an impaired capability to acclimate to normal light irradiance when PSA2 was silenced. Moreover, we also observed an abnormal assembly of grana thylakoids and poorly developed stroma thylakoids in psa2 chloroplasts. Taken together, our results demonstrate that PSA2 is a member of the DnaJ-like zinc finger domain protein family that affects light acclimation and chloroplast development.

Correlation between dental maturity and cervical vertebral maturity.

Science.gov (United States)

Chen, Jianwei; Hu, Haikun; Guo, Jing; Liu, Zeping; Liu, Renkai; Li, Fan; Zou, Shujuan

2010-12-01

The aim of this study was to investigate the association between dental and skeletal maturity. Digital panoramic radiographs and lateral skull cephalograms of 302 patients (134 boys and 168 girls, ranging from 8 to 16 years of age) were examined. Dental maturity was assessed by calcification stages of the mandibular canines, first and second premolars, and second molars, whereas skeletal maturity was estimated by the cervical vertebral maturation (CVM) stages. The Spearman rank-order correlation coefficient was used to measure the association between CVM stage and dental calcification stage of individual teeth. The mean chronologic age of girls was significantly lower than that of boys in each CVM stage. The Spearman rank-order correlation coefficients between dental maturity and cervical vertebral maturity ranged from 0.391 to 0.582 for girls and from 0.464 to 0.496 for boys (P cervical vertebral maturation stage. The development of the mandibular second molar in females and that of the mandibular canine in males had the strongest correlations with cervical vertebral maturity. Therefore, it is practical to consider the relationship between dental and skeletal maturity when planning orthodontic treatment. Copyright © 2010 Mosby, Inc. All rights reserved.

RADX interacts with single-stranded DNA to promote replication fork stability

DEFF Research Database (Denmark)

Schubert, Lisa; Ho, Teresa; Hoffmann, Saskia

2017-01-01

Single-stranded DNA (ssDNA) regions form as an intermediate in many DNA-associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide-binding (OB) fold domain. The heterotrimeric, multi-OB fold domain-containing Replication Protein A (RPA) complex...... ssDNA-binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA-binding proteins....

Participation of ATM in cellular response to DNA damage induced by ionizing radiation

International Nuclear Information System (INIS)

Meng Xiangbing; Song Yi; Mao Jianping; Gong Bo; Dong Yan; Liu Bin; Sun Zhixian

2000-01-01

Objective: To clone ATM full length cDNA and cDNA fragments containing some functional domains and to identify proteins that interact with ATM and mediate DNA damage signal transduction in cellular response to DNA damage. Methods: ATM cDNA was amplified from MarthomTM-Ready cDNA kit of human leukocytes by LD-PCR. ATM-interacting proteins were screened by yeast two hybrid system. Results: ATM full-length cDNA and cDNA fragments containing PI3K kinase domain, leucine zipper and proline rich region were amplified from human cDNAs. Several candidate clones that interacted with ATM PI3K domain were identified. Conclusion: ATM mediates DNA damage signal transduction by interacting with many proteins

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.

Science.gov (United States)

Trejo, Sebastián A; López, Laura M I; Caffini, Néstor O; Natalucci, Claudia L; Canals, Francesc; Avilés, Francesc X

2009-07-01

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

Far-infrared vibrational modes of DNA components studied by terahertz time-domain spectroscopy

International Nuclear Information System (INIS)

Fischer, B M; Walther, M; Jepsen, P Uhd

2002-01-01

The far-infrared dielectric function of a wide range of organic molecules is dominated by vibrations involving a substantial fraction of the atoms forming the molecule and motion associated with intermolecular hydrogen bond vibrations. Due to their collective nature such modes are highly sensitive to the intra- and intermolecular structure and thus provide a unique fingerprint of the conformational state of the molecule and effects of its environment. We demonstrate the use of terahertz time-domain spectroscopy (THz-TDS) for recording the far-infrared (0.5-4.0 THz) dielectric function of the four nucleobases and corresponding nucleosides forming the building blocks of deoxyribose nucleic acid (DNA). We observe numerous distinct spectral features with large differences between the molecules in both frequency-dependent absorption coefficient and index of refraction. Assisted by results from density-functional calculations we interpret the origin of the observed resonances as vibrations of hydrogen bonds between the molecules

Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea.

Science.gov (United States)

Petitjean, Céline; Moreira, David; López-García, Purificación; Brochier-Armanet, Céline

2012-11-26

In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer) domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants). Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs) to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

Mutations of amino acids in the DNA-recognition domain of Epstein-Barr virus ZEBRA protein alter its sub-nuclear localization and affect formation of replication compartments

International Nuclear Information System (INIS)

Park, Richard; Heston, Lee; Shedd, Duane; Delecluse, Henri-Jacques; Miller, George

2008-01-01

foci. The speckled appearance of R179A and Y180E was more regular and clearly defined in EBV-positive than in EBV-negative 293 cells. The Y180E late-mutant induced EA-D, but prevented EA-D from localizing to globular replication compartments. These results show that individual amino acids within the basic domain influence localization of the ZEBRA protein and its capacity to induce EA-D to become located in mature viral replication compartments. Furthermore, these mutant ZEBRA proteins delineate several stages in the processes of nuclear re-organization which accompany lytic EBV replication

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

Science.gov (United States)

Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

2015-01-01

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation. PMID:25934799

Conformational Dynamics of Thermus aquaticus DNA Polymerase I during Catalysis

Science.gov (United States)

Suo, Zucai

2014-01-01

Despite the fact that DNA polymerases have been investigated for many years and are commonly used as tools in a number of molecular biology assays, many details of the kinetic mechanism they use to catalyze DNA synthesis remain unclear. Structural and kinetic studies have characterized a rapid, pre-catalytic open-to-close conformational change of the Finger domain during nucleotide binding for many DNA polymerases including Thermus aquaticus DNA polymerase I (Taq Pol), a thermostable enzyme commonly used for DNA amplification in PCR. However, little has been done to characterize the motions of other structural domains of Taq Pol or any other DNA polymerase during catalysis. Here, we used stopped-flow Förster resonance energy transfer (FRET) to investigate the conformational dynamics of all five structural domains of the full-length Taq Pol relative to the DNA substrate during nucleotide binding and incorporation. Our study provides evidence for a rapid conformational change step induced by dNTP binding and a subsequent global conformational transition involving all domains of Taq Pol during catalysis. Additionally, our study shows that the rate of the global transition was greatly increased with the truncated form of Taq Pol lacking the N-terminal domain. Finally, we utilized a mutant of Taq Pol containing a de novo disulfide bond to demonstrate that limiting protein conformational flexibility greatly reduced the polymerization activity of Taq Pol. PMID:24931550

Chimeric TALE recombinases with programmable DNA sequence specificity.

Science.gov (United States)

Mercer, Andrew C; Gaj, Thomas; Fuller, Roberta P; Barbas, Carlos F

2012-11-01

Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.

The Relationship between Defense Patterns and DSM-5 Maladaptive Personality Domains

Science.gov (United States)

Granieri, Antonella; La Marca, Luana; Mannino, Giuseppe; Giunta, Serena; Guglielmucci, Fanny; Schimmenti, Adriano

2017-01-01

Aim: Research has extensively examined the relationship between defense mechanisms (DM) and personality traits. However, no study to date has explored if specific defenses (alone or in combination) are able to predict dysfunctional variants of personality domains, as conceived in the alternative DSM-5 model for personality disorders. This study aimed to investigate the relationship between DMs and DSM-5 maladaptive personality domains among adults. Materials and Methods: Three hundred and twenty-eight adults aged between 18 and 64 years old completed measures on DMs and maladapive personality domains. Regression analyses were performed to determine which DMs predicted the maladaptive personality domains of negative affectivity, detachment, antagonism, disinhibition, and psychoticism. Results: According to psychoanalytic literature, results showed that immature defenses positively predicted maladaptive personality domain scores, whereas mature defenses were generally related with better personality functioning. Moreover, different defense patterns emerged as significant predictors of the maladaptive personality domains comprised in the alternative DSM-5 model for personality disorder. Discussion: Our findings support the view that defense patterns represent core components of personality and its disorders, and suggest that an increased use of immature defenses and a reduced use of mature defenses have a negative impact on the development of personality. PMID:29163301

DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.

Science.gov (United States)

Zheng, Tao; Jiang, Pan; Cao, Bo; Cheng, Qiuxiang; Kong, Lingxin; Zheng, Xiaoqing; Hu, Qinghai; You, Delin

2016-01-15

Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared with canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as a DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e. the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strand separation and intense ATP hydrolysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

RPA-1 from Leishmania amazonensis (LaRPA-1) structurally differs from other eukaryote RPA-1 and interacts with telomeric DNA via its N-terminal OB-fold domain.

Science.gov (United States)

Pavani, R S; Fernandes, C; Perez, A M; Vasconcelos, E J R; Siqueira-Neto, J L; Fontes, M R; Cano, M I N

2014-12-20

Replication protein A-1 (RPA-1) is a single-stranded DNA-binding protein involved in DNA metabolism. We previously demonstrated the interaction between LaRPA-1 and telomeric DNA. Here, we expressed and purified truncated mutants of LaRPA-1 and used circular dichroism measurements and molecular dynamics simulations to demonstrate that the tertiary structure of LaRPA-1 differs from human and yeast RPA-1. LaRPA-1 interacts with telomeric ssDNA via its N-terminal OB-fold domain, whereas RPA from higher eukaryotes show different binding modes to ssDNA. Our results show that LaRPA-1 is evolutionary distinct from other RPA-1 proteins and can potentially be used for targeting trypanosomatid telomeres. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

cDNA cloning of a snake venom metalloproteinase from the eastern diamondback rattlesnake (Crotalus adamanteus), and the expression of its disintegrin domain with anti-platelet effects

Science.gov (United States)

Suntravat, Montamas; Jia, Ying; Lucena, Sara E.; Sánchez, Elda E.; Pérez, John C.

2013-01-01

A 5′ truncated snake venom metalloproteinase was identified from a cDNA library constructed from venom glands of an eastern diamondback rattlesnake (Crotalus adamanteus). The 5′-rapid amplification of cDNA ends (RACE) was used to obtain the 1865 bp full-length cDNA sequence of a snake venom metalloproteinase (CamVMPII). CamVMPII encodes an open reading frame of 488 amino acids, which includes a signal peptide, a pro-domain, a metalloproteinase domain, a spacer, and an RGD-disintegrin domain. The predicted amino acid sequence of CamVMPII showed a 91%, 90%, 83%, and 82% sequence homology to the P-II class enzymes of C. adamanteus metalloproteinase 2, C. atrox CaVMP-II, Gloydius halys agkistin, and Protobothrops jerdonii jerdonitin, respectively. Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. Therefore, the disintegrin domain (Cam-dis) of CamVMPII was amplified by PCR, cloned into a pET-43.1a vector, and expressed in Escherichia coli BL21. Affinity purified recombinantly modified Cam-dis (r-Cam-dis) with a yield of 8.5 mg/L culture medium was cleaved from the fusion tags by enterokinase cleavage. r-Cam-dis was further purified by two-step chromatography consisting of HiTrap™ Benzamidine FF column, followed by Talon Metal affinity column with a final yield of 1 mg/L culture. r-Cam-dis was able to inhibit all three processes of platelet thrombus formation including platelet adhesion with an estimated IC50 of 1 nM, collagen- and ADP-induced platelet aggregation with the estimated IC50s of 18 and 6 nM, respectively, and platelet function on clot retraction. It is a potent anti-platelet inhibitor, which should be further investigated for drug discovery to treat stroke patients or patients with thrombotic disorders. PMID:23313448

The C-terminal domain of the bacteriophage T4 terminase docks on the prohead portal clip region during DNA packaging

Science.gov (United States)

Dixit, Aparna Banerjee; Ray, Krishanu; Thomas, Julie A.; Black, Lindsay W.

2013-01-01

Bacteriophage ATP-based packaging motors translocate DNA into a pre-formed prohead through a dodecameric portal ring channel to high density. We investigated portal–terminase docking interactions at specifically localized residues within a terminase-interaction region (aa279–316) in the phage T4 portal protein gp20 equated to the clip domain of the SPP1 portal crystal structure by 3D modeling. Within this region, three residues allowed A to C mutations whereas three others did not, consistent with informatics analyses showing the tolerated residues are not strongly conserved evolutionarily. About 7.5 nm was calculated by FCS-FRET studies employing maleimide Alexa488 dye labeled A316C proheads and gp17 CT-ReAsH supporting previous work docking the C-terminal end of the T4 terminase (gp17) closer to the N-terminal GFP-labeled portal (gp20) than the N-terminal end of the terminase. Such a terminase–portal orientation fits better to a proposed “DNA crunching” compression packaging motor and to portal determined DNA headful cutting. PMID:24074593

Maturity and maturity models in lean construction

Directory of Open Access Journals (Sweden)

Claus Nesensohn

2014-03-01

Full Text Available In recent years there has been an increasing interest in maturity models in management-related disciplines; which reflects a growing recognition that becoming more mature and having a model to guide the route to maturity can help organisations in managing major transformational change. Lean Construction (LC is an increasingly important improvement approach that organisations seek to embed. This study explores how to apply the maturity models to LC. Hence the attitudes, opinions and experiences of key industry informants with high levels of knowledge of LC were investigated. To achieve this, a review of maturity models was conducted, and data for the analysis was collected through a sequential process involving three methods. First a group interview with seven key informants. Second a follow up discussion with the same individuals to investigate some of the issues raised in more depth. Third an online discussion held via LinkedIn in which members shared their views on some of the results. Overall, we found that there is a lack of common understanding as to what maturity means in LC, though there is general agreement that the concept of maturity is a suitable one to reflect the path of evolution for LC within organisations.

Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes.

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Monica Poggianella

Full Text Available Dengue virus (DENV infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well.

Genomic DNA extraction from sapwood of Pinus roxburghii for ...

African Journals Online (AJOL)

Ashish

2013-02-22

Feb 22, 2013 ... A method for extraction of genomic DNA from sapwood tissues of mature tall trees of Pinus roxburghii, .... DNA as a template. PCR was performed on a thermal cycler. (Biorad, Mycycler) incorporating 10 ng genomic DNA to a 25 µl reaction mix containing 1X Taq buffer, 3 mM MgCl2, 0.2 mM each of dNTPs ...

Directed evolution of the TALE N-terminal domain for recognition of all 5' bases.

Science.gov (United States)

Lamb, Brian M; Mercer, Andrew C; Barbas, Carlos F

2013-11-01

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5'-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5' T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5' T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes.

Promoting the Affective Domain within Online Education

Science.gov (United States)

Roche, Stephen H.

2013-01-01

In the past decade Higher Education Institutions have experienced tremendous growth in enrollments. To meet this demand, many higher education institutions have embraced online education and its requisite technologies. Online education has matured, and studies focusing on the cognitive domain indicate that distance education is as effective as the…

Structure-based design of a disulfide-linked oligomeric form of the simian virus 40 (SV40) large T antigen DNA-binding domain

International Nuclear Information System (INIS)

Meinke, Gretchen; Phelan, Paul; Fradet-Turcotte, Amélie; Archambault, Jacques; Bullock, Peter A.

2011-01-01

With the aim of forming the ‘lock-washer’ conformation of the origin-binding domain of SV40 large T antigen in solution, using structure-based analysis an intermolecular disulfide bridge was engineered into the origin-binding domain to generate higher order oligomers in solution. The 1.7 Å resolution structure shows that the mutant forms a spiral in the crystal and has the de novo disulfide bond at the protein interface, although structural rearrangements at the interface are observed relative to the wild type. The modular multifunctional protein large T antigen (T-ag) from simian virus 40 orchestrates many of the events needed for replication of the viral double-stranded DNA genome. This protein assembles into single and double hexamers on specific DNA sequences located at the origin of replication. This complicated process begins when the origin-binding domain of large T antigen (T-ag ODB) binds the GAGGC sequences in the central region (site II) of the viral origin of replication. While many of the functions of purified T-ag OBD can be studied in isolation, it is primarily monomeric in solution and cannot assemble into hexamers. To overcome this limitation, the possibility of engineering intermolecular disulfide bonds in the origin-binding domain which could oligomerize in solution was investigated. A recent crystal structure of the wild-type T-ag OBD showed that this domain forms a left-handed spiral in the crystal with six subunits per turn. Therefore, we analyzed the protein interface of this structure and identified two residues that could potentially support an intermolecular disulfide bond if changed to cysteines. SDS–PAGE analysis established that the mutant T-ag OBD formed higher oligomeric products in a redox-dependent manner. In addition, the 1.7 Å resolution crystal structure of the engineered disulfide-linked T-ag OBD is reported, which establishes that oligomerization took place in the expected manner

Horizontal gene transfer of a chloroplast DnaJ-Fer protein to Thaumarchaeota and the evolutionary history of the DnaK chaperone system in Archaea

Directory of Open Access Journals (Sweden)

Petitjean Céline

2012-11-01

Full Text Available Abstract Background In 2004, we discovered an atypical protein in metagenomic data from marine thaumarchaeotal species. This protein, referred as DnaJ-Fer, is composed of a J domain fused to a Ferredoxin (Fer domain. Surprisingly, the same protein was also found in Viridiplantae (green algae and land plants. Because J domain-containing proteins are known to interact with the major chaperone DnaK/Hsp70, this suggested that a DnaK protein was present in Thaumarchaeota. DnaK/Hsp70, its co-chaperone DnaJ and the nucleotide exchange factor GrpE are involved, among others, in heat shocks and heavy metal cellular stress responses. Results Using phylogenomic approaches we have investigated the evolutionary history of the DnaJ-Fer protein and of interacting proteins DnaK, DnaJ and GrpE in Thaumarchaeota. These proteins have very complex histories, involving several inter-domain horizontal gene transfers (HGTs to explain the contemporary distribution of these proteins in archaea. These transfers include one from Cyanobacteria to Viridiplantae and one from Viridiplantae to Thaumarchaeota for the DnaJ-Fer protein, as well as independent HGTs from Bacteria to mesophilic archaea for the DnaK/DnaJ/GrpE system, followed by HGTs among mesophilic and thermophilic archaea. Conclusions We highlight the chimerical origin of the set of proteins DnaK, DnaJ, GrpE and DnaJ-Fer in Thaumarchaeota and suggest that the HGT of these proteins has played an important role in the adaptation of several archaeal groups to mesophilic and thermophilic environments from hyperthermophilic ancestors. Finally, the evolutionary history of DnaJ-Fer provides information useful for the relative dating of the diversification of Archaeplastida and Thaumarchaeota.

Signatures of DNA target selectivity by ETS transcription factors.

Science.gov (United States)

Poon, Gregory M K; Kim, Hye Mi

2017-05-27

The ETS family of transcription factors is a functionally heterogeneous group of gene regulators that share a structurally conserved, eponymous DNA-binding domain. DNA target specificity derives from combinatorial interactions with other proteins as well as intrinsic heterogeneity among ETS domains. Emerging evidence suggests molecular hydration as a fundamental feature that defines the intrinsic heterogeneity in DNA target selection and susceptibility to epigenetic DNA modification. This perspective invokes novel hypotheses in the regulation of ETS proteins in physiologic osmotic stress, their pioneering potential in heterochromatin, and the effects of passive and pharmacologic DNA demethylation on ETS regulation.

Different Binding Properties and Function of CXXC Zinc Finger Domains in Dnmt1 and Tet1

Science.gov (United States)

Meilinger, Daniela; Bultmann, Sebastian; Fellinger, Karin; Hasenöder, Stefan; Wang, Mengxi; Qin, Weihua; Söding, Johannes; Spada, Fabio; Leonhardt, Heinrich

2011-01-01

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1−/− embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1−/− ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites. PMID:21311766

Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1.

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Carina Frauer

2011-02-01

Full Text Available Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1⁻/⁻ embryonic stem cells (ESCs just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1⁻/⁻ ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.

Combining crystallography and EPR: crystal and solution structures of the multidomain cochaperone DnaJ

Energy Technology Data Exchange (ETDEWEB)

Barends, Thomas R. M., E-mail: thomas.barends@mpimf-heidelberg.mpg.de [MPI for Medical Research, Heidelberg (Germany); Brosi, Richard W. W. [Freie Universitat Berlin, Berlin (Germany); Steinmetz, Andrea; Scherer, Anna; Hartmann, Elisabeth; Eschenbach, Jessica; Lorenz, Thorsten [MPI for Medical Research, Heidelberg (Germany); Seidel, Ralf [MPI for Molecular Physiology, Dortmund (Germany); Shoeman, Robert L.; Zimmermann, Sabine [MPI for Medical Research, Heidelberg (Germany); Bittl, Robert [Freie Universitat Berlin, Berlin (Germany); Schlichting, Ilme; Reinstein, Jochen [MPI for Medical Research, Heidelberg (Germany)

2013-08-01

The crystal structure of the N-terminal part of T. thermophilus DnaJ unexpectedly showed an ordered GF domain and guided the design of a construct enabling the first structure determination of a complete DnaJ cochaperone molecule. By combining the crystal structures with spin-labelling EPR and cross-linking in solution, a dynamic view of this flexible molecule was developed. Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin-labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.

The kinase domain residue serine 173 of Schizosaccharomyces pombe Chk1 kinase is critical for the response to DNA replication stress

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Naomi Coulton

2017-12-01

Full Text Available While mammalian Chk1 kinase regulates replication origins, safeguards fork integrity and promotes fork progression, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A in the kinase domain of fission yeast Chk1 abolishes the G1-M and S-M checkpoints with little impact on the G2-M arrest. This separation-of-function mutation strongly reduces the Rad3-dependent phosphorylation of Chk1 at serine 345 during logarithmic growth, but not when cells experience exogenous DNA damage. Loss of S173 lowers the restrictive temperature of a catalytic DNA polymerase epsilon mutant (cdc20.M10 and is epistatic with a mutation in DNA polymerase delta (cdc6.23 when DNA is alkylated by methyl-methanesulfate (MMS. The chk1-S173A allele is uniquely sensitive to high MMS concentrations where it displays a partial checkpoint defect. A complete checkpoint defect occurs only when DNA replication forks break in cells without the intra-S phase checkpoint kinase Cds1. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 (cdc10.V50 is impaired. We conclude that serine 173, which is equivalent to lysine 166 in the activation loop of human Chk1, is only critical in DNA polymerase mutants or when forks collapse in the absence of Cds1.

The disordered C-terminal domain of human DNA glycosylase NEIL1 contributes to its stability via intramolecular interactions.

Science.gov (United States)

Hegde, Muralidhar L; Tsutakawa, Susan E; Hegde, Pavana M; Holthauzen, Luis Marcelo F; Li, Jing; Oezguen, Numan; Hilser, Vincent J; Tainer, John A; Mitra, Sankar

2013-07-10

NEIL1 [Nei (endonuclease VIII)-like protein 1], one of the five mammalian DNA glycosylases that excise oxidized DNA base lesions in the human genome to initiate base excision repair, contains an intrinsically disordered C-terminal domain (CTD; ~100 residues), not conserved in its Escherichia coli prototype Nei. Although dispensable for NEIL1's lesion excision and AP lyase activities, this segment is required for efficient in vivo enzymatic activity and may provide an interaction interface for many of NEIL1's interactions with other base excision repair proteins. Here, we show that the CTD interacts with the folded domain in native NEIL1 containing 389 residues. The CTD is poised for local folding in an ordered structure that is induced in the purified fragment by osmolytes. Furthermore, deletion of the disordered tail lacking both Tyr and Trp residues causes a red shift in NEIL1's intrinsic Trp-specific fluorescence, indicating a more solvent-exposed environment for the Trp residues in the truncated protein, which also exhibits reduced stability compared to the native enzyme. These observations are consistent with stabilization of the native NEIL1 structure via intramolecular, mostly electrostatic, interactions that were disrupted by mutating a positively charged (Lys-rich) cluster of residues (amino acids 355-360) near the C-terminus. Small-angle X-ray scattering (SAXS) analysis confirms the flexibility and dynamic nature of NEIL1's CTD, a feature that may be critical to providing specificity for NEIL1's multiple, functional interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Science.gov (United States)

Pitard, Bruno; Aguerre, Olivier; Airiau, Marc; Lachagès, Anne-Marie; Boukhnikachvili, Tsiala; Byk, Gérardo; Dubertret, Catherine; Herviou, Christian; Scherman, Daniel; Mayaux, Jean-François; Crouzet, Joël

1997-01-01

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains. PMID:9405626

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.

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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

Exonuclease 1 and its versatile roles in DNA repair

DEFF Research Database (Denmark)

Keijzers, Guido; Liu, Dekang; Rasmussen, Lene Juel

2016-01-01

Exonuclease 1 (EXO1) is a multifunctional 5' → 3' exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin...... maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions...

Differential recruitment of DNA Ligase I and III to DNA repair sites

Science.gov (United States)

Mortusewicz, Oliver; Rothbauer, Ulrich; Cardoso, M. Cristina; Leonhardt, Heinrich

2006-01-01

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair. PMID:16855289

The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

Energy Technology Data Exchange (ETDEWEB)

Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

2005-11-01

The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

DNA sequence explains seemingly disordered methylation levels in partially methylated domains of Mammalian genomes.

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Dimos Gaidatzis

2014-02-01

Full Text Available For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs, recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40% of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS. Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2 of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.

Transcription initiation complex structures elucidate DNA opening.

Science.gov (United States)

Plaschka, C; Hantsche, M; Dienemann, C; Burzinski, C; Plitzko, J; Cramer, P

2016-05-19

Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE 'extended winged helix' domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE 'E-ribbon' domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein-protein and protein-DNA contacts.

Cloning of the cDNA for murine von Willebrand factor and identification of orthologous genes reveals the extent of conservation among diverse species.

Science.gov (United States)

Chitta, Mohan S; Duhé, Roy J; Kermode, John C

2007-05-01

Interaction of von Willebrand factor (VWF) with circulating platelets promotes hemostasis when a blood vessel is injured. The A1 domain of VWF is responsible for the initial interaction with platelets and is well conserved among species. Knowledge of the cDNA and genomic DNA sequences for human VWF allowed us to predict the cDNA sequence for murine VWF in silico and amplify its entire coding region by RT-PCR. The murine VWF cDNA has an open reading frame of 8,442 bp, encoding a protein of 2,813 amino acid residues with 83% identity to human pre-pro-VWF. The same strategy was used to predict in silico the cDNA sequence for the ortholog of VWF in a further six species. Many of these predictions diverged substantially from the putative Reference Sequences derived by ab initio methods. Our predicted sequences indicated that the VWF gene has a conserved structure of 52 exons in all seven mammalian species examined, as well as in the chicken. There is a minor structural variation in the pufferfish Takifugu rubripes insofar as the VWF gene in this species has 53 exons. Comparison of the translated amino acid sequences also revealed a high degree of conservation. In particular, the cysteine residues are conserved precisely throughout both the pro-peptide and the mature VWF sequence in all species, with a minor exception in the pufferfish VWF ortholog where two adjacent cysteine residues are omitted. The marked conservation of cysteine residues emphasizes the importance of the intricate pattern of disulfide bonds in governing the structure of pro-VWF and regulating the function of the mature VWF protein. It should also be emphasized that many of the conserved features of the VWF gene and protein were obscured when the comparison among species was based on the putative Reference Sequences instead of our predicted cDNA sequences.

Effect of Hyaluronan on Developmental Competence and Quality of Oocytes and Obtained Blastocysts from In Vitro Maturation of Bovine Oocytes

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Jolanta Opiela

2014-01-01

Full Text Available The objective of the present study was to evaluate the effect of hyaluronan (HA during IVM on meiotic maturation, embryonic development, and the quality of oocytes, granulosa cells (GC, and obtained blastocysts. COCs were matured in vitro in control medium and medium with additional 0.035% or 0.07% of exogenous HA. The meiotic maturity did not differ between the analysed groups. The best rate and the highest quality of obtained blastocysts were observed when 0.07% HA was used. A highly significant difference (P<0.001 was noted in the mean number of apoptotic nuclei per blastocyst and in the DCI between the 0.07% HA and the control blastocysts (P<0.01. Our results suggest that addition of 0.035% HA and 0.07% HA to oocyte maturation media does not affect oocyte nuclear maturation and DNA fragmentation. However, the addition of 0.07% HA during IVM decreases the level of blastocysts DNA fragmentation. Finally, our results suggest that it may be risky to increase the HA concentration during IVM above 0.07% as we found significantly higher Bax mRNA expression levels in GC cultured with 0.07% HA. The final concentration of HA being supplemented to oocyte maturation media is critical for the success of the IVP procedure.

Agarose Gel Electrophoresis Reveals Structural Fluidity of a Phage T3 DNA Packaging Intermediate

Science.gov (United States)

Serwer, Philip; Wright, Elena T.

2012-01-01

We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (1) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for stabilization of structure and then (2) determining of effective radius by two-dimensional agarose gel electrophoresis (2d-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2d-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. PMID:22222979

DNA residence time is a regulatory factor of transcription repression

Science.gov (United States)

Clauß, Karen; Popp, Achim P.; Schulze, Lena; Hettich, Johannes; Reisser, Matthias; Escoter Torres, Laura; Uhlenhaut, N. Henriette

2017-01-01

Abstract Transcription comprises a highly regulated sequence of intrinsically stochastic processes, resulting in bursts of transcription intermitted by quiescence. In transcription activation or repression, a transcription factor binds dynamically to DNA, with a residence time unique to each factor. Whether the DNA residence time is important in the transcription process is unclear. Here, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs) and varying the number of nucleotide-recognizing repeat domains. We characterized the DNA residence times of our repressors in living cells using single molecule tracking. The residence times depended non-linearly on the number of repeat domains and differed by more than a factor of six. The factors provoked a residence time-dependent decrease in transcript level of the glucocorticoid receptor-activated gene SGK1. Down regulation of transcription was due to a lower burst frequency in the presence of long binding repressors and is in accordance with a model of competitive inhibition of endogenous activator binding. Our single molecule experiments reveal transcription factor DNA residence time as a regulatory factor controlling transcription repression and establish TALE-DNA binding domains as tools for the temporal dissection of transcription regulation. PMID:28977492

The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

DEFF Research Database (Denmark)

Lafuente-Barquero, Juan; Luke-Glaser, Sarah; Graf, Marco

2017-01-01

of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA hybrids and RNA-DNA hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA hybrids...

Multivalent Chromatin Engagement and Inter-domain Crosstalk Regulate MORC3 ATPase

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Forest H. Andrews

2016-09-01

Full Text Available MORC3 is linked to inflammatory myopathies and cancer; however, the precise role of MORC3 in normal cell physiology and disease remains poorly understood. Here, we present detailed genetic, biochemical, and structural analyses of MORC3. We demonstrate that MORC3 is significantly upregulated in Down syndrome and that genetic abnormalities in MORC3 are associated with cancer. The CW domain of MORC3 binds to the methylated histone H3K4 tail, and this interaction is essential for recruitment of MORC3 to chromatin and accumulation in nuclear bodies. We show that MORC3 possesses intrinsic ATPase activity that requires DNA, but it is negatively regulated by the CW domain, which interacts with the ATPase domain. Natively linked CW impedes binding of the ATPase domain to DNA, resulting in a decrease in the DNA-stimulated enzymatic activity. Collectively, our studies provide a molecular framework detailing MORC3 functions and suggest that its modulation may contribute to human disease.

Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 Å resolution

Energy Technology Data Exchange (ETDEWEB)

Tu, Chao [Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702 (United States); Tan, Yu-Hong [Department of Molecular Biology and Biochemistry, University of California at Irvine, Irvine, CA 92697 (United States); Shaw, Gary [Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702 (United States); Zhou, Zheng; Bai, Yawen [Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, Bethesda, MD 20892 (United States); Luo, Ray [Department of Molecular Biology and Biochemistry, University of California at Irvine, Irvine, CA 92697 (United States); Ji, Xinhua, E-mail: jix@ncifcrf.gov [Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702 (United States)

2008-05-01

The impact of hotspot mutation R282Q on the structure of human p53 DNA-binding domain has been characterized by X-ray crystallography and molecular-dynamics simulations. Tumor suppressor p53 is a sequence-specific DNA-binding protein and its central DNA-binding domain (DBD) harbors six hotspots (Arg175, Gly245, Arg248, Arg249, Arg273 and Arg282) for human cancers. Here, the crystal structure of a low-frequency hotspot mutant, p53DBD(R282Q), is reported at 1.54 Å resolution together with the results of molecular-dynamics simulations on the basis of the structure. In addition to eliminating a salt bridge, the R282Q mutation has a significant impact on the properties of two DNA-binding loops (L1 and L3). The L1 loop is flexible in the wild type, but it is not flexible in the mutant. The L3 loop of the wild type is not flexible, whereas it assumes two conformations in the mutant. Molecular-dynamics simulations indicated that both conformations of the L3 loop are accessible under biological conditions. It is predicted that the elimination of the salt bridge and the inversion of the flexibility of L1 and L3 are directly or indirectly responsible for deactivating the tumor suppressor p53.

Structure of the DNA-bound BRCA1 C-terminal region from human replication factor C p140 and model of the protein-DNA complex

NARCIS (Netherlands)

Kobayashi, M.; AB, E.; Bonvin, A.M.J.J.; Siegal, G.

2010-01-01

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA

Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

Directory of Open Access Journals (Sweden)

Garabaya Cecilia

2006-03-01

Full Text Available Abstract Background We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity. Results Following our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the α-chain of fibrinogen as well as pro

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

Science.gov (United States)

Odell, M; Shuman, S

1999-05-14

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.

Promoter-enhancer interactions identified from Hi-C data using probabilistic models and hierarchical topological domains.

Science.gov (United States)

Ron, Gil; Globerson, Yuval; Moran, Dror; Kaplan, Tommy

2017-12-21

Proximity-ligation methods such as Hi-C allow us to map physical DNA-DNA interactions along the genome, and reveal its organization into topologically associating domains (TADs). As the Hi-C data accumulate, computational methods were developed for identifying domain borders in multiple cell types and organisms. Here, we present PSYCHIC, a computational approach for analyzing Hi-C data and identifying promoter-enhancer interactions. We use a unified probabilistic model to segment the genome into domains, which we then merge hierarchically and fit using a local background model, allowing us to identify over-represented DNA-DNA interactions across the genome. By analyzing the published Hi-C data sets in human and mouse, we identify hundreds of thousands of putative enhancers and their target genes, and compile an extensive genome-wide catalog of gene regulation in human and mouse. As we show, our predictions are highly enriched for ChIP-seq and DNA accessibility data, evolutionary conservation, eQTLs and other DNA-DNA interaction data.

Cloning and expression of cDNA coding for bouganin.

Science.gov (United States)

den Hartog, Marcel T; Lubelli, Chiara; Boon, Louis; Heerkens, Sijmie; Ortiz Buijsse, Antonio P; de Boer, Mark; Stirpe, Fiorenzo

2002-03-01

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.

Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

DEFF Research Database (Denmark)

Wewer, U M; Gerecke, D R; Durkin, M E

1994-01-01

or other known laminin genes. Immunostaining showed that the beta 2 chain is localized to the smooth muscle basement membranes of the arteries, while the homologous beta 1 chain is confined to the subendothelial basement membranes. The beta 2 chain was found in the basement membranes of ovarian carcinomas......Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... beta 2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human beta 2 chain is predicted to have all of the seven structural domains typical of the beta chains of laminin, including the short cysteine-rich alpha region. The amino acid sequence of human beta 2...

Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain

NARCIS (Netherlands)

Hernandez-Garcia, Armando; Estrich, Nicole A.; Werten, Marc W.T.; Maarel, van der Johan R.C.; Labean, Thomas H.; Wolf, de Frits A.; Cohen Stuart, Martien A.; Vries, de Renko

2017-01-01

Emerging DNA-based nanotechnologies would benefit from the ability to modulate the properties (e.g., solubility, melting temperature, chemical stability) of diverse DNA templates (single molecules or origami nanostructures) through controlled, self-assembling coatings. We here introduce a DNA

Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate.

Science.gov (United States)

Serwer, Philip; Wright, Elena T

2012-01-01

We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expanding the landscape of chromatin modification (CM-related functional domains and genes in human.

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Shuye Pu

2010-11-01

Full Text Available Chromatin modification (CM plays a key role in regulating transcription, DNA replication, repair and recombination. However, our knowledge of these processes in humans remains very limited. Here we use computational approaches to study proteins and functional domains involved in CM in humans. We analyze the abundance and the pair-wise domain-domain co-occurrences of 25 well-documented CM domains in 5 model organisms: yeast, worm, fly, mouse and human. Results show that domains involved in histone methylation, DNA methylation, and histone variants are remarkably expanded in metazoan, reflecting the increased demand for cell type-specific gene regulation. We find that CM domains tend to co-occur with a limited number of partner domains and are hence not promiscuous. This property is exploited to identify 47 potentially novel CM domains, including 24 DNA-binding domains, whose role in CM has received little attention so far. Lastly, we use a consensus Machine Learning approach to predict 379 novel CM genes (coding for 329 proteins in humans based on domain compositions. Several of these predictions are supported by very recent experimental studies and others are slated for experimental verification. Identification of novel CM genes and domains in humans will aid our understanding of fundamental epigenetic processes that are important for stem cell differentiation and cancer biology. Information on all the candidate CM domains and genes reported here is publicly available.

Dendritic cell activation and maturation induced by recombinant calreticulin fragment 39-272.

Science.gov (United States)

Li, Yue; Zeng, Xiaoli; He, Lijuan; Yuan, Hui

2015-01-01

Dendritic cells (DC) are the most potent antigen-presenting cells for initiating immune responses. DC maturation can be induced by exposing of immature DC to pathogen products or pro-inflammatory factor, which dramatically enhances the ability of DC to activate Ag-specific T cells. In this study, a recombinant calreticulin fragment 39-272 (rCRT/39-272) covering the lectin-like N domain and partial P domain of murine CRT has been expressed and purified in Escherichia coli. Functional analysis studies revealed that rCRT/39-272 has potent immunostimulatory activities in both activating human monocytes and B cells to secrete cytokines. rCRT/39-272 can drive the activation of bone marrow derived DC in TLR4/CD14 dependent way, as indicated by secretion of cytokines IL-12/IL-23 (p40) and IL-1β. Exposure of DC to rCRT/39-272 induces P-Akt, suggesting that rCRT/39-272 induces maturation of DC through PI3K/Akt signaling pathway. The results suggest that soluble rCRT/39-272 is a potent stimulatory agent to DC maturation in TLR4/CD14 and PI3K/Akt dependent pathway. It may play important roles in initiating cellular immunity in vivo and the T cell response in vitro. Thus it could be used for study of DC-based tumor vaccines.

Directed evolution of the TALE N-terminal domain for recognition of all 5′ bases

Science.gov (United States)

Lamb, Brian M.; Mercer, Andrew C.; Barbas, Carlos F.

2013-01-01

Transcription activator-like effector (TALE) proteins can be designed to bind virtually any DNA sequence. General guidelines for design of TALE DNA-binding domains suggest that the 5′-most base of the DNA sequence bound by the TALE (the N0 base) should be a thymine. We quantified the N0 requirement by analysis of the activities of TALE transcription factors (TALE-TF), TALE recombinases (TALE-R) and TALE nucleases (TALENs) with each DNA base at this position. In the absence of a 5′ T, we observed decreases in TALE activity up to >1000-fold in TALE-TF activity, up to 100-fold in TALE-R activity and up to 10-fold reduction in TALEN activity compared with target sequences containing a 5′ T. To develop TALE architectures that recognize all possible N0 bases, we used structure-guided library design coupled with TALE-R activity selections to evolve novel TALE N-terminal domains to accommodate any N0 base. A G-selective domain and broadly reactive domains were isolated and characterized. The engineered TALE domains selected in the TALE-R format demonstrated modularity and were active in TALE-TF and TALEN architectures. Evolved N-terminal domains provide effective and unconstrained TALE-based targeting of any DNA sequence as TALE binding proteins and designer enzymes. PMID:23980031

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols

International Nuclear Information System (INIS)

Hoy, C.A.; Lewis, E.D.; Schimke, R.T.

1990-01-01

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [ 3 H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [ 3 H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [ 3 H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations

C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

International Nuclear Information System (INIS)

Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

2013-01-01

Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ

Evolutionistic or revolutionary paths? A PACS maturity model for strategic situational planning.

Science.gov (United States)

van de Wetering, Rogier; Batenburg, Ronald; Lederman, Reeva

2010-07-01

While many hospitals are re-evaluating their current Picture Archiving and Communication System (PACS), few have a mature strategy for PACS deployment. Furthermore, strategies for implementation, strategic and situational planning methods for the evolution of PACS maturity are scarce in the scientific literature. Consequently, in this paper we propose a strategic planning method for PACS deployment. This method builds upon a PACS maturity model (PMM), based on the elaboration of the strategic alignment concept and the maturity growth path concept previously developed in the PACS domain. First, we review the literature on strategic planning for information systems and information technology and PACS maturity. Secondly, the PMM is extended by applying four different strategic perspectives of the Strategic Alignment Framework whereupon two types of growth paths (evolutionistic and revolutionary) are applied that focus on a roadmap for PMM. This roadmap builds a path to get from one level of maturity and evolve to the next. An extended method for PACS strategic planning is developed. This method defines eight distinctive strategies for PACS strategic situational planning that allow decision-makers in hospitals to decide which approach best suits their hospitals' current situation and future ambition and what in principle is needed to evolve through the different maturity levels. The proposed method allows hospitals to strategically plan for PACS maturation. It is situational in that the required investments and activities depend on the alignment between the hospital strategy and the selected growth path. The inclusion of both strategic alignment and maturity growth path concepts make the planning method rigorous, and provide a framework for further empirical research and clinical practice.

The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

Science.gov (United States)

Henke, Sarah K; Cronan, John E

2016-11-01

Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity. © 2016 John Wiley & Sons Ltd.

A Proteomic Approach to Identify Phosphorylation-Dependent Targets of BRCT Domains

National Research Council Canada - National Science Library

Songyang, Zhou

2007-01-01

.... In addition, BRCT domains are found in many proteins that regulate DNA damage repair, cell cycle, and genome stability, implying a more global role of BRCT domains in genome stability surveillance...

UV laser-induced histone-DNA crosslinking proceeds via the N-terminal tails

International Nuclear Information System (INIS)

Stefanovski, V.; Dimitrov, S.; Angelov, D.; Keskinova, E.; Pashev, I.

1990-01-01

The covalent crosslinking of histones to DNA by UV laser irradiation is accomplished solely via the N-terminal part of the molecule. Irradiated isolated calfthymus nuclei are treated with clostripain. The crosslinked protein-DNA complexes are isolated and the presence of each core histone analyzed by dot-immunoassay using antibodies, specific to the central globular domain of the respective histone. The reaction is negative for all core histones i.e. the globular domain is absent. It means that this domain has not been crosslinked to DNA and, once cleaved by clostripain, it has been stripped from DNA during the centrigugation in CsCl. This peculiar property of the crosslinked procedure makes it particularly useful in addressing some yet unanswered questions concerning histone-DNA interactions, such as the interaction of the N-terminal tails with linker DNA, the effect of the transient postsynthetic histone acetylation on its interaction with DNA, etc. These questions are now under study. 1 fig., 6 refs

Maturity group classification and maturity locus genotyping of early-maturing soybean varieties from high-latitude cold regions.

Science.gov (United States)

Jia, Hongchang; Jiang, Bingjun; Wu, Cunxiang; Lu, Wencheng; Hou, Wensheng; Sun, Shi; Yan, Hongrui; Han, Tianfu

2014-01-01

With the migration of human beings, advances of agricultural sciences, evolution of planting patterns and global warming, soybeans have expanded to both tropical and high-latitude cold regions (HCRs). Unlike other regions, HCRs have much more significant and diverse photoperiods and temperature conditions over seasons or across latitudes, and HCR soybeans released there show rich diversity in maturity traits. However, HCR soybeans have not been as well classified into maturity groups (MGs) as other places. Therefore, it is necessary to identify MGs in HCRs and to genotype the maturity loci. Local varieties were collected from the northern part of Northeast China and the far-eastern region of Russia. Maturity group reference (MGR) soybeans of MGs MG000, MG00, and MG0 were used as references during field experiments. Both local varieties and MGR soybeans were planted for two years (2010-2011) in Heihe (N 50°15', E 127°27', H 168.5 m), China. The days to VE (emergence), R1 (beginning bloom) and R7 (beginning maturity) were recorded and statistically analyzed. Furthermore, some varieties were further genotyped at four molecularly-identified maturity loci E1, E2, E3 and E4. The HCR varieties were classified into MG0 or even more early-maturing. In Heihe, some varieties matured much earlier than MG000, which is the most early-maturing known MG, and clustered into a separate group. We designated the group as MG0000, following the convention of MGs. HCR soybeans had relatively stable days to beginning bloom from emergence. The HCR varieties diversified into genotypes of E1, E2, E3 and E4. These loci had different effects on maturity. HCRs diversify early-maturing MGs of soybean. MG0000, a new MG that matures much earlier than known MGs, was developed. HCR soybean breeding should focus more on shortening post-flowering reproductive growth. E1, E2, E3, and E4 function differentially.

Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

OpenAIRE

Shishmarev, Dmitry; Wang, Yao; Mason, Claire E.; Su, Xun-Cheng; Oakley, Aaron J.; Graham, Bim; Huber, Thomas; Dixon, Nicholas E.; Otting, Gottfried

2013-01-01

Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron den...

Mannosylated biodegradable polyethyleneimine for targeted DNA delivery to dendritic cells

Directory of Open Access Journals (Sweden)

Sun X

2012-06-01

Full Text Available Xun Sun, Simu Chen, Jianfeng Han, Zhirong ZhangKey Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of ChinaBackground: To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG and a series of its mannosylated derivatives.Methods: PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs using flow cytometry.Results: PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation.Conclusion: These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system.Keywords: dendritic cells, DCs, mannose, polyethyleneimine, PEI, gene delivery

Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication.

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Fabio Lapenta

Full Text Available DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics.

Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus

DEFF Research Database (Denmark)

Peng, Wenfang; Li, Huan; Hallstrøm, Søren

2013-01-01

-adjacent motif (PAM)-dependent DNA targeting activity and mature CRISPR RNA (crRNA) production in this organism, mutants deleting individual genes of the type IA system or removing each of other Cas modules were constructed. Characterization of these mutants revealed that Cas7, Cas5, Cas6, Cas3' and Cas3......" are essential for PAM-dependent DNA targeting activity, whereas Csa5, along with all other Cas modules, is dispensable for the targeting in the crenarchaeon. Cas6 is implicated as the only enzyme for pre-crRNA processing and the crRNA maturation is independent of the DNA targeting activity. Importantly, we show...

Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

DEFF Research Database (Denmark)

Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

2017-01-01

involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner...... and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1....

Dissection of the IgNAR V domain: molecular scanning and orthologue database mining define novel IgNAR hallmarks and affinity maturation mechanisms.

Science.gov (United States)

Fennell, B J; Darmanin-Sheehan, A; Hufton, S E; Calabro, V; Wu, L; Müller, M R; Cao, W; Gill, D; Cunningham, O; Finlay, W J J

2010-07-09

natural nurse shark cDNA sequences. These findings will aid future V(NAR) engineering and optimisation studies towards the development of V(NAR) single-domain proteins as viable biotherapeutics. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 domain bound to a palindromic SBE DNA element

Science.gov (United States)

Baburajendran, Nithya; Palasingam, Paaventhan; Ng, Calista Keow Leng; Jauch, Ralf; Kolatkar, Prasanna R.

2009-01-01

The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 Å resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-­propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 Å, α = β = γ = 90°. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit. PMID:19923727

DNA topology and transcription

Science.gov (United States)

Kouzine, Fedor; Levens, David; Baranello, Laura

2014-01-01

Chromatin is a complex assembly that compacts DNA inside the nucleus while providing the necessary level of accessibility to regulatory factors conscripted by cellular signaling systems. In this superstructure, DNA is the subject of mechanical forces applied by variety of molecular motors. Rather than being a rigid stick, DNA possesses dynamic structural variability that could be harnessed during critical steps of genome functioning. The strong relationship between DNA structure and key genomic processes necessitates the study of physical constrains acting on the double helix. Here we provide insight into the source, dynamics, and biology of DNA topological domains in the eukaryotic cells and summarize their possible involvement in gene transcription. We emphasize recent studies that might inspire and impact future experiments on the involvement of DNA topology in cellular functions. PMID:24755522

Recruitment of DNA methyltransferase I to DNA repair sites

Science.gov (United States)

Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M. Cristina; Leonhardt, Heinrich

2005-01-01

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair. PMID:15956212

Structural and dynamic studies of the dimerization and DNA-binding domains of the transcription factors v-Myc and Max

International Nuclear Information System (INIS)

Fieber, W.

2001-05-01

In the present work, solution structural and dynamic properties of the dimerization and DNA binding domains of the transcription factors v-Myc and Max were characterized by NMR and CD spectroscopy. It could be demonstrated that v-Myc in the absence of its authentic binding partner Max does not homodimerize, but exists in a monomeric and prestructured form. Two separated α-helical regions in the leucine zipper region and in the basic-H1 region, respectively, could be identified, while the latter appeared to be less stable. Both helices lack stabilizing tertiary side chain interactions and represent exceptional examples for loosely coupled, structured segments in a native protein. The structure of v-Myc is dynamic and can be described as a distribution of conformational substates. Motion within the substates comprise fast (picosecond to nanosecond) local backbone fluctuations like helical fraying, whereas motion between the substates comprise the relative orientation of the two helices and occur at larger time scales (microsecond to millisecond). The preformation of the specific protein and DNA binding sites, leucine zipper and the basic region, presumably allows rapid and accurate recognition of the respective binding partners. v-Myc-Max and Max-Max protein preparations were shown to form stable dimers. Thermodynamic analysis of the dissociation reactions of v-Myc-Max revealed a significant higher stability of the heterodimer than of the Max-Max homodimer over the whole temperature range. It could be demonstrated that the restricted conformational space of the v-Myc bHLHZip domain reduces the entropy penalty associated with dimerization and contributes to the preference of Max to form heterodimers with v-Myc rather than homodimers. (author)

A cargo-sorting DNA robot.

Science.gov (United States)

Thubagere, Anupama J; Li, Wei; Johnson, Robert F; Chen, Zibo; Doroudi, Shayan; Lee, Yae Lim; Izatt, Gregory; Wittman, Sarah; Srinivas, Niranjan; Woods, Damien; Winfree, Erik; Qian, Lulu

2017-09-15

Two critical challenges in the design and synthesis of molecular robots are modularity and algorithm simplicity. We demonstrate three modular building blocks for a DNA robot that performs cargo sorting at the molecular level. A simple algorithm encoding recognition between cargos and their destinations allows for a simple robot design: a single-stranded DNA with one leg and two foot domains for walking, and one arm and one hand domain for picking up and dropping off cargos. The robot explores a two-dimensional testing ground on the surface of DNA origami, picks up multiple cargos of two types that are initially at unordered locations, and delivers them to specified destinations until all molecules are sorted into two distinct piles. The robot is designed to perform a random walk without any energy supply. Exploiting this feature, a single robot can repeatedly sort multiple cargos. Localization on DNA origami allows for distinct cargo-sorting tasks to take place simultaneously in one test tube or for multiple robots to collectively perform the same task. Copyright © 2017, American Association for the Advancement of Science.

Cross-Genome Comparisons of Newly Identified Domains in Mycoplasma gallisepticum and Domain Architectures with Other Mycoplasma species

Directory of Open Access Journals (Sweden)

Chandra Sekhar Reddy Chilamakuri

2011-01-01

Full Text Available Accurate functional annotation of protein sequences is hampered by important factors such as the failure of sequence search methods to identify relationships and the inherent diversity in function of proteins related at low sequence similarities. Earlier, we had employed intermediate sequence search approach to establish new domain relationships in the unassigned regions of gene products at the whole genome level by taking Mycoplasma gallisepticum as a specific example and established new domain relationships. In this paper, we report a detailed comparison of the conservation status of the domain and domain architectures of the gene products that bear our newly predicted domains amongst 14 other Mycoplasma genomes and reported the probable implications for the organisms. Some of the domain associations, observed in Mycoplasma that afflict humans and other non-human primates, are involved in regulation of solute transport and DNA binding suggesting specific modes of host-pathogen interactions.

Conversion of DNA gyrase into a conventional type II topoisomerase

DEFF Research Database (Denmark)

Kampranis, S C; Maxwell, A

1996-01-01

DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. We have demonstrated that deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA but relaxes DNA in an ATP-depend...

Vitrification affects nuclear maturation and gene expression of immature human oocytes

Directory of Open Access Journals (Sweden)

Abbas Shahedi

2017-02-01

Full Text Available Background: Vitrification of oocytes is a fast-freezing technique, which may affect the quality of the human oocyte, and consequently affects the embryo development, pregnancy and birth. The aim of the current study was to investigate the consequence of in-vitro vitrification on maturation status of immature human oocytes, additionally, expression levels of stress, and apoptosis related genes. Materials and Methods: The total of 213 human immature oocytes which routinely discarded from assisted reproduction clinics were collected and divided into two groups including: (I fresh germinal vesicle (GV oocytes (n=106 (matured in-vitro  (fIVM , and  (II GV oocytes (n=107 that initially vitrified, then matured in  in-vitro (vIVM. After 36 hours of incubation, the oocytes were evaluated for nuclear maturation and expression level of DNA methyltransferase (DNMT1, stress related genes (Sod1 and Hsp70, and apoptotic related genes (Bax and Bcl-2 by quantitative Real-Time PCR. Results: Oocyte maturation rates were reduced in vIVM compared to fIVM oocytes (P=0.001. The expression of stress (Sod1 and Hsp70, and apoptotic-related genes (Bax and Bcl-2 in vIVM were significantly higher compared to the fIVM group. Additionally, pro-apoptotic gene up-regulated 4.3 times more than anti-apoptotic gene in vIVM oocyte. However, DNMT1 gene expression was reduced in vIVM oocyte (P = 0.047. Conclusions: The low survival rate of vitrified In-vitro matured GV oocytes could definitely be explained by the alterations of their gene expression profile. 

Length quantization of DNA partially expelled from heads of a bacteriophage T3 mutant

Energy Technology Data Exchange (ETDEWEB)

Serwer, Philip, E-mail: serwer@uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (United States); Wright, Elena T. [Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (United States); Liu, Zheng; Jiang, Wen [Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 (United States)

2014-05-15

DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNA missing 3.7–12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection. - Graphical abstract: Highlights: • We implement directed evolution- and DNA-sequencing-based phage assembly genetics. • We purify stable, mutant phage heads with a partially leaked mature DNA molecule. • Native gels and DNase-protection show leaked DNA segments to have quantized lengths. • Native gels after DNase I-removal of leaked DNA reveal the capsids to vary in radius. • Thus, we hypothesize leaked DNA quantization via variably quantized capsid radius.

Mutagenic effect of radionuclides incorporated into DNA of drosphila melanogaster. Progress report, 1976--1977

International Nuclear Information System (INIS)

Lee, W.R.

1977-01-01

Progress is reported on studies of the genetic effect of tritium incorporated into DNA of Drosophila melanogaster. Following a short pulse exposure to tritium-labeled uridine most of the radioactivity appeared in RNA; however, the turnover of radioactivity in tritium-labeled RNA was rapid whereas there was no exchange of tritium from the labeled DNA during spermatogenesis. Furthermore, most of the cytoplasm and most of the RNA in primary spermatocytes was lost during spermatogenesis and thus the mature sperm cell was left with only the DNA labeled. Preliminary experiments did not show a significant level of labeled RNA remaining in the mature sperm cell, whereas labeled DNA was verified after extraction and purification with phenol and enzyme digestion. Preliminary results of genetic tests on the progeny of normal females inseminated with tritium-labeled sperm cells are reported

Recognition of 5-Hydroxymethylcytosine by the Uhrf1 SRA Domain

Science.gov (United States)

Bultmann, Sebastian; Casa, Valentina; Cardoso, M. Cristina; Antes, Iris; Leonhardt, Heinrich

2011-01-01

Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group. PMID:21731699

Mycobacterium tuberculosis class II apurinic/apyrimidinic-endonuclease/3'-5' exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β-clamp.

Science.gov (United States)

Khanam, Taran; Rai, Niyati; Ramachandran, Ravishankar

2015-10-01

The class-II AP-endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β-clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel 239 QLRFPKK245 motif in the DNA-binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding-groove (PBG) and the C-terminal of β-clamp located on different domains interact with XthA. The β-clamp-XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β-clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β-clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β-clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β-clamp is important for interactions with XthA, while the C-terminal domain predominantly mediates functional interactions in the substrate's presence. © 2015 John Wiley & Sons Ltd.

Global conformational dynamics of a Y-family DNA polymerase during catalysis.

Directory of Open Access Journals (Sweden)

Cuiling Xu

2009-10-01

Full Text Available Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4 from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.

Structural similarities in DNA packaging and delivery apparatuses in Herpesvirus and dsDNA bacteriophages.

Science.gov (United States)

Rixon, Frazer J; Schmid, Michael F

2014-04-01

Structural information can inform our understanding of virus origins and evolution. The herpesviruses and tailed bacteriophages constitute two large families of dsDNA viruses which infect vertebrates and prokaryotes respectively. A relationship between these disparate groups was initially suggested by similarities in their capsid assembly and DNA packaging strategies. This relationship has now been confirmed by a range of studies that have revealed common structural features in their capsid proteins, and similar organizations and sequence conservation in their DNA packaging machinery and maturational proteases. This concentration of conserved traits in proteins involved in essential and primordial capsid/packaging functions is evidence that these structures are derived from an ancient, common ancestor and is in sharp contrast to the lack of such evidence for other virus functions. Copyright © 2014. Published by Elsevier B.V.

A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

Science.gov (United States)

Eden, Thomas; Menzel, Stephan; Wesolowski, Janusz; Bergmann, Philine; Nissen, Marion; Dubberke, Gudrun; Seyfried, Fabienne; Albrecht, Birte; Haag, Friedrich; Koch-Nolte, Friedrich

2018-01-01

Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic

Functional properties and structural requirements of the plasmid pMV158-encoded MobM relaxase domain.

Science.gov (United States)

Fernández-López, Cris; Pluta, Radoslaw; Pérez-Luque, Rosa; Rodríguez-González, Lorena; Espinosa, Manuel; Coll, Miquel; Lorenzo-Díaz, Fabián; Boer, D Roeland

2013-07-01

A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriTpMV158, whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N- and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself.

Crystallization and X-ray diffraction analysis of the HMG domain of the chondrogenesis master regulator Sox9 in complex with a ChIP-Seq-identified DNA element

Energy Technology Data Exchange (ETDEWEB)

Vivekanandan, Saravanan; Moovarkumudalvan, Balasubramanian; Lescar, Julien; Kolatkar, Prasanna R.

2015-10-30

Sox9 is a fundamental sex-determining gene and the master regulator of chondrogenesis, and is involved in the development of various vital organs such as testes, kidney, heart and brain, and in skeletal development. Similar to other known Sox transcription factors, Sox9 recognizes and binds DNA with the consensus sequence C(T/A)TTG(T/A)(T/A) through the highly conserved HMG domain. Nonetheless, the molecular basis of the functional specificity of Sox9 in key developmental processes is still unclear. As an initial step towards a mechanistic understanding of Sox9 transcriptional regulation, the current work describes the details of the purification of the mouse Sox9 HMG domain (mSox9HMG), its crystallization in complex with a ChIP-Seq-identified FOXP2 promoter DNA element and the X-ray diffraction data analysis of this complex. The mSox9HMG–FOXP2 promoter DNA complex was crystallized by the hanging-drop vapour-diffusion method using 20% PEG 3350 in 200 mMsodium/potassium phosphate with 100 mMbis-tris propane at pH 8.5. The crystals diffracted to 2.7 Å resolution and the complex crystallized in the tetragonal space groupP4₁2₁2, with unit-cell parametersa=b= 99.49,c= 45.89 Å. Crystal-packing parameters revealed that asymmetric unit contained one mSox9HMG–FOXP2 promoter DNA complex with an estimated solvent content of 64%.

Regulation of TCF ETS-domain transcription factors by helix-loop-helix motifs.

Science.gov (United States)

Stinson, Julie; Inoue, Toshiaki; Yates, Paula; Clancy, Anne; Norton, John D; Sharrocks, Andrew D

2003-08-15

DNA binding by the ternary complex factor (TCF) subfamily of ETS-domain transcription factors is tightly regulated by intramolecular and intermolecular interactions. The helix-loop-helix (HLH)-containing Id proteins are trans-acting negative regulators of DNA binding by the TCFs. In the TCF, SAP-2/Net/ERP, intramolecular inhibition of DNA binding is promoted by the cis-acting NID region that also contains an HLH-like motif. The NID also acts as a transcriptional repression domain. Here, we have studied the role of HLH motifs in regulating DNA binding and transcription by the TCF protein SAP-1 and how Cdk-mediated phosphorylation affects the inhibitory activity of the Id proteins towards the TCFs. We demonstrate that the NID region of SAP-1 is an autoinhibitory motif that acts to inhibit DNA binding and also functions as a transcription repression domain. This region can be functionally replaced by fusion of Id proteins to SAP-1, whereby the Id moiety then acts to repress DNA binding in cis. Phosphorylation of the Ids by cyclin-Cdk complexes results in reduction in protein-protein interactions between the Ids and TCFs and relief of their DNA-binding inhibitory activity. In revealing distinct mechanisms through which HLH motifs modulate the activity of TCFs, our results therefore provide further insight into the role of HLH motifs in regulating TCF function and how the inhibitory properties of the trans-acting Id HLH proteins are themselves regulated by phosphorylation.

CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems

Directory of Open Access Journals (Sweden)

Kira eMakarova

2014-04-01

Full Text Available CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold. Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and effector domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.

Next-Generation DNA Sequencing of VH/VL Repertoires: A Primer and Guide to Applications in Single-Domain Antibody Discovery.

Science.gov (United States)

Henry, Kevin A

2018-01-01

Immunogenetic analyses of expressed antibody repertoires are becoming increasingly common experimental investigations and are critical to furthering our understanding of autoimmunity, infectious disease, and cancer. Next-generation DNA sequencing (NGS) technologies have now made it possible to interrogate antibody repertoires to unprecedented depths, typically by sequencing of cDNAs encoding immunoglobulin variable domains. In this chapter, we describe simple, fast, and reliable methods for producing and sequencing multiplex PCR amplicons derived from the variable regions (V H , V H H or V L ) of rearranged immunoglobulin heavy and light chain genes using the Illumina MiSeq platform. We include complete protocols and primer sets for amplicon sequencing of V H /V H H/V L repertoires directly from human, mouse, and llama lymphocytes as well as from phage-displayed V H /V H H/V L libraries; these can be easily be adapted to other types of amplicons with little modification. The resulting amplicons are diverse and representative, even using as few as 10 3 input B cells, and their generation is relatively inexpensive, requiring no special equipment and only a limited set of primers. In the absence of heavy-light chain pairing, single-domain antibodies are uniquely amenable to NGS analyses. We present a number of applications of NGS technology useful in discovery of single-domain antibodies from phage display libraries, including: (i) assessment of library functionality; (ii) confirmation of desired library randomization; (iii) estimation of library diversity; and (iv) monitoring the progress of panning experiments. While the case studies presented here are of phage-displayed single-domain antibody libraries, the principles extend to other types of in vitro display libraries.

Chromatin challenges during DNA replication and repair

DEFF Research Database (Denmark)

Groth, Anja; Rocha, Walter; Verreault, Alain

2007-01-01

Inheritance and maintenance of the DNA sequence and its organization into chromatin are central for eukaryotic life. To orchestrate DNA-replication and -repair processes in the context of chromatin is a challenge, both in terms of accessibility and maintenance of chromatin organization. To meet...... the challenge of maintenance, cells have evolved efficient nucleosome-assembly pathways and chromatin-maturation mechanisms that reproduce chromatin organization in the wake of DNA replication and repair. The aim of this Review is to describe how these pathways operate and to highlight how the epigenetic...... landscape may be stably maintained even in the face of dramatic changes in chromatin structure....

Growth, maturation, functional capacities and sport-specific skills in 12-13 year-old- basketball players

NARCIS (Netherlands)

Coelho e Silva, M. J.; Moreira Carvalho, H.; Goncalves, C. E.; Figueiredo, A. J.; Elferink-Gemser, M. T.; Philippaerts, R. M.; Malina, R. M.

Aim. The influence of maturity status on body size, functional capacities and basketball-specific skills was evaluated and multivariate relationships between domains of variables were examined in 80 male basketball players 12.0-13.9 years. Methods. Height, body mass and two skinfolds were measured.

Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain.

Science.gov (United States)

Rust, K; Grosso, L; Zhang, V; Chang, D; Persson, A; Longmore, W; Cai, G Z; Crouch, E

1991-10-01

Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.

Melatonin-induced increase of lipid droplets accumulation and in vitro maturation in porcine oocytes is mediated by mitochondrial quiescence.

Science.gov (United States)

He, Bin; Yin, Chao; Gong, Yabin; Liu, Jie; Guo, Huiduo; Zhao, Ruqian

2018-01-01

Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA-encoded genes was significantly down-regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin-treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up-regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin-induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence. © 2017 Wiley Periodicals, Inc.

Heterochromatinization associated with cell differentiation as a model to study DNA double strand break induction and repair in the context of higher-order chromatin structure

International Nuclear Information System (INIS)

Falk, Martin; Lukášová, Emilie; Štefančíková, Lenka; Baranová, Elena; Falková, Iva; Ježková, Lucie; Davídková, Marie; Bačíková, Alena; Vachelová, Jana; Michaelidesová, Anna; Kozubek, Stanislav

2014-01-01

Cell differentiation is associated with extensive gene silencing, heterochromatinization and potentially decreasing need for repairing DNA double-strand breaks (DSBs). Differentiation stages of blood cells thus represent an excellent model to study DSB induction, repair and misrepair in the context of changing higher-order chromatin structure. We show that immature granulocytes form γH2AX and 53BP1 foci, contrary to the mature cells; however, these foci colocalize only rarely and DSB repair is inefficient. Moreover, specific chromatin structure of granulocytes probably influences DSB induction. - Highlights: ► DSB repair is absent in mature granulocytes with condensed chromatin. ► Repair proteins and γH2AX appear in immature stages but rarely colocalize. ► γH2AX persist long times in these cells and DSB repair is inefficient. ► Even though, γH2AX foci “move” out of the dense chromatin. ► 53BP1 enters HP1β domains only after their decondensation

In silico studies on structure-function of DNA GCC- box binding domain of brassica napus DREB1 protein

International Nuclear Information System (INIS)

Qamarunnisa, S.; Hussain, M.

2012-01-01

DREB1 is a transcriptional factor, which selectively binds with the promoters of the genes involved in stress response in the plants. Homology of DREB protein and its binding element have been detected in the genome of many plants. However, only a few reports exist that discusses the binding properties of this protein with the gene (s) promoter. In the present study, we have undertaken studies exploring the structure-function relationship of Brassica napus DREB1. Multiple sequence alignment, protein homology modeling and intermolecular docking of GCC-box binding domain (GBD) of the said protein was carried out using atomic coordinates of GBD from Arabdiopsis thaliana and GCC-box containing DNA respectively. Similarities and/or identities in multiple, sequence alignment, particularly at the functionally important amino acids, strongly suggested the binding specificity of B. napus DREB1 to GCC-box. Similarly, despite 56% sequence homology, tertiary structures of both template and modeled protein were found to be extremely similar as indicated by root mean square deviation of 0.34 A. More similarities were established between GBD of both A. thaliana and B. napus DREB1 by conducting protein docking with the DNA containing GCC-box. It appears that both proteins interact through their beta-sheet with the major DNA groove including both nitrogen bases and phosphate and sugar moieties. Additionally, in most cases the interacting residues were also found to be identical. Briefly, this study attempts to elucidate the molecular basis of DREB1 interaction with its target sequence in the promoter. (author)

Toward The Reconstitution of the Maturation of Okazaki Fragments Multiprotein Complex in Human At The Single Molecule Level

KAUST Repository

Joudeh, Luay

2017-01-01

The maturation of Okazaki fragments on the lagging strand in eukaryotes is mediated by a highly coordinated multistep process involving several proteins that ensure the accurate and efficient replication of genomic DNA. Human proliferating cell

Cell-penetrating DNA-binding protein as a safe and efficient naked DNA delivery carrier in vitro and in vivo

International Nuclear Information System (INIS)

Kim, Eun-Sung; Yang, Seung-Woo; Hong, Dong-Ki; Kim, Woo-Taek; Kim, Ho-Guen; Lee, Sang-Kyou

2010-01-01

Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS, and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 μg of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.

Cell-penetrating DNA-binding protein as a safe and efficient naked DNA delivery carrier in vitro and in vivo

Energy Technology Data Exchange (ETDEWEB)

Kim, Eun-Sung; Yang, Seung-Woo [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Hong, Dong-Ki; Kim, Woo-Taek [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Ho-Guen [Department of Pathology, Yonsei Medical School, Seoul 120-752 (Korea, Republic of); Lee, Sang-Kyou, E-mail: sjrlee@yonsei.ac.kr [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

2010-01-29

Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS, and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 {mu}g of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.

The Bacteriophage lambdaDNA packaging enzyme: Identification of four structural domains of the gpNu1 subunit using limited proteolysis

Directory of Open Access Journals (Sweden)

PAMELA ARAYA

2001-01-01

Full Text Available Lambda DNA terminase, the enzyme that cleaves virion-length chromosomes from multigenomic concatemers and packages them into the bacteriophage head, is composed of two subunits, gpNu1 and gpA. Direct determination of the structure of gpNu1, the smaller subunit, has not been possible because of its insolubility in aqueous solutions. Therefore, to identify smaller and potentially water-soluble domains of gpNu1, we analyzed the nature of the products obtained by limited digestion of the protein with several proteases. The gpNu1 subunit was obtained from E.coli cells transfected with the plasmid pH6-Nu1 that overproduces the protein. Incubation of gpNu1 solubized in 2.5 M guanidinium chloride with chymotrypsin resulted in the formation of at least eight discrete protein bands, while treatment with endoproteinase glu-C and bromelain yielded three and one major bands, respectively. The peptides generated by digestion with the various proteases were separated by two-dimensional gel electrophoresis and transferred to Immobilon membranes. Amino acid sequencing of the peptides allowed for the precise assignment of their N-terminal amino acid, while their estimated molecular weights permitted the identification of their C-terminal ends. The results reveal that in the presence of 2.5 M guanidinium chloride, gpNu1 is partially folded in at least four distinct structural domains that correspond to functional domains as determined by previously reported genetic experiments. This information is key to design new plasmids to overproduce these domains for further structural analysis.

Clustering of double strand break-containing chromosome domains is not inhibited by inactivation of major repair proteins

International Nuclear Information System (INIS)

Krawczyk, P. M.; Stap, C.; Van Oven, C.; Hoebe, R.; Aten, J. A.

2006-01-01

For efficient repair of DNA double strand breaks (DSBs) cells rely on a process that involves the Mre11/Rad50/Nbs1 complex, which may help to protect non-repaired DNA ends from separating until they can be rejoined by DNA repair proteins. It has been observed that as a secondary effect, this process can lead to unintended clustering of multiple, initially separate, DSB-containing chromosome domains. This work demonstrates that neither inactivation of the major repair proteins XRCC3 and the DNA-dependent protein kinase (DNA-PK) nor inhibition of DNA-PK by vanillin influences the aggregation of DSB-containing chromosome domains. (authors)

Isolation and characterization of a PUF-domain of pumilio gene from ...

African Journals Online (AJOL)

STORAGESEVER

2009-03-20

Mar 20, 2009 ... study, a partial pumilio gene with complete PUF-domain in Bombyx mori has been ... Key words: Bombyx mori, pumilio, PUF-domain, RACE, germline stem cell. .... The first-strand cDNA was synthesized from 2 ug of total. RNA.

A hepatocellular carcinoma cell line producing mature hepatitis B viral particles

International Nuclear Information System (INIS)

Fellig, Yakov; Almogy, Gidon; Galun, Eithan; Ketzinel-Gilad, Mali

2004-01-01

Current in vitro models for hepatitis B virus (HBV) are based on human hepatoblastoma cell lines transfected with HBV genome. The objective of this work was to develop an in vitro, hepatocellular carcinoma (HCC)-based system supporting HBV full replication and producing mature viral particles. The FLC4 human HCC cell line was stably transfected with a plasmid carrying a head-to-tail dimer of the adwHBV genome. One of the clones, FLC4A10 II , exhibited prolonged expression of HBV, as was demonstrated by secreted levels of HBsAg, HBeAg, and HBV DNA in the culture medium of the growing cells. Furthermore, the cells produced HBV particles that were detected by a cesium chloride density gradient performed on the culture medium. Analysis by Southern blot revealed that HBV DNA has integrated into the FLC4A10 II cell genome. The presence of HBV in the FLC4A10 II cells did not cause alterations in cell morphology and the cells continued to resemble mature hepatocytes. They do exhibit a high mitotic activity. The new HBV stably transfected cell line, FLC4A10 II , can serve as an important tool for further exploration of HBV host-pathogen interaction, viral life cycle, and for assessing new antiviral agents

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

Science.gov (United States)

Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

2008-08-22

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor

Science.gov (United States)

Leavitt, Justin C.; Gilcrease, Eddie B.; Wilson, Kassandra; Casjens, Sherwood R.

2013-01-01

Bacteriophage Sf6 DNA packaging series initiate at many locations across a 2 kbp region. Our in vivo studies that show that Sf6 small terminase subunit (TerS) protein recognizes a specific packaging (pac) site near the center of this region, that this site lies within the portion of the Sf6 gene that encodes the DNA-binding domain of TerS protein, that this domain of the TerS protein is responsible for the imprecision in Sf6 packaging initiation, and that the DNA-binding domain of TerS must be covalently attached to the domain that interacts with the rest of the packaging motor. The TerS DNA-binding domain is self-contained in that it apparently does not interact closely with the rest of the motor and it binds to a recognition site that lies within the DNA that encodes the domain. This arrangement has allowed the horizontal exchange of terS genes among phages to be very successful. PMID:23562538

Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages

Energy Technology Data Exchange (ETDEWEB)

Sun, Siyang; Gao, Song; Kondabagil, Kiran; Xiang, Ye; Rossmann, Michael G.; Rao, Venigalla B. (CUA); (Purdue)

2012-04-04

Tailed DNA bacteriophages assemble empty procapsids that are subsequently filled with the viral genome by means of a DNA packaging machine situated at a special fivefold vertex. The packaging machine consists of a 'small terminase' and a 'large terminase' component. One of the functions of the small terminase is to initiate packaging of the viral genome, whereas the large terminase is responsible for the ATP-powered translocation of DNA. The small terminase subunit has three domains, an N-terminal DNA-binding domain, a central oligomerization domain, and a C-terminal domain for interacting with the large terminase. Here we report structures of the central domain in two different oligomerization states for a small terminase from the T4 family of phages. In addition, we report biochemical studies that establish the function for each of the small terminase domains. On the basis of the structural and biochemical information, we propose a model for DNA packaging initiation.

DNA structure modulates the oligomerization properties of the AAV initiator protein Rep68.

Directory of Open Access Journals (Sweden)

Jorge Mansilla-Soto

2009-07-01

Full Text Available Rep68 is a multifunctional protein of the adeno-associated virus (AAV, a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3 helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag and bovine papillomavirus (BPV E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID and an AAA(+ motor domain. The AAA(+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA(+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration.

Acrolein inhibits cytokine gene expression by alkylating cysteine and arginine residues in the NF-kappaB1 DNA binding domain.

Science.gov (United States)

Lambert, Cherie; Li, Jimei; Jonscher, Karen; Yang, Teng-Chieh; Reigan, Philip; Quintana, Megan; Harvey, Jean; Freed, Brian M

2007-07-06

Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The alpha,beta-unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of interleukin-2 (IL-2), IL-10, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha by human T cells but did not inhibit production of IL-8. The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) in cigarette smoke were inactive. Acrolein inhibited induction of NF-kappaB DNA binding activity after mitogenic stimulation of T cells but had no effect on induction of NFAT or AP-1. Acrolein inhibited NF-kappaB1 (p50) binding to the IL-2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2000-fold more potent than crotonaldehyde in blocking DNA binding to an NF-kappaB consensus sequence. Matrix-assisted laser desorption/ionization time-of-flight and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys-61 and Arg-307) in the DNA binding domain. Crotonaldehyde reacted with Cys-61, but not Arg-307, whereas the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor.

Binding of the N-Terminal Domain of the Lactococcal Bacteriophage TP901-1 CI Repressor to Its Target DNA: A Crystallography, Small Angle Scattering, and Nuclear Magnetic Resonance Study

DEFF Research Database (Denmark)

Frandsen, Kristian Erik Høpfner; Rasmussen, Kim K.; Jensen, Malene Ringkjøbing

2013-01-01

In most temperate bacteriophages, regulation of the choice of lysogenic or lytic life cycle is controlled by a CI repressor protein. Inhibition of transcription is dependent on a helix–turn–helix motif, often located in the N-terminal domain (NTD), which binds to specific DNA sequences (operator ...

SOXE transcription factors form selective dimers on non-compact DNA motifs through multifaceted interactions between dimerization and high-mobility group domains.

Science.gov (United States)

Huang, Yong-Heng; Jankowski, Aleksander; Cheah, Kathryn S E; Prabhakar, Shyam; Jauch, Ralf

2015-05-27

The SOXE transcription factors SOX8, SOX9 and SOX10 are master regulators of mammalian development directing sex determination, gliogenesis, pancreas specification and neural crest development. We identified a set of palindromic SOX binding sites specifically enriched in regulatory regions of melanoma cells. SOXE proteins homodimerize on these sequences with high cooperativity. In contrast to other transcription factor dimers, which are typically rigidly spaced, SOXE group proteins can bind cooperatively at a wide range of dimer spacings. Using truncated forms of SOXE proteins, we show that a single dimerization (DIM) domain, that precedes the DNA binding high mobility group (HMG) domain, is sufficient for dimer formation, suggesting that DIM : HMG rather than DIM:DIM interactions mediate the dimerization. All SOXE members can also heterodimerize in this fashion, whereas SOXE heterodimers with SOX2, SOX4, SOX6 and SOX18 are not supported. We propose a structural model where SOXE-specific intramolecular DIM:HMG interactions are allosterically communicated to the HMG of juxtaposed molecules. Collectively, SOXE factors evolved a unique mode to combinatorially regulate their target genes that relies on a multifaceted interplay between the HMG and DIM domains. This property potentially extends further the diversity of target genes and cell-specific functions that are regulated by SOXE proteins.

SOXE transcription factors form selective dimers on non-compact DNA motifs through multifaceted interactions between dimerization and high-mobility group domains

Science.gov (United States)

Huang, Yong-Heng; Jankowski, Aleksander; Cheah, Kathryn S. E.; Prabhakar, Shyam; Jauch, Ralf

2015-01-01

The SOXE transcription factors SOX8, SOX9 and SOX10 are master regulators of mammalian development directing sex determination, gliogenesis, pancreas specification and neural crest development. We identified a set of palindromic SOX binding sites specifically enriched in regulatory regions of melanoma cells. SOXE proteins homodimerize on these sequences with high cooperativity. In contrast to other transcription factor dimers, which are typically rigidly spaced, SOXE group proteins can bind cooperatively at a wide range of dimer spacings. Using truncated forms of SOXE proteins, we show that a single dimerization (DIM) domain, that precedes the DNA binding high mobility group (HMG) domain, is sufficient for dimer formation, suggesting that DIM : HMG rather than DIM:DIM interactions mediate the dimerization. All SOXE members can also heterodimerize in this fashion, whereas SOXE heterodimers with SOX2, SOX4, SOX6 and SOX18 are not supported. We propose a structural model where SOXE-specific intramolecular DIM:HMG interactions are allosterically communicated to the HMG of juxtaposed molecules. Collectively, SOXE factors evolved a unique mode to combinatorially regulate their target genes that relies on a multifaceted interplay between the HMG and DIM domains. This property potentially extends further the diversity of target genes and cell-specific functions that are regulated by SOXE proteins. PMID:26013289

Crystal Structure of the HEAT Domain from the Pre-mRNA Processing Factor Symplekin

Energy Technology Data Exchange (ETDEWEB)

Kennedy, Sarah A.; Frazier, Monica L.; Steiniger, Mindy; Mast, Ann M.; Marzluff, William F.; Redinbo, Matthew R.; (UNC)

2010-09-30

The majority of eukaryotic pre-mRNAs are processed by 3'-end cleavage and polyadenylation, although in metazoa the replication-dependent histone mRNAs are processed by 3'-end cleavage but not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the {approx} 1160-residue protein Symplekin. Secondary-structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain were investigated to begin elucidating the role Symplekin plays in mRNA maturation. The crystal structure of the Drosophila melanogaster Symplekin HEAT domain was determined to 2.4 {angstrom} resolution with single-wavelength anomalous dispersion phasing methods. The structure exhibits five canonical HEAT repeats along with an extended 31-amino-acid loop (loop 8) between the fourth and fifth repeat that is conserved within closely related Symplekin sequences. Molecular dynamics simulations of this domain show that the presence of loop 8 dampens correlated and anticorrelated motion in the HEAT domain, therefore providing a neutral surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts. The Symplekin HEAT region not only structurally aligns with several established scaffolding proteins, but also has been reported to contact proteins essential for regulating 3'-end processing. Together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process.

Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition.

Science.gov (United States)

Izsvák, Zsuzsanna; Khare, Dheeraj; Behlke, Joachim; Heinemann, Udo; Plasterk, Ronald H; Ivics, Zoltán

2002-09-13

Sleeping Beauty (SB) is the most active Tc1/mariner-like transposon in vertebrate species. Each of the terminal inverted repeats (IRs) of SB contains two transposase-binding sites (DRs). This feature, termed the IR/DR structure, is conserved in a group of Tc1-like transposons. The DNA-binding region of SB transposase, similar to the paired domain of Pax proteins, consists of two helix-turn-helix subdomains (PAI + RED = PAIRED). The N-terminal PAI subdomain was found to play a dominant role in contacting the DRs. Transposase was able to bind to mutant sites retaining the 3' part of the DRs; thus, primary DNA binding is not sufficient to determine the specificity of the transposition reaction. The PAI subdomain was also found to bind to a transpositional enhancer-like sequence within the left IR of SB, and to mediate protein-protein interactions between transposase subunits. A tetrameric form of the transposase was detected in solution, consistent with an interaction between the IR/DR structure and a transposase tetramer. We propose a model in which the transpositional enhancer and the PAI subdomain stabilize complexes formed by a transposase tetramer bound at the IR/DR. These interactions may result in enhanced stability of synaptic complexes, which might explain the efficient transposition of Sleeping Beauty in vertebrate cells.

Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

KAUST Repository

Li, Lixin; Atef, Ahmed; Piatek, Agnieszka Anna; Ali, Zahir; Piatek, Marek J.; Aouida, Mustapha; Sharakuu, Altanbadralt; Mahjoub, Ali; Wang, Guangchao; Khan, Mohammad Suhail; Fedoroff, Nina V.; Zhu, Jiankang; Mahfouz, Magdy M.

2013-01-01

, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA

Structure of the C-terminal heme-binding domain of THAP domain containing protein 4 from Homo sapiens

Energy Technology Data Exchange (ETDEWEB)

Bianchetti, Christopher M.; Bingman, Craig A.; Phillips, Jr., George N. (UW)

2012-03-15

The thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence-specific DNA-binding domain that contains a C2-CH (Cys-Xaa{sub 2-4}-Cys-Xaa{sub 35-50}-Cys-Xaa{sub 2}-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila. THAP-containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus. To date, there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 identified human THAP domain-containing proteins (THAP0-11). In all human THAP protein, the THAP domain is located at the N-terminus and is {approx}90 residues in length. Although all of the human THAP-containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein. Even though the exact function of these THAP proteins is not well defined, there is evidence that they play a role in cell proliferation, apoptosis, cell cycle modulation, chromatin modification, and transcriptional regulation. THAP-containing proteins have also been implicated in a number of human disease states including heart disease, neurological defects, and several types of cancers. Human THAP4 is a 577-residue protein of unknown function that is proposed to bind DNA in a sequence-specific manner similar to THAP1 and has been found to be upregulated in response to heat shock. THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all known THAP4 orthologs. Several single-domain proteins lacking a THAP domain are found in plants and bacteria and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding*

Science.gov (United States)

Duan, Xiao-Lei; Liu, Na-Nv; Yang, Yan-Tao; Li, Hai-Hong; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

2015-01-01

The evolutionarily conserved G-quadruplexes (G4s) are faithfully inherited and serve a variety of cellular functions such as telomere maintenance, gene regulation, DNA replication initiation, and epigenetic regulation. Different from the Watson-Crick base-pairing found in duplex DNA, G4s are formed via Hoogsteen base pairing and are very stable and compact DNA structures. Failure of untangling them in the cell impedes DNA-based transactions and leads to genome instability. Cells have evolved highly specific helicases to resolve G4 structures. We used a recombinant nuclear form of Saccharomyces cerevisiae Pif1 to characterize Pif1-mediated DNA unwinding with a substrate mimicking an ongoing lagging strand synthesis stalled by G4s, which resembles a replication origin and a G4-structured flap in Okazaki fragment maturation. We find that the presence of G4 may greatly stimulate the Pif1 helicase to unwind duplex DNA. Further studies reveal that this stimulation results from G4-enhanced Pif1 dimerization, which is required for duplex DNA unwinding. This finding provides new insights into the properties and functions of G4s. We discuss the observed activation phenomenon in relation to the possible regulatory role of G4s in the rapid rescue of the stalled lagging strand synthesis by helping the replicator recognize and activate the replication origin as well as by quickly removing the G4-structured flap during Okazaki fragment maturation. PMID:25627683

Efficiency and Fidelity of Human DNA Polymerases λ and β during Gap-Filling DNA Synthesis

Science.gov (United States)

Brown, Jessica A.; Pack, Lindsey R.; Sanman, Laura E.; Suo, Zucai

2010-01-01

The base excision repair (BER) pathway coordinates the replacement of 1 to 10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase β (Pol β), as it filled 1- to 10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol β did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol β, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5′-template base. Our results suggested that both Pol λ and Pol β would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol β, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER. PMID:20961817

DNA fusion gene vaccines

DEFF Research Database (Denmark)

Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup

2010-01-01

DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

Directory of Open Access Journals (Sweden)

Philip Serwer

2017-05-01

Full Text Available Adenosine triphosphate (ATP cleavage powers packaging of a double-stranded DNA (dsDNA molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

Decision support methods for finding phenotype--disorder associations in the bone dysplasia domain.

Directory of Open Access Journals (Sweden)

Razan Paul

Full Text Available A lack of mature domain knowledge and well established guidelines makes the medical diagnosis of skeletal dysplasias (a group of rare genetic disorders a very complex process. Machine learning techniques can facilitate objective interpretation of medical observations for the purposes of decision support. However, building decision support models using such techniques is highly problematic in the context of rare genetic disorders, because it depends on access to mature domain knowledge. This paper describes an approach for developing a decision support model in medical domains that are underpinned by relatively sparse knowledge bases. We propose a solution that combines association rule mining with the Dempster-Shafer theory (DST to compute probabilistic associations between sets of clinical features and disorders, which can then serve as support for medical decision making (e.g., diagnosis. We show, via experimental results, that our approach is able to provide meaningful outcomes even on small datasets with sparse distributions, in addition to outperforming other Machine Learning techniques and behaving slightly better than an initial diagnosis by a clinician.

One-pot synthesis of strongly fluorescent DNA-CuInS2 quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

International Nuclear Information System (INIS)

Liu, Ziping; Su, Xingguang

2016-01-01

Herein, high quality DNA-CuInS 2 QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS 2 QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS 2 QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS 2 QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I 0 (I 0 is the original fluorescence intensity of DNA-CuInS 2 QDs, and I is the fluorescence intensity of DNA-CuInS 2 QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L −1 with a detection limit of 0.013 nmol L −1 . The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS 2 QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were obtained. • This method was utilized to detect

DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

OpenAIRE

Kristie, T M; Roizman, B

1986-01-01

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

DNA adducts in senescent cells

International Nuclear Information System (INIS)

Gaubatz, J.W.

1987-01-01

Perturbations in DNA repair and other metabolic processes during development and aging might affect the steady-state level of genomic damage. The persistence or accumulation of DNA lesions in postmitotic cells could have a significant impact on proper cellular function, interfering with gene regulation for example. To test the notion that DNA damage increases as a function of age in non-dividing cells, DNA was purified from heart tissue of C57BL/6Nia mice at different ages and analyzed by post labeling techniques to detect DNA adducts. In the present experiments, four-dimensional, thin-layer chromatography was used to isolate aromatic adducts that were labeled with carrier-free (γ- 32 P) ATP under DNA-P excess conditions. The complexity and frequency of aromatic adducts varied between DNA samples. Several adducts were present in all preparations and were clearly more abundant in nucleotide maps of mature and old heart DNA. However, a direct correlation with age was not observed. In contrast, experiments in which aromatic adducts were first isolated by phase-transfer to 1-butanol, then labeled with excess (γ- 32 P)ATP indicated that there was an age-related increase in these adducts. The results are consistent with their earlier studies that showed alkyl adducts increased during aging of mouse myocardium and suggest that a common repair pathway might be involved

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

Science.gov (United States)

Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

2014-04-16

Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

Assessment of cortical maturation with prenatal MRI. Part I: normal cortical maturation

Energy Technology Data Exchange (ETDEWEB)

Fogliarini, Celine [Faculte Timone, Centre de Resonance Magnetique Biologique et Medicale, Marseille (France); Chaumoitre, Katia [Hopital Nord, Department of Radiology, Marseille (France); Chapon, Frederique; Levrier, Olivier; Girard, Nadine [Hopital Timone, Department of Neuroradiology, Marseille Cedex 5 (France); Fernandez, Carla; Figarella-Branger, Dominique [Hopital Timone, Department of Pathology, Marseille (France)

2005-08-01

Cortical maturation, especially gyral formation, follows a temporospatial schedule and is a good marker of fetal maturation. Although ultrasonography is still the imaging method of choice to evaluate fetal anatomy, MRI has an increasingly important role in the detection of brain abnormalities, especially of cortical development. Knowledge of MRI techniques in utero with the advantages and disadvantages of some sequences is necessary, in order to try to optimize the different magnetic resonance sequences to be able to make an early diagnosis. The different steps of cortical maturation known from histology represent the background necessary for the understanding of maturation in order to be then able to evaluate brain maturation through neuroimaging. Illustrations of the normal cortical maturation are given for each step accessible to MRI for both the cerebral hemispheres and the posterior fossa. (orig.)

Assessment of cortical maturation with prenatal MRI. Part I: normal cortical maturation

International Nuclear Information System (INIS)

Fogliarini, Celine; Chaumoitre, Katia; Chapon, Frederique; Levrier, Olivier; Girard, Nadine; Fernandez, Carla; Figarella-Branger, Dominique

2005-01-01

Cortical maturation, especially gyral formation, follows a temporospatial schedule and is a good marker of fetal maturation. Although ultrasonography is still the imaging method of choice to evaluate fetal anatomy, MRI has an increasingly important role in the detection of brain abnormalities, especially of cortical development. Knowledge of MRI techniques in utero with the advantages and disadvantages of some sequences is necessary, in order to try to optimize the different magnetic resonance sequences to be able to make an early diagnosis. The different steps of cortical maturation known from histology represent the background necessary for the understanding of maturation in order to be then able to evaluate brain maturation through neuroimaging. Illustrations of the normal cortical maturation are given for each step accessible to MRI for both the cerebral hemispheres and the posterior fossa. (orig.)

Expansion of protein domain repeats.

Directory of Open Access Journals (Sweden)

Asa K Björklund

2006-08-01

Full Text Available Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.

DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation.

Science.gov (United States)

Huang, Shijiao; Xu, Xiaowei; Wang, Guopeng; Lu, Guoliang; Xie, Wenbing; Tao, Wei; Zhang, Hongyin; Jiang, Qing; Zhang, Chuanmao

2016-04-01

RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells. © 2016. Published by The Company of Biologists Ltd.

ORGANIZATIONAL PROJECT MANAGEMENT MATURITY

Directory of Open Access Journals (Sweden)

Yana Derenskaya

2017-11-01

Full Text Available The present article is aimed at developing a set of recommendations for achieving a higher level of organizational project maturity at a given enterprise. Methodology. For the purposes of the current research, the available information sources on the components of project management system are analysed; the essence of “organizational maturity” and the existing models of organizational maturity are studied. The method of systemic and structural analysis, as well as the method of logical generalization, are employed in order to study the existing models of organizational maturity, to describe levels of organizational maturity, and finally to develop a set of methodological recommendations for achieving a higher level of organizational project maturity at a given enterprise. The results of the research showed that the core elements of project management system are methodological, organizational, programtechnical, and motivational components. Project management encompasses a wide range of issues connected with organizational structure, project team, communication management, project participants, etc. However, the fundamental basis for developing project management concept within a given enterprise starts with defining its level of organizational maturity. The present paper describes various models of organizational maturity (staged, continuous, petal-shaped and their common types (H. Кеrzner Organizational Maturity Model, Berkeley PM Maturity Model, Organizational Project Management Maturity Model, Portfolio, Program & Project Management Maturity Model. The analysis of available theoretic works showed that the notion “organizational project maturity” refers to the capability of an enterprise to select projects and manage them with the intention of achieving its strategic goals in the most effective way. Importantly, the level of maturity can be improved by means of formalizing the acquired knowledge, regulating project-related activities

The herpes viral transcription factor ICP4 forms a novel DNA recognition complex

Science.gov (United States)

Tunnicliffe, Richard B.; Lockhart-Cairns, Michael P.; Levy, Colin; Mould, A. Paul; Jowitt, Thomas A.; Sito, Hilary; Baldock, Clair; Sandri-Goldin, Rozanne M.

2017-01-01

Abstract The transcription factor ICP4 from herpes simplex virus has a central role in regulating the gene expression cascade which controls viral infection. Here we present the crystal structure of the functionally essential ICP4 DNA binding domain in complex with a segment from its own promoter, revealing a novel homo-dimeric fold. We also studied the complex in solution by small angle X-Ray scattering, nuclear magnetic resonance and surface-plasmon resonance which indicated that, in addition to the globular domain, a flanking intrinsically disordered region also recognizes DNA. Together the data provides a rationale for the bi-partite nature of the ICP4 DNA recognition consensus sequence as the globular and disordered regions bind synergistically to adjacent DNA motifs. Therefore in common with its eukaryotic host, the viral transcription factor ICP4 utilizes disordered regions to enhance the affinity and tune the specificity of DNA interactions in tandem with a globular domain. PMID:28505309

Polynucleotide Phosphorylase, RNase E/G, and YbeY Are Involved in the Maturation of 4.5S RNA in Corynebacterium glutamicum.

Science.gov (United States)

Maeda, Tomoya; Tanaka, Yuya; Wachi, Masaaki; Inui, Masayuki

2017-03-01

Corynebacterium glutamicum has been applied for the industrial production of various metabolites, such as amino acids. To understand the biosynthesis of the membrane protein in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP is found in all three domains of life and plays an important role in the membrane insertion of proteins. SRP RNA is initially transcribed as precursor molecules; however, relatively little is known about its maturation. In C. glutamicum , SRP consists of the Ffh protein and 4.5S RNA lacking an Alu domain. In this study, we found that 3'-to-5' exoribonuclease, polynucleotide phosphorylase (PNPase), and two endo-type RNases, RNase E/G and YbeY, are involved in the 3' maturation of 4.5S RNA in C. glutamicum The mature form of 4.5S RNA was inefficiently formed in Δ rneG Δ pnp mutant cells, suggesting the existence of an alternative pathway for the 3' maturation of 4.5S RNA. Primer extension analysis also revealed that the 5' mature end of 4.5S RNA corresponds to that of the transcriptional start site. Immunoprecipitated Ffh protein contained immature 4.5S RNA in Δ pnp , Δ rneG , and Δ ybeY mutants, suggesting that 4.5S RNA precursors can interact with Ffh. These results imply that the maturation of 4.5S RNA can be performed in the 4.5S RNA-Ffh complex. IMPORTANCE Overproduction of a membrane protein, such as a transporter, is useful for engineering of strains of Corynebacterium glutamicum , which is a workhorse of amino acid production. To understand membrane protein biogenesis in this bacterium, we investigated the process of signal recognition particle (SRP) assembly. SRP contains the Ffh protein and SRP RNA and plays an important role in the membrane insertion of proteins. Although SRP RNA is highly conserved among the three domains of life, relatively little is known about its maturation. We show that PNPase, RNase E/G, and YbeY are involved in the 3' maturation of the SRP RNA (4.5S RNA) in

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

KAUST Repository

Diaz Galicia, Miriam Escarlet

2018-01-01

is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain

One-pot synthesis of strongly fluorescent DNA-CuInS{sub 2} quantum dots for label-free and ultrasensitive detection of anthrax lethal factor DNA

Energy Technology Data Exchange (ETDEWEB)

Liu, Ziping; Su, Xingguang, E-mail: suxg@jlu.edu.cn

2016-10-26

Herein, high quality DNA-CuInS{sub 2} QDs are facilely synthesized through a one-pot hydrothermal method with fluorescence quantum yield as high as 23.4%, and the strongly fluorescent DNA-CuInS{sub 2} QDs have been utilized as a novel fluorescent biosensor for label-free and ultrasensitive detection of anthrax lethal factor DNA. L-Cysteine (L-Cys) and a specific-sequence DNA are used as co-ligands to stabilize the CuInS{sub 2} QDs. The specific-sequence DNA consists of two domains: phosphorothiolates domain (sulfur-containing variants of the usual phosphodiester backbone) controls the nanocrystal passivation and serves as a ligand, and the functional domain (non-phosphorothioates) controls the biorecognition. The as-prepared DNA-CuInS{sub 2} QDs have high stability, good water-solubility and low toxicity. Under the optimized conditions, a linear correlation was established between the fluorescence intensity ratio I/I{sub 0} (I{sub 0} is the original fluorescence intensity of DNA-CuInS{sub 2} QDs, and I is the fluorescence intensity of DNA-CuInS{sub 2} QDs/GO with the addition of various concentrations of anthrax lethal factor DNA) and the concentration of anthrax lethal factor DNA in the range of 0.029–0.733 nmol L{sup −1} with a detection limit of 0.013 nmol L{sup −1}. The proposed method has been successfully applied to the determination of anthrax lethal factor DNA sequence in human serum samples with satisfactory results. Because of low toxicity and fine biocompatibility, DNA-CuInS{sub 2} QDs also hold potential applications in bioimaging. - Highlights: • Strongly fluorescent DNA-QDs were successfully prepared by a one-pot hydrothermal method with quantum yield up to 23.4%. • A biosensor for label-free detection of anthrax lethal factor DNA was established based on the as-prepared DNA-QDs. • The DNA sensor took advantage of the feature that ssDNA binds to GO with significantly higher affinity than dsDNA. • Good sensitivity and selectivity were