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Sample records for activates dna polymerase

  1. DNA polymerase activity of tomato fruit chromoplasts.

    Science.gov (United States)

    Serra, E C; Carrillo, N

    1990-11-26

    DNA polymerase activity was measured in chromoplasts of ripening tomato fruits. Plastids isolated from young leaves or mature red fruits showed similar DNA polymerase activities. The same enzyme species was present in either chloroplasts or chromoplasts as judged by pH and temperature profiles, sensitivities towards different inhibitors and relative molecular mass (Mr 88 kDa). The activities analyzed showed the typical behaviour of plastid-type polymerases. The results presented here suggest that chromoplast maintain their DNA synthesis potential in fruit tissue at chloroplast levels. Consequently, the sharp decrease of the plastid chromosome transcription observed at the onset of fruit ripening could not be due to limitations in the availability of template molecules. Other mechanisms must be involved in the inhibition of chromoplast RNA synthesis.

  2. 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.

  3. Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

    OpenAIRE

    1991-01-01

    The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product s...

  4. DNA Polymerase e - More Than a Polymerase

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    Helmut Pospiech

    2003-01-01

    Full Text Available This paper presents a comprehensive review of the structure and function of DNA polymerase e. Together with DNA polymerases a and d, this enzyme replicates the nuclear DNA in the eukaryotic cell. During this process, DNA polymerase a lays down RNA-DNA primers that are utilized by DNA polymerases d and e for the bulk DNA synthesis. Attempts have been made to assign these two enzymes specifically to the synthesis of the leading and the lagging strand. Alternatively, the two DNA polymerases may be needed to replicate distinct regions depending on chromatin structure. Surprisingly, the essential function of DNA polymerase e does not depend on its catalytic activity, but resides in the nonenzymatic carboxy-terminal domain. This domain not only mediates the interaction of the catalytic subunit with the three smaller regulatory subunits, but also links the replication machinery to the S phase checkpoint. In addition to its role in DNA replication, DNA polymerase e fulfils roles in the DNA synthesis step of nucleotide excision and base excision repair, and has been implicated in recombinational processes in the cell.

  5. Developmental activity variations of DNA polymerase α,δ,ε in mouse forebrains and spleens

    Institute of Scientific and Technical Information of China (English)

    杨荣武; 陆长德

    1995-01-01

    The levels of DNA polymerase α,δ,ε were examined in the neonatal mouse forebrains andspleens.The levels of DNA polymerase α were determined by the difference of polymerase activity in theabsence and the presence of α specific inhibitor,BuPdGTP,or its monoclonal antibody.The levels of DNApolymerase δ were determined in H · A fractions after separating it from the other two enzymes.The levelsof DNA polymerase ε were identified in H · A fractions by the use of α-monoclonal antibody or BuPdGTP.Results showed that in the mouse forebrain DNA polymerase α,δ,ε activities are the highest before birth,decline sharply following birth and are very low on the 8th day and hardly detectable on the 17th day;as forthe mouse spleen,however,DNA polymerase α,δ,ε activities are the lowest at birth,increase rapidly afterbirth and reach their maxima on the 8th day and then decline gradually but remain in higher levels.Theseresults not only prove that DNA polymerase α and δ take part in cell DNA replication but also suggest thatDNA polymerase ε is involved in DNA replication.

  6. Sulfolobus Replication Factor C stimulates the activity of DNA Polymerase B1

    DEFF Research Database (Denmark)

    Xing, Xuanxuan; Zhang, Likui; Guo, Li;

    2014-01-01

    Replication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from...... with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication....... the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3'-5' exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent...

  7. The exonuclease activity of DNA polymerase γ is required for ligation during mitochondrial DNA replication

    Science.gov (United States)

    Macao, Bertil; Uhler, Jay P.; Siibak, Triinu; Zhu, Xuefeng; Shi, Yonghong; Sheng, Wenwen; Olsson, Monica; Stewart, James B.; Gustafsson, Claes M.; Falkenberg, Maria

    2015-01-01

    Mitochondrial DNA (mtDNA) polymerase γ (POLγ) harbours a 3′–5′ exonuclease proofreading activity. Here we demonstrate that this activity is required for the creation of ligatable ends during mtDNA replication. Exonuclease-deficient POLγ fails to pause on reaching a downstream 5′-end. Instead, the enzyme continues to polymerize into double-stranded DNA, creating an unligatable 5′-flap. Disease-associated mutations can both increase and decrease exonuclease activity and consequently impair DNA ligation. In mice, inactivation of the exonuclease activity causes an increase in mtDNA mutations and premature ageing phenotypes. These mutator mice also contain high levels of truncated, linear fragments of mtDNA. We demonstrate that the formation of these fragments is due to impaired ligation, causing nicks near the origin of heavy-strand DNA replication. In the subsequent round of replication, the nicks lead to double-strand breaks and linear fragment formation. PMID:26095671

  8. A novel type of replicative enzyme harbouring ATPase, primase and DNA polymerase activity

    Science.gov (United States)

    Lipps, Georg; Röther, Susanne; Hart, Christina; Krauss, Gerhard

    2003-01-01

    Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers ∼8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved indepen dently from the eubacterial and archaeal/eukaryal proteins of DNA replication. PMID:12743045

  9. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    Science.gov (United States)

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  10. Label-free molecular beacon for real-time monitoring of DNA polymerase activity.

    Science.gov (United States)

    Ma, Changbei; Liu, Haisheng; Wang, Jun; Jin, Shunxin; Wang, Kemin

    2016-05-01

    Traditional methods for assaying DNA polymerase activity are discontinuous, time consuming, and laborious. Here, we report a new approach for label-free and real-time monitoring of DNA polymerase activity using a Thioflavin T (ThT) probe. In the presence of DNA polymerase, the DNA primer could be elongated through polymerase reaction to open MB1, leading to the release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation. It exhibits a satisfying detection result for the activity of DNA polymerase with a low detection limit of 0.05 unit/ml. In addition, no labeling with a fluorophore or a fluorophore-quencher pair is required; this method is fairly simple, fast, and low cost. Furthermore, the proposed method was also applied to assay the inhibition of DNA polymerase activity. This approach may offer potential applications in drug screening, clinical diagnostics, and some other related biomedical research.

  11. Study of the activity of DNA polymerases β and λ using 5-formyluridine containing DNA substrates

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    Lavrik O. I.

    2012-06-01

    Full Text Available Aim. To investigate the TLS-activity of human DNA polymerases β and λ (pols β and λ using 5-formyluridine (5-foU containing DNA duplexes which are imitating the intermediates during replication of the leading DNA strand, and to study the influence of replication factors hRPA and hPCNA on this activity. Methods. The EMSA and the methods of enzyme’s kinetics were used. Results. The capability of pols β and λ to catalyze DNA synthesis across 5-foU was investigated and the kinetic characteristics of this process in the presence and in the absence of protein factors hRPA and hPCNA were evaluated. Conclusions. It was shown that: (i both proteins are able to catalyze TLS on used DNA substrates regardless of the reaction conditions, however, pol λ was more accurate enzyme; (ii hRPA can stimulate the efficacy of the nonmutagenic TLS catalyzed by pol at the nucleotide incorporation directly opposite of 5-foU, at the same time it doesn’t influence the incorporation efficacy if the damage displaced into the duplex; (iii hPCNA doesn’t influence the efficacy of TLS catalyzed by both enzymes.

  12. Polymerase/DNA interactions and enzymatic activity: multi-parameter analysis with electro-switchable biosurfaces

    Science.gov (United States)

    Langer, Andreas; Schräml, Michael; Strasser, Ralf; Daub, Herwin; Myers, Thomas; Heindl, Dieter; Rant, Ulrich

    2015-07-01

    The engineering of high-performance enzymes for future sequencing and PCR technologies as well as the development of many anticancer drugs requires a detailed analysis of DNA/RNA synthesis processes. However, due to the complex molecular interplay involved, real-time methodologies have not been available to obtain comprehensive information on both binding parameters and enzymatic activities. Here we introduce a chip-based method to investigate polymerases and their interactions with nucleic acids, which employs an electrical actuation of DNA templates on microelectrodes. Two measurement modes track both the dynamics of the induced switching process and the DNA extension simultaneously to quantitate binding kinetics, dissociation constants and thermodynamic energies. The high sensitivity of the method reveals previously unidentified tight binding states for Taq and Pol I (KF) DNA polymerases. Furthermore, the incorporation of label-free nucleotides can be followed in real-time and changes in the DNA polymerase conformation (finger closing) during enzymatic activity are observable.

  13. Effect of novel benzoylphenylurea derivatives on DNA polymerase alpha activity using the synthesome-based in vitro model system.

    Science.gov (United States)

    Abdel-Aziz, Waleed; Hickey, Robert; Edelman, Martin; Malkas, Linda

    2003-11-01

    Six benzoylphenylurea (BPU) derivatives have been synthesized in Japan and extensively evaluated by the U.S. National Cancer Institute. They demonstrated potent antitumor activity in vitro against several cancer cell lines as well as in vivo against several tumor models. One of these agents, NSC639829, has now entered clinical trials. Studies have shown that these compounds are effective inhibitors of in vitro tubulin polymerization. The parent compound, NSC624548 (HO-221), has been shown to inhibit calf thymus DNA polymerase alpha activity. In this study we examined the effects of four BPU derivatives (NSC624548, NSC639828, NSC639829, and NSC654259) on the activity of the synthesome-associated DNA polymerase alpha, Escherichia coli DNA polymerase I, and calf thymus DNA polymerase alpha. Among the compounds tested, only NSC624548 and NSC639828 inhibited the activities of E. coli DNA polymerase I and calf thymus DNA polymerase alpha. Excess DNA polymerase I or DNA polymerase alpha dramatically reduced the inhibition produced by these compounds. NSC624548 and NSC639828 also showed inhibitory effects of the synthesome-associated DNA polymerase alpha similar to that produced upon using the purified E. coli and calf thymus enzymes. All of the four compounds did not show inhibitory effect on DNA polymerase delta. The similar pattern of inhibition these compounds exert on both the purified calf thymus and the synthesome-associated DNA polymerase alpha offers further support for the validity of the DNA synthesome as a novel in vitro model system for studying anticancer drug action.

  14. Unfaithful DNA polymerase caught in the act

    OpenAIRE

    2004-01-01

    The 3D structures of all 12 mispairs formed in the active site of a DNA polymerase help explain their differential effects on polymerase stalling and on translocation of the primer terminus to the enzyme's proofreading site.

  15. Exploring the mechanism of DNA polymerases by analyzing the effect of mutations of active site acidic groups in Polymerase β.

    Science.gov (United States)

    Matute, Ricardo A; Yoon, Hanwool; Warshel, Arieh

    2016-11-01

    Elucidating the catalytic mechanism of DNA polymerase is crucial for a progress in the understanding of the control of replication fidelity. This work tries to advance the mechanistic understanding by analyzing the observed effect of mutations of the acidic groups in the active site of Polymerase β as well as the pH effect on the rate constant. The analysis involves both empirical valence bond (EVB) free energy calculations and considerations of the observed pH dependence of the reaction. The combined analysis indicates that the proton transfer (PT) from the nucleophilic O3' has two possible pathways, one to D256 and the second to the bulk. We concluded based on calculations and the experimental pH profile that the most likely path for the wild-type (WT) and the D256E and D256A mutants is a PT to the bulk, although the WT may also use a PT to Asp 256. Our analysis highlights the need for very extensive sampling in the calculations of the activation barrier and also clearly shows that ab initio QM/MM calculations that do not involve extensive sampling are unlikely to give a clear quantitative picture of the reaction mechanism. Proteins 2016; 84:1644-1657. © 2016 Wiley Periodicals, Inc.

  16. Insights into the conformation of aminofluorene-deoxyguanine adduct in a DNA polymerase active site.

    Science.gov (United States)

    Vaidyanathan, Vaidyanathan G; Liang, Fengting; Beard, William A; Shock, David D; Wilson, Samuel H; Cho, Bongsup P

    2013-08-09

    The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase β (pol β) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.

  17. Structure of the 2-Aminopurine-Cytosine Base Pair Formed in the Polymerase Active Site of the RB69 Y567A-DNA Polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Reha-Krantz, Linda J.; Hariharan, Chithra; Subuddhi, Usharani; Xia, Shuangluo; Zhao, Chao; Beckman, Jeff; Christian, Thomas; Konigsberg, William (Yale); (Alberta)

    2011-11-21

    The adenine base analogue 2-aminopurine (2AP) is a potent base substitution mutagen in prokaryotes because of its enhanceed ability to form a mutagenic base pair with an incoming dCTP. Despite more than 50 years of research, the structure of the 2AP-C base pair remains unclear. We report the structure of the 2AP-dCTP base pair formed within the polymerase active site of the RB69 Y567A-DNA polymerase. A modified wobble 2AP-C base pair was detected with one H-bond between N1 of 2AP and a proton from the C4 amino group of cytosine and an apparent bifurcated H-bond between a proton on the 2-amino group of 2-aminopurine and the ring N3 and O2 atoms of cytosine. Interestingly, a primer-terminal region rich in AT base pairs, compared to GC base pairs, facilitated dCTP binding opposite template 2AP. We propose that the increased flexibility of the nucleotide binding pocket formed in the Y567A-DNA polymerase and increased 'breathing' at the primer-terminal junction of A+T-rich DNA facilitate dCTP binding opposite template 2AP. Thus, interactions between DNA polymerase residues with a dynamic primer-terminal junction play a role in determining base selectivity within the polymerase active site of RB69 DNA polymerase.

  18. Bacteriophage T7 DNA polymerase — Sequenase

    Directory of Open Access Journals (Sweden)

    Bin eZhu

    2014-04-01

    Full Text Available An ideal DNA polymerase for chain-terminating DNA sequencing should possess the following features: 1 incorporate dideoxy- and other modified nucleotides at an efficiency similar to that of the cognate deoxynucleotides; 2 high processivity; 3 high fidelity in the absence of proofreading/exonuclease activity; and 4 production of clear and uniform signals for detection. The DNA polymerase encoded by bacteriophage T7 is naturally endowed with or can be engineered to have all these characteristics. The chemically or genetically modified enzyme (Sequenase expedited significantly the development of DNA sequencing technology. This article reviews the history of studies on T7 DNA polymerase with emphasis on the serial key steps leading to its use in DNA sequencing. Lessons from the study and development of T7 DNA polymerase have and will continue to enlighten the characterization of novel DNA polymerases from newly discovered microbes and their modification for use in biotechnology.

  19. Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase alpha activity.

    Science.gov (United States)

    Simbulan, C M; Taki, T; Tamiya-Koizumi, K; Suzuki, M; Savoysky, E; Shoji, M; Yoshida, S

    1994-03-16

    The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase alpha were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase alpha, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 microM of CSE, 50 microM of SPG or GM3, and 80 microM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, the ceramide backbone was also found to be necessary for maximal inhibition since the inhibition was largely abolished by substituting the lipid backbone with cholesterol. Increasing the number of sialic acid moieties per molecule further enhanced the inhibition, while elongating the sugar chain diminished it. It was clearly shown that the N-acetyl residue of the sialic acid moiety is particularly essential for inhibition by both SPG and GM3 because the loss of this residue or substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase alpha activity.

  20. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  1. DNA polymerase-α regulates the activation of type I interferons through cytosolic RNA:DNA synthesis.

    Science.gov (United States)

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J; Xing, Chao; Wang, Richard C; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R; Burstein, Ezra

    2016-05-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.

  2. Clostridium difficile DNA polymerase IIIC: basis for activity of antibacterial compounds.

    Science.gov (United States)

    Torti, Andrea; Lossani, Andrea; Savi, Lida; Focher, Federico; Wright, George Edward; Brown, Neal Curtis; Xu, Wei-Chu

    2011-10-01

    Based on the finding that aerobic Gram-positive antibacterials that inhibit DNA polymerase IIIC (pol IIIC) were potent inhibitors of the growth of anaerobic Clostridium difficile (CD) strains, we chose to clone and express the gene for pol IIIC from this organism. The properties of the recombinant enzyme are similar to those of related pol IIICs from Gram-positive aerobes, e.g. B. subtilis. Inhibitors of the CD enzyme also inhibited B. subtilis pol IIIC, and were competitive with respect to the cognate substrate 2'-deoxyguanosine 5'-triphosphate (dGTP). Significantly, several of these inhibitors of the CD pol IIIC had potent activity against the growth of CD clinical isolates in culture.

  3. Inhibitory effect of monogalactosyldiacylglycerol, extracted from spinach using supercritical CO2, on mammalian DNA polymerase activity.

    Science.gov (United States)

    Iijima, Hiroshi; Musumi, Keiichi; Hada, Takahiko; Maeda, Naoki; Yonezawa, Yuko; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2006-03-08

    We investigated the effective extraction of monogalactosyldiacylglycerol (MGDG) from dried spinach (Spinacia oleracea) using supercritical fluid carbon dioxide (SC-CO(2)) with a modifier/entrainer. The yield of MGDG in the SC-CO(2) extract was not influenced by increasing temperature at a constant pressure, although the total extract yield was decreased. The total extract yield and MGDG yield in the extract from commercially purchased spinach (unknown subspecies), were greatly influenced by lower pressure. In a modifier (i.e., ethanol) concentration range of 2.5-20%, both the extract and MGDG yield increased as the ethanol concentration rose. The highest total extract yield (69.5 mg/g of spinach) and a good MGDG yield (16.3 mg/g of spinach) were obtained at 80 degrees C, 25 MPa, and 20% ethanol. The highest MGDG concentration (76.0% in the extract) was obtained at 80 degrees C, 25 MPa, and 2.5% ethanol, although the total extract yield under these conditions was low (5.2 mg/g of spinach). The optimal conditions for the extraction of MGDG were 80 degrees C, 20 MPa, and 10% ethanol. Of the 11 subspecies of spinach tested under these conditions, "Ujyou" had the highest concentration of MGDG. The total extract yield and MGDG concentration of Ujyou were 20.4 mg of the extract/g of spinach and 70.5%, respectively. The concentration of MGDG was higher in the SC-CO(2) extract than in the extract obtained using solvents such as methanol and n-hexane. The extract of Ujyou, which was the optimal subspecies for the extraction of MGDG, inhibited the activity of calf DNA polymerase alpha with IC(50) values of 145 microg/mL but was not effective against DNA polymerase beta.

  4. The arabidopsis DNA polymerase δ has a role in the deposition of transcriptionally active epigenetic marks, development and flowering.

    Directory of Open Access Journals (Sweden)

    Francisco M Iglesias

    2015-02-01

    Full Text Available DNA replication is a key process in living organisms. DNA polymerase α (Polα initiates strand synthesis, which is performed by Polε and Polδ in leading and lagging strands, respectively. Whereas loss of DNA polymerase activity is incompatible with life, viable mutants of Polα and Polε were isolated, allowing the identification of their functions beyond DNA replication. In contrast, no viable mutants in the Polδ polymerase-domain were reported in multicellular organisms. Here we identify such a mutant which is also thermosensitive. Mutant plants were unable to complete development at 28°C, looked normal at 18°C, but displayed increased expression of DNA replication-stress marker genes, homologous recombination and lysine 4 histone 3 trimethylation at the SEPALLATA3 (SEP3 locus at 24°C, which correlated with ectopic expression of SEP3. Surprisingly, high expression of SEP3 in vascular tissue promoted FLOWERING LOCUS T (FT expression, forming a positive feedback loop with SEP3 and leading to early flowering and curly leaves phenotypes. These results strongly suggest that the DNA polymerase δ is required for the proper establishment of transcriptionally active epigenetic marks and that its failure might affect development by affecting the epigenetic control of master genes.

  5. Binding of Mn-deoxyribonucleoside Triphosphates to the Active Site of the DNA Polymerase of Bacteriophage T7

    Energy Technology Data Exchange (ETDEWEB)

    B Akabayov; C Richardson

    2011-12-31

    Divalent metal ions are crucial as cofactors for a variety of intracellular enzymatic activities. Mg{sup 2+}, as an example, mediates binding of deoxyribonucleoside 5'-triphosphates followed by their hydrolysis in the active site of DNA polymerase. It is difficult to study the binding of Mg{sup 2+} to an active site because Mg{sup 2+} is spectroscopically silent and Mg{sup 2+} binds with low affinity to the active site of an enzyme. Therefore, we substituted Mg{sup 2+} with Mn{sup 2+}:Mn{sup 2+} that is not only visible spectroscopically but also provides full activity of the DNA polymerase of bacteriophage T7. In order to demonstrate that the majority of Mn{sup 2+} is bound to the enzyme, we have applied site-directed titration analysis of T7 DNA polymerase using X-ray near edge spectroscopy. Here we show how X-ray near edge spectroscopy can be used to distinguish between signal originating from Mn{sup 2+} that is free in solution and Mn{sup 2+} bound to the active site of T7 DNA polymerase. This method can be applied to other enzymes that use divalent metal ions as a cofactor.

  6. Inhibition of DNA polymerase λ and associated inflammatory activities of extracts from steamed germinated soybeans.

    Science.gov (United States)

    Mizushina, Yoshiyuki; Kuriyama, Isoko; Yoshida, Hiromi

    2014-04-01

    During the screening of selective DNA polymerase (pol) inhibitors from more than 50 plant food materials, we found that the extract from steamed germinated soybeans (Glycine max L.) inhibited human pol λ activity. Among the three processed soybean samples tested (boiled soybeans, steamed soybeans, and steamed germinated soybeans), both the hot water extract and organic solvent extract from the steamed germinated soybeans had the strongest pol λ inhibition. We previously isolated two glucosyl compounds, a cerebroside (glucosyl ceramide, AS-1-4, compound ) and a steroidal glycoside (eleutheroside A, compound ), from dried soybean, and these compounds were prevalent in the extracts of the steamed germinated soybeans as pol inhibitors. The hot water and organic solvent extracts of the steamed germinated soybeans and compounds and selectively inhibited the activity of eukaryotic pol λ in vitro but did not influence the activities of other eukaryotic pols, including those from the A-family (pol γ), B-family (pols α, δ, and ε), and Y-family (pols η, ι, and κ), and also showed no effect on the activity of pol β, which is of the same family (X) as pol λ. The tendency for in vitro pol λ inhibition by these extracts and compounds showed a positive correlation with the in vivo suppression of TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation in mouse ear. These results suggest that steamed germinated soybeans, especially the glucosyl compound components, may be useful for their anti-inflammatory properties.

  7. A model for transition of 5'-nuclease domain of DNA polymerase I from inert to active modes.

    Directory of Open Access Journals (Sweden)

    Ping Xie

    Full Text Available Bacteria contain DNA polymerase I (PolI, a single polypeptide chain consisting of ∼930 residues, possessing DNA-dependent DNA polymerase, 3'-5' proofreading and 5'-3' exonuclease (also known as flap endonuclease activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must ultimately produce a double-stranded substrate with a nick suitable for DNA ligase to seal. PolI's activities must be highly coordinated both temporally and spatially otherwise uncontrolled 5'-nuclease activity could attack a nick and produce extended gaps leading to potentially lethal double-strand breaks. To investigate the mechanism of how PolI efficiently produces these nicks, we present theoretical studies on the dynamics of two possible scenarios or models. In one the flap DNA substrate can transit from the polymerase active site to the 5'-nuclease active site, with the relative position of the two active sites being kept fixed; while the other is that the 5'-nuclease domain can transit from the inactive mode, with the 5'-nuclease active site distant from the cleavage site on the DNA substrate, to the active mode, where the active site and substrate cleavage site are juxtaposed. The theoretical results based on the former scenario are inconsistent with the available experimental data that indicated that the majority of 5'-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data. We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities. Moreover, predicted results for the latter model are presented.

  8. The inhibition of P338 lymphocytic leukemia DNA polymerase alpha activity by cis-diamminedichloroplatinum(II) and related derivatives.

    Science.gov (United States)

    Oswald, C B; Hall, I H

    1989-01-01

    Cis-platinum derivatives were observed to inhibit the activity of DNA polymerase alpha of P388 lymphocytic leukemia cells. A 600g nuclear preparation of the polymerase alpha was inhibited by cis-diamminedichloroplatinum(II) [cDDP], diamminemalonatoplatinum(II) [MAL], (1,2-diaminocyclohexane)-dichloroplatinum(II) [DACH-Pt-CL2], and (1,2-diaminocyclohexane)malonato-platinum(II) [DACH-Pt-MAL]. cDDP was a more potent inhibitor of the enzyme activity which was positively correlated with the observed inhibition of DNA synthesis of P388 cells in vivo and in vitro. The inhibition of the 600g preparation by cDDP could be partially reversed by the addition of exogenous ctDNA, but 35% inhibition was not retreivable by adding new template. Isolation of the P388 DNA polymerase alpha enzyme by DEAE column chromatography led to an enzyme with 100 fold purification, which was sensitive to N-ethyl maleimide at 0.1 mM concentration. cDDP inhibited the activity of this enzyme in a dose dependent manner. However, MAL, DACH-Pt-Cl2 and DACH-Pt-MAL afforded no inhibition, nor did the latter two derivatives bind to the enzyme. cDDP inhibition of the activity of purified enzyme was partially reversed by the addition of exogenous ctDNA and by the addition of dGTP, whereas addition of other d(NTP)s had no effect on the recovery of the enzyme activity. These studies suggest that cDDP inhibits DNA polymerase alpha activity and that the inhibition is not the sole mechanism of the action of the drug in suppression of DNA synthesis and cell death. Preliminary studies suggest that the drug may bind to the apoprotein of the enzyme in a competitive manner with dGTP.

  9. In vitro effect of aflatoxin B1 on the transcriptional activity of DNA template, chromatin and soluble DNA-dependent RNA polymerases in buffalo liver.

    Science.gov (United States)

    Mashaly, R I; Habib, S L; Salem, M H; el Deeb, S A; Safwat, M M; Sarhan, F

    1988-04-01

    The effect of aflatoxin B1 on the DNA template and DNA-dependent RNA polymerases in buffalo liver was studied. Aflatoxin B1 inhibited both Mg2+- and Mn2+-activated RNA polymerases in a dose-dependent manner. At 10 micrograms the inhibition of both enzymes was almost complete. The inhibitory effect on the solubilized enzymes was higher than the chromatin-bound, suggesting a direct effect at the enzyme level. On the other hand, incubating DNA or deoxyribonucleoprotein (DNP) with 2 micrograms aflatoxin reduces its transcriptional capacity with a greater effect on the Mg2+-activated RNA polymerase than the Mn2+-activated enzyme. These results suggest that aflatoxin B1 inhibits in vitro transcription in buffalo liver at both enzyme and template levels.

  10. Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii.

    Science.gov (United States)

    Shen, Y; Tang, X-F; Matsui, E; Matsui, I

    2004-04-01

    Family D DNA polymerase (PolD) has recently been found in the Euryarchaeota subdomain of Archaea. Its genes are adjacent to several other genes related to DNA replication, repair and recombination in the genome, suggesting that this enzyme may be the major DNA replicase in Euryarchaeota. We successfully cloned, expressed, and purified the family D DNA polymerase from Pyrococcus horikoshii (PolDPho). By site-directed mutagenesis, we identified amino acid residues Asp-1122 and Asp-1124 of a large subunit as the essential residues responsible for DNA-polymerizing activity. We analysed the domain structure using proteins truncated at the N- and C-termini of both small and large subunits (DP1Pho and DP2Pho), and identified putative regions responsible for subunit interaction, oligomerization and regulation of the 3'-5' exonuclease activity in PolDPho. It was also found that the internal region of the putative zinc finger motif (cysteine cluster II) at the C-terminal of DP2Pho is involved in the 3'-5' exonuclease activity. Using gel filtration analysis, we determined the molecular masses of the recombinant PolDPho and the N-terminal putative dimerization domain of the large subunit, and proposed that PolD from P. horikoshii probably forms a heterotetrameric structure in solution. Based on these results, a model regarding the subunit interaction and regulation of activity of PolDPho is proposed.

  11. DNA polymerase beta can substitute for DNA polymerase I in the initiation of plasmid DNA replication.

    OpenAIRE

    1995-01-01

    We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant ...

  12. Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase

    Directory of Open Access Journals (Sweden)

    R.S. Diaz

    1998-10-01

    Full Text Available For certain applications of the polymerase chain reaction (PCR, it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA™ DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA™ and Taq, respectively, after reading about 3,000 bases each. We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.

  13. Replicative DNA polymerase mutations in cancer.

    Science.gov (United States)

    Heitzer, Ellen; Tomlinson, Ian

    2014-02-01

    Three DNA polymerases - Pol α, Pol δ and Pol ɛ - are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson-Crick base pairing and 3'exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to 'polymerase proofreading associated polyposis' (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an 'ultramutator' phenotype, with a dramatic increase in base substitutions.

  14. 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.

  15. DnaA-stimulated transcriptional activation of oriλ: Escherichia coli RNA polymerase β subunit as a transcriptional activator contact site

    OpenAIRE

    Szalewska-Palasz, Agnieszka; Wegrzyn, Alicja; Blaszczak, Adam; Taylor, Karol; Wegrzyn, Grzegorz

    1998-01-01

    We present evidence that Escherichia coli RNA polymerase β subunit may be a transcriptional activator contact site. Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage λ. We found that DnaA activates the pR promoter in vitro. Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of λ plasmids; this suppression was allele-specific. When a pote...

  16. Effects of Intermediates between Vitamins K2 and K3 on Mammalian DNA Polymerase Inhibition and Anti-Inflammatory Activity

    Directory of Open Access Journals (Sweden)

    Takeshi Azuma

    2011-02-01

    Full Text Available Previously, we reported that vitamin K3 (VK3, but not VK1 or VK2 (=MK-4, inhibits the activity of human DNA polymerase γ (pol γ. In this study, we chemically synthesized three intermediate compounds between VK2 and VK3, namely MK-3, MK-2 and MK-1, and investigated the inhibitory effects of all five compounds on the activity of mammalian pols. Among these compounds, MK-2 was the strongest inhibitor of mammalian pols α, κ and λ, which belong to the B, Y and X families of pols, respectively; whereas VK3 was the strongest inhibitor of human pol γ, an A-family pol. MK-2 potently inhibited the activity of all animal species of pol tested, and its inhibitory effect on pol λ activity was the strongest with an IC50 value of 24.6 μM. However, MK-2 did not affect the activity of plant or prokaryotic pols, or that of other DNA metabolic enzymes such as primase of pol α, RNA polymerase, polynucleotide kinase or deoxyribonuclease I. Because we previously found a positive relationship between pol λ inhibition and anti-inflammatory action, we examined whether these compounds could inhibit inflammatory responses. Among the five compounds tested, MK-2 caused the greatest reduction in 12-O-tetradecanoylphorbol-13-acetate (TPA-induced acute inflammation in mouse ear. In addition, in a cell culture system using mouse macrophages, MK-2 displayed the strongest suppression of the production of tumor necrosis factor (TNF-α induced by lipopolysaccharide (LPS. Moreover, MK-2 was found to inhibit the action of nuclear factor (NF-κB. In an in vivo mouse model of LPS-evoked acute inflammation, intraperitoneal injection of MK-2 in mice led to suppression of TNF-α production in serum. In conclusion, this study has identified VK2 and VK3 intermediates, such as MK-2, that are promising anti-inflammatory candidates.

  17. Threonine 79 is a hinge residue that governs the fidelity of DNA polymerase beta by helping to position the DNA within the active site.

    Science.gov (United States)

    Maitra, Mausumi; Gudzelak, Andrew; Li, Shu-Xia; Matsumoto, Yoshihiro; Eckert, Kristin A; Jager, Joachim; Sweasy, Joann B

    2002-09-20

    DNA polymerase beta (pol beta) is an ideal system for studying the role of its different amino acid residues in the fidelity of DNA synthesis. In this study, the T79S variant of pol beta was identified using an in vivo genetic screen. T79S is located in the N-terminal 8-kDa domain of pol beta and has no contact with either the DNA template or the incoming dNTP substrate. The T79S protein produced 8-fold more multiple mutations in the herpes simplex virus type 1-thymidine kinase assay than wild-type pol beta. Surprisingly, T79S is a misincorporation mutator only when using a 3'-recessed primer-template. In the presence of a single nucleotide-gapped DNA substrate, T79S displays an antimutator phenotype when catalyzing DNA synthesis opposite template C and has similar fidelity as wild type opposite templates A, G, or T. Threonine 79 is located directly between two helix-hairpin-helix motifs located within the 8-kDa and thumb domains of pol beta. As the pol beta enzyme closes into its active form, the helix-hairpin-helix motifs appear to assist in the production and stabilization of a 90 degrees bend of the DNA. The function of the bent DNA is to present the templating base to the incoming nucleotide substrate. We propose that Thr-79 is part of a hydrogen bonding network within the helix-hairpin-helix motifs that is important for positioning the DNA within the active site. We suggest that alteration of Thr-79 to Ser disrupts this hydrogen bonding network and results in an enzyme that is unable to bend the DNA into the proper geometry for accurate DNA synthesis.

  18. Proofreading activity of DNA polymerase Pol2 mediates 3'-end processing during nonhomologous end joining in yeast.

    Directory of Open Access Journals (Sweden)

    Shun-Fu Tseng

    2008-04-01

    Full Text Available Genotoxic agents that cause double-strand breaks (DSBs often generate damage at the break termini. Processing enzymes, including nucleases and polymerases, must remove damaged bases and/or add new bases before completion of repair. Artemis is a nuclease involved in mammalian nonhomologous end joining (NHEJ, but in Saccharomyces cerevisiae the nucleases and polymerases involved in NHEJ pathways are poorly understood. Only Pol4 has been shown to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends. We previously developed a chromosomal DSB assay in yeast to study factors involved in NHEJ. Here, we use this system to examine DNA polymerases required for NHEJ in yeast. We demonstrate that Pol2 is another major DNA polymerase involved in imprecise end joining. Pol1 modulates both imprecise end joining and more complex chromosomal rearrangements, and Pol3 is primarily involved in NHEJ-mediated chromosomal rearrangements. While Pol4 is the major polymerase to fill the gap that may form by imprecise pairing of overhanging 3' DNA ends, Pol2 is important for the recession of 3' flaps that can form during imprecise pairing. Indeed, a mutation in the 3'-5' exonuclease domain of Pol2 dramatically reduces the frequency of end joins formed with initial 3' flaps. Thus, Pol2 performs a key 3' end-processing step in NHEJ.

  19. DNA polymerase preference determines PCR priming efficiency

    Science.gov (United States)

    2014-01-01

    Background Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3’ hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Results Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3’ end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. Conclusions DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification

  20. 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

    2009-01-01

    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 Mg2+/Mn2+-dependent DNA/RNA polymerase, Mn2+-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 Mn2+-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. PMID:19211662

  1. Structure and associated DNA-helicase activity of a general transcription initiation factor that binds to RNA polymerase II.

    Science.gov (United States)

    Sopta, M; Burton, Z F; Greenblatt, J

    1989-10-05

    RAP30/74 is a heteromeric general transcription initiation factor which binds to RNA polymerase II. Here we report that preparations of RAP30/74 contain an ATP-dependent DNA helicase whose probable function is to melt the DNA at transcriptional start sites. The sequence of the RAP30 subunit of RAP30/74 indicates that RAP30 may be distantly related to bacterial sigma factors.

  2. Replicative DNA polymerase mutations in cancer☆

    Science.gov (United States)

    Heitzer, Ellen; Tomlinson, Ian

    2014-01-01

    Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions. PMID:24583393

  3. Kinetics and thermodynamics of DNA polymerases with exonuclease proofreading

    Science.gov (United States)

    Gaspard, Pierre

    2016-04-01

    Kinetic theory and thermodynamics are applied to DNA polymerases with exonuclease activity, taking into account the dependence of the rates on the previously incorporated nucleotide. The replication fidelity is shown to increase significantly thanks to this dependence at the basis of the mechanism of exonuclease proofreading. In particular, this dependence can provide up to a 100-fold lowering of the error probability under physiological conditions. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.

  4. Macromolecular crowding increases binding of DNA polymerase to DNA: an adaptive effect

    Energy Technology Data Exchange (ETDEWEB)

    Zimmerman, S.B.; Harrison, B.

    1987-05-01

    Macromolecular crowding extends the range of ionic conditions supporting high DNA polymerase reaction rates. Reactions tested were nick translation and gap-filling by DNA polymerase I of E. coli, nuclease and polymerase activities of the large fragment of that polymerase, and polymerization by the T4 DNA polymerase. For all of these reactions, high concentrations of nonspecific polymers increased enzymatic activity under otherwise inhibitory conditions resulting from relatively high ionic strength. The primary mechanism of the polymer effect seems to be to increase the binding of polymerase to DNA. They suggest that this effect of protein-DNA complexes is only one example of a general metabolic buffering action of crowded solutions on a variety of macromolecular interactions.

  5. Detection of DNA polymerase λ activity during seed germination and enhancement after salinity stress and dehydration in the plumules of indica rice (Oryza sativa L.

    Science.gov (United States)

    Sihi, Sayantani; Bakshi, Sankar; Sengupta, Dibyendu Narayan

    2015-02-01

    DNA polymerase λ (DNA pol λ) is the only reported X-family DNA polymerases in plants and has been shown to play a significant role in dry quiescent seeds, growth, development and nuclear DNA repair. cDNA for DNA pol λ has been reported in Arabidopsis and japonica rice cultivar and has been characterized from E. coli expressed protein, but very little is known about its activity at protein level in plants. The enzymatic activity of DNA pol λ was studied in dry, imbibed and during different germination stages of indica rice IR-8 (salt sensitive) by in-gel activity assay to determine its physiological role in important stages of growth and development. The upstream sequence was also analyzed using plantCARE database and was found to contain several cis-acting elements, including light responsive elements, dehydration responsive elements, Myb binding sites, etc. Hence, 4-day-old germinating seedlings of IR29, a salt-sensitive, but high yielding indica rice cultivar and Nonabokra, a salt-tolerant, but low yielding cultivar were treated with water (control) or 250 mM NaCl or 20% polyethyleneglycol-6000 for 4 and 8 h. The protein was analyzed by in vitro DNA pol λ activity assay, in-gel activity assay and Western blot analysis. DNA pol λ was not detected in dry seeds, but enhanced after imbibition and detectable from low level to high level during subsequent germination steps. Both salinity and dehydration stress led to the enhancement of the activity and protein level of DNA pol λ, as compared to control tissues. This is the first evidence of the salinity or dehydration stress induced enhancement of DNA pol λ activity in the plumules of rice (Oryza sativa L.) cultivars.

  6. Atomistic Molecular Dynamics Simulations of Mitochondrial DNA Polymerase γ

    DEFF Research Database (Denmark)

    Euro, Liliya; Haapanen, Outi; Róg, Tomasz

    2017-01-01

    DNA polymerase γ (Pol γ) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site...... of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol γ replicative complex. Our simulation data show that DNA binding triggers remarkable...

  7. NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Aruna S Jaiswal

    Full Text Available Recently approved chemotherapeutic agents to treat colorectal cancer (CRC have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β activity. Temozolomide (TMZ, an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715. In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

  8. Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase x sigma 54 holoenzyme by scanning force microscopy.

    Science.gov (United States)

    Rippe, K; Guthold, M; von Hippel, P H; Bustamante, C

    1997-07-11

    Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase x sigma 54 (RNAP x sigma 54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP x sigma 54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP x sigma 54 at the glnA promoter; (3) association between DNA bound RNAP x sigma 54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP x sigma 54. Measurements of the DNA bending angle of RNAP x sigma 54 closed promoter complexes yielded an apparent bending angle of 49(+/-24) degrees. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50(+/-24) degrees and 114(+/-18) degrees, suggesting that the transition from the RNAP x sigma 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.

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

    OpenAIRE

    2003-01-01

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

  10. Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies.

    Science.gov (United States)

    Sharma, Amit; Leach, Robert N; Gell, Christopher; Zhang, Nan; Burrows, Patricia C; Shepherd, Dale A; Wigneshweraraj, Sivaramesh; Smith, David Alastair; Zhang, Xiaodong; Buck, Martin; Stockley, Peter G; Tuma, Roman

    2014-04-01

    Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ(70) or σ(54), that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ(54) version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ(70) and σ(54), the domain movements of the latter have evolved to require an activator ATPase.

  11. 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; 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.

  12. A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

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    Agata Jacewicz

    Full Text Available Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A, that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

  13. Engineered DNA Polymerase Improves PCR Results for Plastid DNA

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    Melanie Schori

    2013-02-01

    Full Text Available Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase.

  14. Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol epsilon and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors.

    Science.gov (United States)

    Tahirov, Tahir H; Makarova, Kira S; Rogozin, Igor B; Pavlov, Youri I; Koonin, Eugene V

    2009-03-18

    Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved. We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase epsilon consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol epsilon evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol epsilon parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol epsilon, and the other one to the common ancestor of Pol alpha, Pol delta, and Pol zeta. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polepsilon shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases. Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal

  15. A novel kinetic analysis to calculate nucleotide affinity of proofreading DNA polymerases. Application to phi 29 DNA polymerase fidelity mutants.

    Science.gov (United States)

    Saturno, J; Blanco, L; Salas, M; Esteban, J A

    1995-12-29

    Amino acids Tyr254 and Tyr390 of phi 29 DNA polymerase belong to one of the most conserved regions in eukaryotic-type DNA polymerases. In this paper we report a mutational study of these two residues to address their role in nucleotide selection. This study was carried out by means of a new kinetic analysis that takes advantage of the competition between DNA polymerization and 3'-->5' exonuclease activity to measure the Km values for correct and incorrect nucleotides in steady-state conditions. This method is valid for any 3'-->5' exonuclease-containing DNA polymerase, without any restriction concerning catalytic rates of nucleotide incorporation. The results showed that the discrimination factor achieved by phi 29 DNA polymerase in the nucleotide binding step of DNA polymerization is 2.4 x 10(3), that is, a wrong nucleotide is bound with a 2.4 x 10(3)-fold lower affinity than the correct one. Mutants Y254F, Y390F, and Y390S showed discrimination values of 7.0 x 10(2), > 1.9 x 10(3), and 2.9 x 10(2), respectively. The reduced accuracy of nucleotide binding produced by mutations Y254F and Y390S lead us to propose that phi 29 DNA polymerase residues Tyr254 and Tyr390, highly conserved in eukaryotic-type DNA polymerases, are involved in nucleotide binding selection, thus playing a crucial role in the fidelity of DNA replication. Comparison of the discrimination factors of mutants Y390S and Y390F strongly suggests that the phenyl ring of Tyr390 is directly involved in checking base-pairing correctness of the incoming nucleotide.

  16. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    Science.gov (United States)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  17. Molecular architecture and function of adenovirus DNA polymerase

    NARCIS (Netherlands)

    Brenkman, A.B. (Arjan Bernard)

    2003-01-01

    Central to this thesis is the role of adenovirus DNA polymerase (Ad pol) in adenovirus DNA replication. Ad pol is a member of the family B DNA polymerases but belongs to a distinct subclass of polymerases that use a protein as primer. As Ad pol catalyses both the initiation and elongation phases and

  18. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

    Science.gov (United States)

    Tashjian, Tommy F.; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M.; Godoy, Veronica G.

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB’s fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability.

  19. Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η.

    Science.gov (United States)

    Su, Yan; Egli, Martin; Guengerich, F Peter

    2016-02-19

    Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10(3)-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.

  20. DNA polymerase delta is required for early mammalian embryogenesis.

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    Arikuni Uchimura

    Full Text Available BACKGROUND: In eukaryotic cells, DNA polymerase delta (Poldelta, whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Poldelta in mammalian development has not been thoroughly investigated. METHODOLOGY/PRINCIPAL FINDINGS: To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Poldelta-null (Pold1(-/- mice and D400A exchanged Poldelta (Pold1(exo/exo mice. The D400A exchange caused deficient 3'-5' exonuclease activity in the Poldelta protein. In Poldelta-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1(-/- mice suffered from peri-implantation lethality. Although Pold1(-/- blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Poldelta participates in DNA replication during mouse embryogenesis. In Pold1(exo/exo mice, although heterozygous Pold1(exo/+ mice were normal and healthy, Pold1(exo/exo and Pold1(exo/- mice suffered from tumorigenesis. CONCLUSIONS: These results clearly demonstrate that DNA polymerase delta is essential for mammalian early embryogenesis and that the 3'-5' exonuclease activity of DNA polymerase delta is dispensable for normal development but necessary to suppress tumorigenesis.

  1. Effects of essential oils from herbal plants and citrus fruits on DNA polymerase inhibitory, cancer cell growth inhibitory, antiallergic, and antioxidant activities.

    Science.gov (United States)

    Mitoshi, Mai; Kuriyama, Isoko; Nakayama, Hiroto; Miyazato, Hironari; Sugimoto, Keiichiro; Kobayashi, Yuko; Jippo, Tomoko; Kanazawa, Kazuki; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2012-11-14

    In this study, the biological activity of 20 essential oils (EOs) from herbal plants and citrus fruits were investigated in terms of mammalian DNA polymerase (pol) inhibitory activity, cancer cell (human colon carcinoma, HCT116) growth inhibitory activity, antiallergic activity, as anti-β-hexosaminidase release activity in rat basophilic leukemia RBL-2H3 cells treated with calcium ionophore A23187, and antioxidant activity by a lipophilic-oxygen radical absorbance capacity method. These EOs showed patterns of inhibition of pol α, a DNA replicative pol, similar to their cancer cell growth inhibitory activity, and their inhibitory activity on pol λ, a DNA repair/recombination pol, by the EOs showed correlation with anti-β-hexosaminidase release activity. Among these EOs, chamomile (Matricaria chamomilla L.) was the strongest inhibitor of pols α and λ and showed significant effects on both cancer cell growth and mast cell degranulation. On the basis of these results, chamomile EO can be recommended as a potentially useful, bioactive candidate for therapeutic applications.

  2. A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability.

    Science.gov (United States)

    De, Ananya; Campbell, Colin

    2007-02-15

    The data in the present study show that DNA polymerase gamma and DNA ligase III interact in mitochondrial protein extracts from cultured HT1080 cells. An interaction was also observed between the two recombinant proteins in vitro. Expression of catalytically inert versions of DNA ligase III that bind DNA polymerase gamma was associated with reduced mitochondrial DNA copy number and integrity. In contrast, overexpression of wild-type DNA ligase III had no effect on mitochondrial DNA copy number or integrity. Experiments revealed that wild-type DNA ligase III facilitates the interaction of DNA polymerase gamma with a nicked DNA substrate in vitro, and that the zinc finger domain of DNA ligase III is required for this activity. Mitochondrial protein extracts prepared from cells overexpressing a DNA ligase III protein that lacked the zinc finger domain had reduced base excision repair activity compared with extracts from cells overexpressing the wild-type protein. These data support the interpretation that the interaction of DNA ligase III and DNA polymerase gamma is required for proper maintenance of the mammalian mitochondrial genome.

  3. New Insights into DNA Polymerase Function Revealed by Phosphonoacetic Acid-Sensitive T4 DNA Polymerases.

    Science.gov (United States)

    Zhang, Likui

    2017-09-15

    The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of nonherpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S, and V475W mutations in DNA pol. These amino acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols. We investigated whether the T4 phages expressing the engineered mutant DNA pols were sensitive to the antiviral drug phosphonoacetic acid (PAA) and characterized the in vivo replication fidelity of the phage DNA pols. We found that G466S substitution marginally increased PAA sensitivity, whereas Y460F substitution conferred resistance. The phage expressing a double mutant G466S/Y460F DNA pol was more PAA-sensitive. V475W T4 DNA pol was highly sensitive to PAA, as was the case with V478W RB69 DNA pol. However, DNA replication was severely compromised, which resulted in the selection of phages expressing more robust DNA pols that have strong ability to replicate DNA and contain additional amino acid substitutions that suppress PAA sensitivity. Reduced replication fidelity was observed in all mutant phages expressing PAA-sensitive DNA pols. These observations indicate that PAA sensitivity and fidelity are balanced in DNA pols that can replicate DNA in different environments.

  4. Fidelity and processivity of Saccharomyces cerevisiae DNA polymerase eta.

    Science.gov (United States)

    Washington, M T; Johnson, R E; Prakash, S; Prakash, L

    1999-12-24

    The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol eta, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol eta in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol eta has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10(-2) to 10(-3). Also pol eta has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol eta's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors.

  5. Role for DNA polymerase beta in response to ionizing radiation.

    NARCIS (Netherlands)

    Vermeulen, C.; Verwijs-Janssen, M.; Cramers, P.; Begg, A.C.; Vens, C.

    2007-01-01

    Evidence for a role of DNA polymerase beta in determining radiosensitivity is conflicting. In vitro assays show an involvement of DNA polymerase beta in single strand break repair and base excision repair of oxidative damages, both products of ionizing radiation. Nevertheless the lack of DNA polymer

  6. Micro-RNA quantification using DNA polymerase and pyrophosphate quantification.

    Science.gov (United States)

    Yu, Hsiang-Ping; Hsiao, Yi-Ling; Pan, Hung-Yin; Huang, Chih-Hung; Hou, Shao-Yi

    2011-12-15

    A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20-100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Crystal structure of yeast DNA polymerase ε catalytic domain.

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    Rinku Jain

    Full Text Available DNA polymerase ε (Polε is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2 bound to a nascent G:C base pair (Pol2G:C. Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.

  8. Inhibition of mammalian DNA polymerases and the suppression of inflammatory and allergic responses by tyrosol from used activated charcoal waste generated during sake production.

    Science.gov (United States)

    Mizushina, Yoshiyuki; Ogawa, Yoshiaki; Onodera, Takefumi; Kuriyama, Isoko; Sakamoto, Yuka; Nishikori, Shu; Kamisuki, Shinji; Sugawara, Fumio

    2014-08-06

    The components adsorbed onto activated charcoal following the fermentation process of the Japanese rice wine "sake" have been studied with the aim of identifying suitable applications for this industrial food waste product. The absorbed materials were effectively extracted from the charcoal, and inhibited the activity of several mammalian DNA polymerases (pols). Subsequent purification of the extract afforded tyrosol [4-(2-hydroxyethyl)phenol] as the active component, which selectively inhibited the activity of 11 mammalian pols with IC50 values in the range of 34.3-46.1 μM. In contrast, this compound did not influence the activities of plant or prokaryotic pols or any of the other DNA metabolic enzymes tested. Tyrosol suppressed both anti-inflammatory and antiallergic effects in vivo, including 12-O-tetradecanoylphorbol-13-acetate-induced inflammatory mouse ear edema, and immunoglobulin E-induced passive cutaneous anaphylactic reaction in mice. These results suggested that this byproduct formed during the sake-brewing process could be used as an anti-inflammatory and/or antiallergic agent.

  9. Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

    Science.gov (United States)

    Suzuki, Tetsuya; Grúz, Petr; Honma, Masamitsu; Adachi, Noritaka; Nohmi, Takehiko

    2016-09-01

    Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed.

  10. Amplification of Chloroplast DNA Using the Polymerase Chain Reaction (PCR): A Practical Activity for Secondary School Students

    Science.gov (United States)

    Hamilton, Kenny; Barfoot, Jan; Crawford, Kathleen E.; Simpson, Craig G.; Beaumont, Paul C.; Bownes, Mary

    2006-01-01

    We describe a polymerase chain reaction (PCR) protocol suitable for use in secondary schools and colleges. This PCR protocol can be used to investigate genetic variation between plants. The protocol makes use of primers which are complementary to sequences of nucleotides that are highly conserved across different plant genera. The regions of…

  11. Amplification of Chloroplast DNA Using the Polymerase Chain Reaction (PCR): A Practical Activity for Secondary School Students

    Science.gov (United States)

    Hamilton, Kenny; Barfoot, Jan; Crawford, Kathleen E.; Simpson, Craig G.; Beaumont, Paul C.; Bownes, Mary

    2006-01-01

    We describe a polymerase chain reaction (PCR) protocol suitable for use in secondary schools and colleges. This PCR protocol can be used to investigate genetic variation between plants. The protocol makes use of primers which are complementary to sequences of nucleotides that are highly conserved across different plant genera. The regions of…

  12. Thioredoxin suppresses microscopic hopping of T7 DNA polymerase on duplex DNA

    NARCIS (Netherlands)

    Etson, Candice M.; Hamdan, Samir M.; Richardson, Charles C.; Oijen, Antoine M. van; Richardson, Charles C.

    2010-01-01

    The DNA polymerases involved in DNA replication achieve high processivity of nucleotide incorporation by forming a complex with processivity factors. A model system for replicative DNA polymerases, the bacteriophage T7 DNA polymerase (gp5), encoded by gene 5, forms a tight, 1:1 complex with

  13. Role of DNA Polymerases in Repeat-Mediated Genome Instability

    Directory of Open Access Journals (Sweden)

    Kartik A. Shah

    2012-11-01

    Full Text Available Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich’s ataxia (GAAn repeats in a yeast experimental system. The elementary step of expansion corresponded to ∼160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM. Notably, defects in the replicative DNA polymerases δ and ∊ strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.

  14. Hepatitis B virus DNA polymerase gene polymorphism based ...

    African Journals Online (AJOL)

    2017-09-03

    Sep 3, 2017 ... HBV is distributed into various genotypes based on nucleic acid sequence variation. ... compared to genotype B and higher incidence of HCC in genotype D ... DNA sequencing technology to sequence HBV DNA polymerase ...

  15. Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol ε and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors

    Directory of Open Access Journals (Sweden)

    Pavlov Youri I

    2009-03-01

    Full Text Available Abstract Background Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved. Results We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase ε consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol ε evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol ε parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol ε, and the other one to the common ancestor of Pol α, Pol δ, and Pol ζ. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polε shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases. Conclusion Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We

  16. Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol ε and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors

    Science.gov (United States)

    Tahirov, Tahir H; Makarova, Kira S; Rogozin, Igor B; Pavlov, Youri I; Koonin, Eugene V

    2009-01-01

    Background Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved. Results We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase ε consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol ε evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol ε parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol ε, and the other one to the common ancestor of Pol α, Pol δ, and Pol ζ. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polε shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases. Conclusion Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal

  17. Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

    Energy Technology Data Exchange (ETDEWEB)

    Yang, S.

    1992-01-01

    DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase [alpha] are the molecular targets for two metal ions, Zn[sup 2+] and Cd[sup 2+], and an anticancer drug, F-ara-ATP. The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP. A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex. F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3[prime]-terminus of DNA nick by DNA polymerase [alpha]. All steps of the DNA ligation reaction were inhibited by Zn[sup 2+] and Cd[sup 2+] in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn[sup 2+] and Cd[sup 2+] showed their contradictory effects on the fidelity of the reaction by human DNA polymerase [alpha]. Zn[sup 2+] decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd[sup 2+] increased the frequencies of both misinsertion and mispair extension at very low concentration. The data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported.

  18. DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response.

    Science.gov (United States)

    Colis, Laureen; Peltonen, Karita; Sirajuddin, Paul; Liu, Hester; Sanders, Sara; Ernst, Glen; Barrow, James C; Laiho, Marikki

    2014-06-30

    DNA intercalation is a major therapeutic modality for cancer therapeutic drugs. The therapeutic activity comes at a cost of normal tissue toxicity and genotoxicity. We have recently described a planar heterocyclic small molecule DNA intercalator, BMH-21, that binds ribosomal DNA and inhibits RNA polymerase I (Pol I) transcription. Despite DNA intercalation, BMH-21 does not cause phosphorylation of H2AX, a key biomarker activated in DNA damage stress. Here we assessed whether BMH-21 activity towards expression and localization of Pol I marker proteins depends on DNA damage signaling and repair pathways. We show that BMH-21 effects on the nucleolar stress response were independent of major DNA damage associated PI3-kinase pathways, ATM, ATR and DNA-PKcs. However, testing a series of BMH-21 derivatives with alterations in its N,N-dimethylaminocarboxamide arm showed that several derivatives had acquired the property to activate ATM- and DNA-PKcs -dependent damage sensing and repair pathways while their ability to cause nucleolar stress and affect cell viability was greatly reduced. The data show that BMH-21 is a chemically unique DNA intercalator that has high bioactivity towards Pol I inhibition without activation or dependence of DNA damage stress. The findings also show that interference with DNA and DNA metabolic processes can be exploited therapeutically without causing DNA damage.

  19. White Spot Syndrome Virus Orf514 Encodes a Bona Fide DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Rogerio R. Sotelo-Mundo

    2011-01-01

    Full Text Available White spot syndrome virus (WSSV is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514, which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication.

  20. A euryarchaeal histone modulates strand displacement synthesis by replicative DNA polymerases.

    Science.gov (United States)

    Sun, Fei; Huang, Li

    2016-07-01

    Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase (PolB), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of PolB and PolD from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPfA1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both PolB and PolD were efficient in strand displacement. HPfA1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPfA1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.

  1. Norovirus Proteinase-Polymerase and Polymerase Are Both Active Forms of RNA-Dependent RNA Polymerase

    OpenAIRE

    Belliot, Gaël; Sosnovtsev, Stanislav V.; Chang, Kyeong-Ok; Babu, Vijay; Uche, Uzo; Arnold, Jamie J.; Cameron, Craig E.; Green, Kim Y.

    2005-01-01

    In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro−Pol with an inactivated proteinase domain to prevent autocleavage) and r...

  2. Purification of DNA polymerase II stimulatory factor I, a yeast single-stranded DNA-binding protein.

    OpenAIRE

    1990-01-01

    Incidental to the purification of yeast DNA polymerase II was the observation that various chromatographic fractions contained activities that stimulated synthesis by this polymerase. In this paper we report the purification and initial characterization of one such factor, stimulatory factor I (SFI). SFI, which is associated with an apparent complex of three polypeptides of 66, 37, and 13.5 kDa, binds preferentially to single-stranded DNA, possibly explaining its ability to stimulate DNA poly...

  3. Comparative Analysis of Eubacterial DNA Polymerase Ⅲ Alpha Subunits

    Institute of Scientific and Technical Information of China (English)

    Xiao-Qian Zhao; Jian-Fei Hu; Jun Yu

    2006-01-01

    DNA polymerase Ⅲ is one of the five eubacterial DNA polymerases that is responsible for the replication of DNA duplex. Among the ten subunits of the DNA polymerase Ⅲ core enzyme, the alpha subunit catalyzes the reaction for polymerizing both DNA strands. In this study, we extracted genomic sequences of the alpha subunit from 159 sequenced eubacterial genomes, and carried out sequencebased phylogenetic and structural analyses. We found that all eubacterial genomes have one or more alpha subunits, which form either homodimers or heterodimers.Phylogenetic and domain structural analyses as well as copy number variations of the alpha subunit in each bacterium indicate the classification of alpha subunit into four basic groups: polC, dnaE1, dnaE2, and dnaE3. This classification is of essence in genome composition analysis. We also consolidated the naming convention to avoid further confusion in gene annotations.

  4. Discovery of cyanophage genomes which contain mitochondrial DNA polymerase.

    Science.gov (United States)

    Chan, Yi-Wah; Mohr, Remus; Millard, Andrew D; Holmes, Antony B; Larkum, Anthony W; Whitworth, Anna L; Mann, Nicholas H; Scanlan, David J; Hess, Wolfgang R; Clokie, Martha R J

    2011-08-01

    DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

  5. X-ray-induced mutations in Escherichia coli K-12 strains with altered DNA polymerase I activities

    Energy Technology Data Exchange (ETDEWEB)

    Nagata, Yuki; Kawata, Masakado; Komura, Jun-ichiro; Ono, Tetsuya; Yamamoto, Kazuo

    2003-07-25

    Spectra of ionizing radiation mutagenesis were determined by sequencing X-ray-induced endogenous tonB gene mutations in Escherichia coli polA strains. We used two polA alleles, the polA1 mutation, defective for Klenow domain, and the polA107 mutation, defective for flap domain. We demonstrated that irradiation of 75 and 50 Gy X-rays could induce 3.8- and 2.6-fold more of tonB mutation in polA1 and polA107 strains, respectively, than spontaneous level. The radiation induced spectrum of 51 tonB mutations in polA1 and 51 in polA107 indicated that minus frameshift, A:T{yields}T:A transversion and G:C{yields}T:A transversion were the types of mutations increased. Previously, we have reported essentially the same X-ray-induced tonB mutation spectra in the wild-type strain. These results indicate that (1) X-rays can induce minus frameshift, A:T{yields}T:A transversion and G:C{yields}T:A transversion in E. coli and (2) presence or absence of polymerase I (PolI) of E. coli does not have any effects on the process of X-ray mutagenesis.

  6. The Error-Prone DNA Polymerase κ Promotes Temozolomide Resistance in Glioblastoma through Rad17-Dependent Activation of ATR-Chk1 Signaling.

    Science.gov (United States)

    Peng, Chenghao; Chen, Zhengxin; Wang, Shuai; Wang, Hong-Wei; Qiu, Wenjin; Zhao, Lin; Xu, Ran; Luo, Hui; Chen, Yuanyuan; Chen, Dan; You, Yongping; Liu, Ning; Wang, Huibo

    2016-04-15

    The acquisition of drug resistance is a persistent clinical problem limiting the successful treatment of human cancers, including glioblastoma (GBM). However, the molecular mechanisms by which initially chemoresponsive tumors develop therapeutic resistance remain poorly understood. In this study, we report that Pol κ, an error-prone polymerase that participates in translesion DNA synthesis, was significantly upregulated in GBM cell lines and tumor tissues following temozolomide treatment. Overexpression of Pol κ in temozolomide-sensitive GBM cells conferred resistance to temozolomide, whereas its inhibition markedly sensitized resistant cells to temozolomide in vitro and in orthotopic xenograft mouse models. Mechanistically, depletion of Pol κ disrupted homologous recombination (HR)-mediated repair and restart of stalled replication forks, impaired the activation of ATR-Chk1 signaling, and delayed cell-cycle re-entry and progression. Further investigation of the relationship between Pol κ and temozolomide revealed that Pol κ inactivation facilitated temozolomide-induced Rad17 ubiquitination and proteasomal degradation, subsequently silencing ATR-Chk1 signaling and leading to defective HR repair and the reversal of temozolomide resistance. Moreover, overexpression of Rad17 in Pol κ-depleted GBM cells restored HR efficiency, promoted the clearance of temozolomide-induced DNA breaks, and desensitized cells to the cytotoxic effects of temozolomide observed in the absence of Pol κ. Finally, we found that Pol κ overexpression correlated with poor prognosis in GBM patients undergoing temozolomide therapy. Collectively, our findings identify a potential mechanism by which GBM cells develop resistance to temozolomide and suggest that targeting the DNA damage tolerance pathway may be beneficial for overcoming resistance. Cancer Res; 76(8); 2340-53. ©2016 AACR.

  7. ø29 DNA polymerase residue Lys383, invariant at motif B of DNA-dependent polymerases, is involved in dNTP binding.

    Science.gov (United States)

    Saturno, J; Lázaro, J M; Esteban, F J; Blanco, L; Salas, M

    1997-06-13

    Bacteriophage ø29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. Among them, motif B, characterized by the consensus +x3Kx(6-7)YG (+ being a positively charged amino acid), appears to be specifically conserved in those polymerases that use DNA but not RNA as template. In particular, the lysine residue of this motif is invariant in all members of DNA-dependent polymerases. In this paper we report a mutational analysis of this invariant residue of motif B with the construction and characterization of two mutant proteins in the corresponding residue (Lys383) of ø29 DNA polymerase. Mutant proteins (K383R and K383P) were overexpressed, purified and analyzed under steady-state conditions. In agreement with the modular organization proposed for ø29 DNA polymerase, the exonuclease activity was not affected in either mutant protein. Conversely, mutant K383P showed no detectable capacity to incorporate dNTP substrates using either DNA or TP as primer, although its affinity for DNA was not affected. The conservative substitution of Lys383 by arginine (K383R) resulted in a considerable impairment to use dNTPs, in both processive and non-processive DNA synthesis; the Km for dNTPs being 200-fold higher than that of the wild-type enzyme. Mutant K383R recovered the wild-type polymerase/exonuclease ratio when Mn2+ was used instead of Mg2+ as metal activator, indicating a distorted binding of the [dNTP-metal] chelate at the mutant enzyme active site. The positive charge at residue Lys383 was also critical in the catalysis of deoxynucleotidylation of the terminal protein by ø29 DNA polymerase. The results obtained suggest a direct role for the lysine residue in motif B in forming an evolutionarily conserved DNA templated dNTP binding pocket. Additionally, K383R mutant protein was also affected in the progression from protein-primed initiation to DNA elongation, a switch between two modes of

  8. Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications.

    Science.gov (United States)

    Oscorbin, Igor P; Boyarskikh, Ulyana A; Filipenko, Maksim L

    2015-10-01

    A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA.

  9. Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

    OpenAIRE

    1997-01-01

    The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conse...

  10. Inhibition of DNA restrictive endonucleases and Taq DNA polymerase by trimalonic acid C60

    Institute of Scientific and Technical Information of China (English)

    YANG XinLin; CHEN Zhe; MENG XianMei; LI Bo; TAN Xin

    2007-01-01

    Activities of trimalonic acid fullerene (TMA C60) on DNA restrictive enzymatic reaction were investigated by using two restrictive endonucleases Hind III and EcoR I and plasmid pEGFP-N1 with single restrictive site for both enzymes. Meanwhile,TMA C60 was also tested to clarify its effects on polymerase chain reaction (PCR) with the catalyst of Taq DNA polymerase and the template of plasmid pEGFP-N1. The products from restrictive reactions or PCR were detected by agarose gel electrophoresis. It was found that the product amounts from restrictive reactions or PCR decreased significantly with addition of TMA C60. The inhibition by TMA C60 was dose-dependent and IC50 values for reactions of Hind III,EcoR I and PCR were 16.3,6.0 and 6.0 μmol/L,respectively. Addition of two scavengers of reactive oxygen species (ROS),L-ascorbic acid-2-phosphate ester magnesium and sodium azide at the concentrations of 2―10 mmol/L did not antagonize the activities of TMA C60 against PCR and two restrictive reactions. However,increase of Taq DNA polymerase amounts in PCR system antagonized the activities of TMA C60. These data implied that TMA C60 was able to inhibit the activities of the three above-mentioned enzymes involved in DNA metabolism,and that this inhibition probably did not correlate to ROS.

  11. Kinetics and thermodynamics of exonuclease-deficient DNA polymerases

    Science.gov (United States)

    Gaspard, Pierre

    2016-04-01

    A kinetic theory is developed for exonuclease-deficient DNA polymerases, based on the experimental observation that the rates depend not only on the newly incorporated nucleotide, but also on the previous one, leading to the growth of Markovian DNA sequences from a Bernoullian template. The dependencies on nucleotide concentrations and template sequence are explicitly taken into account. In this framework, the kinetic and thermodynamic properties of DNA replication, in particular, the mean growth velocity, the error probability, and the entropy production are calculated analytically in terms of the rate constants and the concentrations. Theory is compared with numerical simulations for the DNA polymerases of T7 viruses and human mitochondria.

  12. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

    Energy Technology Data Exchange (ETDEWEB)

    1992-01-01

    This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3' to 5' exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.

  13. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

    Energy Technology Data Exchange (ETDEWEB)

    1992-12-31

    This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3` to 5` exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.

  14. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Directory of Open Access Journals (Sweden)

    Tom eKillelea

    2014-05-01

    Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  15. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  16. PCR performance of a thermostable heterodimeric archaeal DNA polymerase.

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  17. Preparation of Phi29 DNA polymerase free of amplifiable DNA using ethidium monoazide, an ultraviolet-free light-emitting diode lamp and trehalose.

    Directory of Open Access Journals (Sweden)

    Hirokazu Takahashi

    Full Text Available We previously reported that multiply-primed rolling circle amplification (MRPCA using modified random RNA primers can amplify tiny amounts of circular DNA without producing any byproducts. However, contaminating DNA in recombinant Phi29 DNA polymerase adversely affects the outcome of MPRCA, especially for negative controls such as non-template controls. The amplified DNA in negative control casts doubt on the result of DNA amplification. Since Phi29 DNA polymerase has high affinity for both single-strand and double-stranded DNA, some amount of host DNA will always remain in the recombinant polymerase. Here we describe a procedure for preparing Phi29 DNA polymerase which is essentially free of amplifiable DNA. This procedure is realized by a combination of host DNA removal using appropriate salt concentrations, inactivation of amplifiable DNA using ethidium monoazide, and irradiation with visible light from a light-emitting diode lamp. Any remaining DNA, which likely exists as oligonucleotides captured by the Phi29 DNA polymerase, is degraded by the 3'-5' exonuclease activity of the polymerase itself in the presence of trehalose, used as an anti-aggregation reagent. Phi29 DNA polymerase purified by this procedure has little amplifiable DNA, resulting in reproducible amplification of at least ten copies of plasmid DNA without any byproducts and reducing reaction volume. This procedure could aid the amplification of tiny amounts DNA, thereby providing clear evidence of contamination from laboratory environments, tools and reagents.

  18. DIFFERENT RESULTS BY DIFFERENT COMMERCIAL TAQ DNA POLYMERASE IN RAPD

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ RAPD (Random Amplified Polymorphic DNA) technique has been widely used in animal, plant, human and microorganism research since it was first established by Williams in 1990[1-3]. But, because of low annealed temperature and short 10-nt primers, the resolution and repetition is low in RAPD. The stability of RAPD is influenced by many factors such as the concentration of template, primers, dNTP, Mg++,and Taq DNA polymerase[4-6]. The influence on amplified products of different commercial Taq DNA polymerase in RAPD was studied in this paper.

  19. A Comparative Analysis of Translesion DNA Synthesis Catalyzed by a High-Fidelity DNA Polymerase.

    Science.gov (United States)

    Dasari, Anvesh; Deodhar, Tejal; Berdis, Anthony J

    2017-07-21

    Translesion DNA synthesis (TLS) is the ability of DNA polymerases to incorporate nucleotides opposite and beyond damaged DNA. TLS activity is an important risk factor for the initiation and progression of genetic diseases such as cancer. In this study, we evaluate the ability of a high-fidelity DNA polymerase to perform TLS with 8-oxo-guanine (8-oxo-G), a highly pro-mutagenic DNA lesion formed by reactive oxygen species. Results of kinetic studies monitoring the incorporation of modified nucleotide analogs demonstrate that the binding affinity of the incoming dNTP is controlled by the overall hydrophobicity of the nucleobase. However, the rate constant for the polymerization step is regulated by hydrogen-bonding interactions made between the incoming nucleotide with 8-oxo-G. Results generated here for replicating the miscoding 8-oxo-G are compared to those published for the replication of the non-instructional abasic site. During the replication of both lesions, binding of the nucleotide substrate is controlled by energetics associated with nucleobase desolvation, whereas the rate constant for the polymerization step is influenced by the physical nature of the DNA lesion, that is, miscoding versus non-instructional. Collectively, these studies highlight the importance of nucleobase desolvation as a key physical feature that enhances the misreplication of structurally diverse DNA lesions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil

    OpenAIRE

    Abellón-Ruiz, Javier; Waldron, Kevin J.; Connolly, Bernard A.

    2016-01-01

    Archaeal family-D DNA polymerases (Pol-D) comprise a small (DP1) proofreading subunit and a large (DP2) polymerase subunit. Pol-D is one of the least studied polymerase families, and this publication investigates the enzyme from Archaeoglobus fulgidus (Afu Pol-D). The C-terminal region of DP2 contains two conserved cysteine clusters, and their roles are investigated using site-directed mutagenesis. The cluster nearest the C terminus is essential for polymerase activity, and the cysteines are ...

  1. Norovirus proteinase-polymerase and polymerase are both active forms of RNA-dependent RNA polymerase.

    Science.gov (United States)

    Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Babu, Vijay; Uche, Uzo; Arnold, Jamie J; Cameron, Craig E; Green, Kim Y

    2005-02-01

    In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro(-)Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro(-)Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis.

  2. Proofreading genotyping assays mediated by high fidelity exo+ DNA polymerases.

    Science.gov (United States)

    Zhang, Jia; Li, Kai; Pardinas, Jose R; Sommer, Steve S; Yao, Kai-Tai

    2005-02-01

    DNA polymerases with 3'-5' proofreading function mediate high fidelity DNA replication but their application for mutation detection was almost completely neglected before 1998. The obstacle facing the use of exo(+) polymerases for mutation detection could be overcome by primer-3'-termini modification, which has been tested using allele-specific primers with 3' labeling, 3' exonuclease-resistance and 3' dehydroxylation modifications. Accordingly, three new types of single nucleotide polymorphism (SNP) assays have been developed to carry out genome-wide genotyping making use of the fidelity advantage of exo(+) polymerases. Such SNP assays might also provide a novel approach for re-sequencing and de novo sequencing. These new mutation detection assays are widely adaptable to a variety of platforms, including real-time PCR, multi-well plate and microarray technologies. Application of exo(+) polymerases to genetic analysis could accelerate the pace of personalized medicine.

  3. The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

    OpenAIRE

    Hirota, Kouji; Yoshikiyo, Kazunori; Guilbaud, Guillaume; Tsurimoto, Toshiki; Murai, Junko; Tsuda, Masataka; Phillips, Lara G.; Narita, Takeo; Nishihara, Kana; Kobayashi, Kaori; Yamada, Kouich; Nakamura, Jun; Pommier, Yves; Lehmann, Alan; Sale, Julian E.

    2015-01-01

    The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with ...

  4. Replication pauses of the wild-type and mutant mitochondrial DNA polymerase gamma: a simulation study.

    Directory of Open Access Journals (Sweden)

    Zhuo Song

    2011-11-01

    Full Text Available The activity of polymerase γ is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-γ might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-γ that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-γ variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-γ (exo(- in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(- polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.

  5. Replication pauses of the wild-type and mutant mitochondrial DNA polymerase gamma: a simulation study.

    Science.gov (United States)

    Song, Zhuo; Cao, Yang; Samuels, David C

    2011-11-01

    The activity of polymerase γ is complicated, involving both correct and incorrect DNA polymerization events, exonuclease activity, and the disassociation of the polymerase:DNA complex. Pausing of pol-γ might increase the chance of deletion and depletion of mitochondrial DNA. We have developed a stochastic simulation of pol-γ that models its activities on the level of individual nucleotides for the replication of mtDNA. This method gives us insights into the pausing of two pol-γ variants: the A467T substitution that causes PEO and Alpers syndrome, and the exonuclease deficient pol-γ (exo(-)) in premature aging mouse models. To measure the pausing, we analyzed simulation results for the longest time for the polymerase to move forward one nucleotide along the DNA strand. Our model of the exo(-) polymerase had extremely long pauses, with a 30 to 300-fold increase in the time required for the longest single forward step compared to the wild-type, while the naturally occurring A467T variant showed at most a doubling in the length of the pauses compared to the wild-type. We identified the cause of these differences in the polymerase pausing time to be the number of disassociations occurring in each forward step of the polymerase.

  6. Baculovirus RNA Polymerase: Activities, Composition, and Evolution

    Institute of Scientific and Technical Information of China (English)

    A.Lorena Passarelli

    2007-01-01

    Baculoviruses are the only nuclear replicating DNA-containing viruses that encode their own DNA-directed RNA polymerase (RNAP). The baculovirus RNAP is specific for the transcription of genes expressed after virus DNA replication. It is composed of four subunits, making it the simplest multisubunit RNAP known. Two subunits contain motifs found at the catalytic center of other RNAPs and a third has capping enzyme functions. The function of the fourth subunit is not known. Structural studies on this unique RNAP will provide new insights into the functions of this enzyme and the regulation of viral genes and may be instrumental to optimize the baculovirus gene expression system.

  7. Characterization of family D DNA polymerase from Thermococcus sp. 9°N.

    Science.gov (United States)

    Greenough, Lucia; Menin, Julie F; Desai, Nirav S; Kelman, Zvi; Gardner, Andrew F

    2014-07-01

    Accurate DNA replication is essential for maintenance of every genome. All archaeal genomes except Crenarchaea, encode for a member of Family B (polB) and Family D (polD) DNA polymerases. Gene deletion studies in Thermococcus kodakaraensis and Methanococcus maripaludis show that polD is the only essential DNA polymerase in these organisms. Thus, polD may be the primary replicative DNA polymerase for both leading and lagging strand synthesis. To understand this unique archaeal enzyme, we report the biochemical characterization of a heterodimeric polD from Thermococcus. PolD contains both DNA polymerase and proofreading 3'-5' exonuclease activities to ensure efficient and accurate genome duplication. The polD incorporation fidelity was determined for the first time. Despite containing 3'-5' exonuclease proofreading activity, polD has a relatively high error rate (95 × 10(-5)) compared to polB (19 × 10(-5)) and at least 10-fold higher than the polB DNA polymerases from yeast (polε and polδ) or Escherichia coli DNA polIII holoenzyme. The implications of polD fidelity and biochemical properties in leading and lagging strand synthesis are discussed.

  8. Overexpression of DNA polymerase zeta reduces the mitochondrial mutability caused by pathological mutations in DNA polymerase gamma in yeast.

    Directory of Open Access Journals (Sweden)

    Enrico Baruffini

    Full Text Available In yeast, DNA polymerase zeta (Rev3 and Rev7 and Rev1, involved in the error-prone translesion synthesis during replication of nuclear DNA, localize also in mitochondria. We show that overexpression of Rev3 reduced the mtDNA extended mutability caused by a subclass of pathological mutations in Mip1, the yeast mitochondrial DNA polymerase orthologous to human Pol gamma. This beneficial effect was synergistic with the effect achieved by increasing the dNTPs pools. Since overexpression of Rev3 is detrimental for nuclear DNA mutability, we constructed a mutant Rev3 isoform unable to migrate into the nucleus: its overexpression reduced mtDNA mutability without increasing the nuclear one.

  9. Inhibition of RNA polymerase by captan at both DNA and substrate binding sites.

    Science.gov (United States)

    Luo, G; Lewis, R A

    1992-12-01

    RNA synthesis carried out in vitro by Escherichia coli RNA polymerase was inhibited irreversibly by captan when T7 DNA was used as template. An earlier report and this one show that captan blocks the DNA binding site on the enzyme. Herein, it is also revealed that captan acts at the nucleoside triphosphate (NTP) binding site, and kinetic relationships of the action of captan at the two sites are detailed. The inhibition by captan via the DNA binding site of the enzyme was confirmed by kinetic studies and it was further shown that [14C]captan bound to the beta' subunit of RNA polymerase. This subunit contains the DNA binding site. Competitive-like inhibition by captan versus UTP led to the conclusion that captan also blocked the NTP binding site. In support of this conclusion, [14C]captan was observed to bind to the beta subunit which contains the NTP binding site. Whereas, preincubation of RNA polymerase with both DNA and NTPs prevented captan inhibition, preincubation with either DNA or NTPs alone was insufficient to protect the enzyme from the action of captan. Furthermore, the interaction of [14C]captan with the beta and beta' subunits was not prevented by a similar preincubation. Captan also bound, to a lesser extent, to the alpha and sigma subunits. Therefore, captan binding appears to involve interaction with RNA polymerase at sites in addition to those for DNA and NTP; however, this action does not inhibit the polymerase activity.

  10. 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

  11. Template-primer analogs as substrates for DNA polymerase.

    OpenAIRE

    1987-01-01

    In order to gain more understanding about the mode of action of DNA polymerase, eight related partially self-complementary "hairpin" shaped oligodeoxynucleotides were prepared. Four of the oligomers contained either 1-beta-D-arabinofuranosyluracil (ara-U) or 1-beta-D-2'deoxyxylofuranosylthymine (dxT) nucleoside analogs at their 3' termini. We investigated the ability of the oligomers to prime DNA synthesis in relationship to the stability of the hybridized region, the nature of the sugar term...

  12. DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

    OpenAIRE

    Arikuni Uchimura; Yuko Hidaka; Takahiro Hirabayashi; Masumi Hirabayashi; Takeshi Yagi

    2009-01-01

    BACKGROUND: In eukaryotic cells, DNA polymerase delta (Poldelta), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Poldelta in mammalian development has not been thoroughly investigated. METHODOLOGY/PRINCIPAL FINDINGS: To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Poldelta-null (Pold1(-/-)) mice and D400A exchang...

  13. Characterization of the mammalian DNA polymerase gene and protein. Annual progress report

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, N.C.

    1992-01-01

    We have purified and characterized at least three DNA polymerases from Chinese hamster ovary (CHO) cell line Kl in order to evaluate the roles of different polymerases in eukaryotic DNA replication. Pol {alpha} was the most abundant among different polymerase activities and it was neutralized by a monoclonal antibody raised against human pol {alpha}. Pol {var_epsilon} was separated from pol {alpha} and pol {delta} activities using DEAE Sephacell, phosphocellulose and hydroxylapatite columns. The enzyme proved to be sensitive to aphidicolin and carbonyldiphosphonate and was not stimulated by PCNA- Pol {delta} was the least abundant among the three enzymes. It was sensitive to aphidicolin and carbonyidiphosphonate and was stimulated by PCNA. it had a preference for template/primer poly (dA-dT). Based on DNA sequence data of different eukaryotic polymerases PCR primers complementary to two neighboring synthesized. In PCR experiments several products were obtained which are assumed to be specific for the CHO polymerases. We have also analyzed a large number of aphidicolin resistant mutants of CHO to identify mutants with altered DNA polymerases.

  14. DNA dependent RNA polymerases from the fungus Aspergillus nidulans

    NARCIS (Netherlands)

    Stunnenberg, H.G.

    1981-01-01

    The aim of the work presented here was the isolation and characterization of the DNA-dependent RNA polymerases from the fungus Aspergillus nidulans, which was a part of a project concerning the regulation of gene expression in this lower eukaryote.The transcription of a genome and the regulation mec

  15. DNA-dependent RNA polymerases from the fungus Aspergillus nidulans

    NARCIS (Netherlands)

    Stunnenberg, H.G.

    1981-01-01

    The aim of the work presented here was the isolation and characterization of the DNA-dependent RNA polymerases from the fungus Aspergillus nidulans, which was a part of a project concerning the regulation of gene expression in this lower eukaryote.

    The transcription of

  16. DNA Polymerases Drive DNA Sequencing-by-Synthesis Technologies: Both Past and Present

    Directory of Open Access Journals (Sweden)

    Cheng-Yao eChen

    2014-06-01

    Full Text Available Next-generation sequencing (NGS technologies have revolutionized modern biological and biomedical research. The engines responsible for this innovation are DNA polymerases; they catalyze the biochemical reaction for deriving template sequence information. In fact, DNA polymerase has been a cornerstone of DNA sequencing from the very beginning. E. coli DNA polymerase I proteolytic (Klenow fragment was originally utilized in Sanger's dideoxy chain terminating DNA sequencing chemistry. From these humble beginnings followed an explosion of organism-specific, genome sequence information accessible via public database. Family A/B DNA polymerases from mesophilic/thermophilic bacteria/archaea were modified and tested in today's standard capillary electrophoresis (CE and NGS sequencing platforms. These enzymes were selected for their efficient incorporation of bulky dye-terminator and reversible dye-terminator nucleotides respectively. Third generation, real-time single molecule sequencing platform requires slightly different enzyme properties. Enterobacterial phage ⱷ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ⱷ29 enzyme has also been utilized in emerging DNA sequencing technologies including nanopore-, and protein-transistor-based sequencing. DNA polymerase is, and will continue to be, a crucial component of sequencing technologies.

  17. Effects of 8-halo-7-deaza-2'-deoxyguanosine triphosphate on DNA synthesis by DNA polymerases and cell proliferation.

    Science.gov (United States)

    Yin, Yizhen; Sasaki, Shigeki; Taniguchi, Yosuke

    2016-08-15

    8-OxodG (8-oxo-2'-deoxyguanosine) is representative of nucleoside damage and shows a genotoxicity. To significantly reveal the contributions of 7-NH and C8-oxygen to the mutagenic effect of 8-oxodG by DNA polymerases, we evaluated the effects of the 8-halo-7-deaza-dG (8-halogenated 7-deaza-2'-deoxyguanosine) derivatives by DNA polymerases. 8-Halo-7-deaza-dGTPs were poorly incorporated by both KF(exo(-)) and human DNA polymerase β opposite dC or dA into the template DNA. Furthermore, it was found that KF(exo(-)) was very sensitive to the introduction of the C8-halogen, while polymerase β can accommodate the C8-halogen resulting in an efficient dCTP insertion opposite the 8-halo-7-deaza-dG in the template DNA. These results indicate that strong hydrogen bonding between 7-NH in the 8-oxo-G nucleobase and 1-N in the adenine at the active site of the DNA polymerase is required for the mutagenic effects. Whereas, I-deaza-dGTP shows an antiproliferative effect for the HeLa cells, suggesting that it could become a candidate as a new antitumor agent.

  18. Translesion Synthesis: Insights into the Selection and Switching of DNA Polymerases

    Directory of Open Access Journals (Sweden)

    Linlin Zhao

    2017-01-01

    Full Text Available DNA replication is constantly challenged by DNA lesions, noncanonical DNA structures and difficult-to-replicate DNA sequences. Two major strategies to rescue a stalled replication fork and to ensure continuous DNA synthesis are: (1 template switching and recombination-dependent DNA synthesis; and (2 translesion synthesis (TLS using specialized DNA polymerases to perform nucleotide incorporation opposite DNA lesions. The former pathway is mainly error-free, and the latter is error-prone and a major source of mutagenesis. An accepted model of translesion synthesis involves DNA polymerase switching steps between a replicative DNA polymerase and one or more TLS DNA polymerases. The mechanisms that govern the selection and exchange of specialized DNA polymerases for a given DNA lesion are not well understood. In this review, recent studies concerning the mechanisms of selection and switching of DNA polymerases in eukaryotic systems are summarized.

  19. A design proposal of the reverse DNA polymerase (3'-5' direction)

    OpenAIRE

    2013-01-01

    DNA Polymerase is an enzyme that participates in the process of DNA replication. In general, forward DNA polymerase moves along the template strand in a 3'-5' direction, and the daughter strand is formed in a 5'-3' direction. This paper theoretically analyzes the mechanism of forward DNA polymerase, and gives the design proposal of a new reverse DNA polymerase (3'-5' direction).

  20. Cloning, expression, and functional characterization of the equine herpesvirus 1 DNA polymerase and its accessory subunit.

    Science.gov (United States)

    Loregian, Arianna; Case, Alessandro; Cancellotti, Enrico; Valente, Carlo; Marsden, Howard S; Palù, Giorgio

    2006-07-01

    We report the expression and characterization of the putative catalytic subunit (pORF30) and accessory protein (pORF18) of equine herpesvirus 1 DNA polymerase, which are encoded by open reading frames 30 and 18 and are homologous to herpes simplex virus type 1 UL30 and UL42, respectively. In vitro transcription-translation of open reading frames 30 and 18 generated proteins of 136 and 45 kDa, respectively. In vitro-expressed pORF30 possessed basal DNA polymerase activity that was stimulated by pORF18, as measured by DNA polymerase assays in vitro. Purified baculovirus-expressed pORF30 exhibited DNA polymerase activity similar to that of the in vitro-expressed protein, and baculovirus-expressed pORF18 could stimulate both nucleotide incorporation and long-chain DNA synthesis by pORF30 in a dose- and time-dependent manner. The salt optima for activity of both pORF30 and the holoenzyme were substantially different from those for other herpesvirus DNA polymerases. As demonstrated by yeast two-hybrid assays, pORF30 and pORF18 could physically interact, most likely with a 1:1 stoichiometry. Finally, by mutational analysis of the 1,220-residue pORF30, we demonstrated that the extreme C terminus of pORF30 is important for physical and functional interaction with the accessory protein, as reported for UL30 and other herpesvirus DNA polymerases. In addition, a C-proximal region of pORF30, corresponding to residues 1114 to 1172, is involved in binding to, and stimulation by, pORF18. Taken together, the results indicate that pORF30 and pORF18 are the equine herpesvirus 1 counterparts of herpes simplex virus type 1 UL30 and UL42 and share many, but not all, of their characteristics.

  1. Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance*

    OpenAIRE

    Schermerhorn, Kelly M.; Gardner, Andrew F.

    2015-01-01

    Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, k pol and Kd , for corre...

  2. Specific incorporation of an artificial nucleotide opposite a mutagenic DNA adduct by a DNA polymerase.

    Science.gov (United States)

    Wyss, Laura A; Nilforoushan, Arman; Eichenseher, Fritz; Suter, Ursina; Blatter, Nina; Marx, Andreas; Sturla, Shana J

    2015-01-14

    The ability to detect DNA modification sites at single base resolution could significantly advance studies regarding DNA adduct levels, which are extremely difficult to determine. Artificial nucleotides that are specifically incorporated opposite a modified DNA site offer a potential strategy for detection of such sites by DNA polymerase-based systems. Here we investigate the action of newly synthesized base-modified benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates on DNA polymerases when performing translesion DNA synthesis past the pro-mutagenic DNA adduct O(6)-benzylguanine (O(6)-BnG). We found that a mutated form of KlenTaq DNA polymerase, i.e., KTqM747K, catalyzed O(6)-BnG adduct-specific processing of the artificial BenziTP in favor of the natural dNTPs. Steady-state kinetic parameters revealed that KTqM747K catalysis of BenziTP is 25-fold more efficient for template O(6)-BnG than G, and 5-fold more efficient than natural dTMP misincorporation in adduct bypass. Furthermore, the nucleotide analogue BenziTP is required for full-length product formation in O(6)-BnG bypass, as without BenziTP the polymerase stalls at the adduct site. By combining the KTqM747K polymerase and BenziTP, a first round of DNA synthesis enabled subsequent amplification of Benzi-containing DNA. These results advance the development of technologies for detecting DNA adducts.

  3. A transposon-derived DNA polymerase from Entamoeba histolytica displays intrinsic strand displacement, processivity and lesion bypass.

    Directory of Open Access Journals (Sweden)

    Guillermo Pastor-Palacios

    Full Text Available Entamoeba histolytica encodes four family B2 DNA polymerases that vary in amino acid length from 813 to 1279. These DNA polymerases contain a N-terminal domain with no homology to other proteins and a C-terminal domain with high amino acid identity to archetypical family B2 DNA polymerases. A phylogenetic analysis indicates that these family B2 DNA polymerases are grouped with DNA polymerases from transposable elements dubbed Polintons or Mavericks. In this work, we report the cloning and biochemical characterization of the smallest family B2 DNA polymerase from E. histolytica. To facilitate its characterization we subcloned its 660 amino acids C-terminal region that comprises the complete exonuclease and DNA polymerization domains, dubbed throughout this work as EhDNApolB2. We found that EhDNApolB2 displays remarkable strand displacement, processivity and efficiently bypasses the DNA lesions: 8-oxo guanosine and abasic site.Family B2 DNA polymerases from T. vaginalis, G. lambia and E. histolytica contain a Terminal Region Protein 2 (TPR2 motif twice the length of the TPR2 from φ29 DNA polymerase. Deletion studies demonstrate that as in φ29 DNA polymerase, the TPR2 motif of EhDNApolB2 is solely responsible of strand displacement and processivity. Interestingly the TPR2 of EhDNApolB2 is also responsible for efficient abasic site bypass. These data suggests that the 21 extra amino acids of the TPR2 motif may shape the active site of EhDNApolB2 to efficiently incorporate and extended opposite an abasic site. Herein we demonstrate that an open reading frame derived from Politons-Mavericks in parasitic protozoa encode a functional enzyme and our findings support the notion that the introduction of novel motifs in DNA polymerases can confer specialized properties to a conserved scaffold.

  4. Structural Basis for Error-free Replication of Oxidatively Damaged DNA by Yeast DNA Polymerase eta

    Energy Technology Data Exchange (ETDEWEB)

    T Silverstein; R Jain; R Johnson; L Prakash; S Prakash; A Aggarwal

    2011-12-31

    7,8-dihydro-8-oxoguanine (8-oxoG) adducts are formed frequently by the attack of oxygen-free radicals on DNA. They are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase {eta} (Pol{eta}). We show that the open active site cleft of Pol{eta} can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and post-insertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Pol to prevent the mutagenic replication of 8-oxoG lesions in cells.

  5. Maintenance of Genome Integrity: How Mammalian Cells Orchestrate Genome Duplication by Coordinating Replicative and Specialized DNA Polymerases

    Directory of Open Access Journals (Sweden)

    Ryan Barnes

    2017-01-01

    Full Text Available Precise duplication of the human genome is challenging due to both its size and sequence complexity. DNA polymerase errors made during replication, repair or recombination are central to creating mutations that drive cancer and aging. Here, we address the regulation of human DNA polymerases, specifically how human cells orchestrate DNA polymerases in the face of stress to complete replication and maintain genome stability. DNA polymerases of the B-family are uniquely adept at accurate genome replication, but there are numerous situations in which one or more additional DNA polymerases are required to complete genome replication. Polymerases of the Y-family have been extensively studied in the bypass of DNA lesions; however, recent research has revealed that these polymerases play important roles in normal human physiology. Replication stress is widely cited as contributing to genome instability, and is caused by conditions leading to slowed or stalled DNA replication. Common Fragile Sites epitomize “difficult to replicate” genome regions that are particularly vulnerable to replication stress, and are associated with DNA breakage and structural variation. In this review, we summarize the roles of both the replicative and Y-family polymerases in human cells, and focus on how these activities are regulated during normal and perturbed genome replication.

  6. Maintenance of Genome Integrity: How Mammalian Cells Orchestrate Genome Duplication by Coordinating Replicative and Specialized DNA Polymerases.

    Science.gov (United States)

    Barnes, Ryan; Eckert, Kristin

    2017-01-06

    Precise duplication of the human genome is challenging due to both its size and sequence complexity. DNA polymerase errors made during replication, repair or recombination are central to creating mutations that drive cancer and aging. Here, we address the regulation of human DNA polymerases, specifically how human cells orchestrate DNA polymerases in the face of stress to complete replication and maintain genome stability. DNA polymerases of the B-family are uniquely adept at accurate genome replication, but there are numerous situations in which one or more additional DNA polymerases are required to complete genome replication. Polymerases of the Y-family have been extensively studied in the bypass of DNA lesions; however, recent research has revealed that these polymerases play important roles in normal human physiology. Replication stress is widely cited as contributing to genome instability, and is caused by conditions leading to slowed or stalled DNA replication. Common Fragile Sites epitomize "difficult to replicate" genome regions that are particularly vulnerable to replication stress, and are associated with DNA breakage and structural variation. In this review, we summarize the roles of both the replicative and Y-family polymerases in human cells, and focus on how these activities are regulated during normal and perturbed genome replication.

  7. COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

    Directory of Open Access Journals (Sweden)

    Masashi Miura

    2013-12-01

    Full Text Available SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

  8. Priming DNA Replication from Triple Helix Oligonucleotides: Possible Threestranded DNA in DNA Polymerases

    Directory of Open Access Journals (Sweden)

    Patrick P. Lestienne

    2011-01-01

    Full Text Available Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second homologous duplex sequence through the formation of a triple helix, which is able to prime further DNA replication. In these case, the d(G-rich Triple Helix Primers (THP bind the homologous strand in a parallel manner, possibly via a RecA-like mechanism. This novel property is shared by all tested DNA polymerases: phage, retrovirus, bacteria, and human. These features may account for illegitimate initiation of replication upon single-strand breakage and annealing to a homologous sequence where priming may occur. Our experiments suggest that DNA polymerases can bind three instead of two polynucleotide strands in their catalytic centre.

  9. Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins.

    Science.gov (United States)

    Cardenas, Paula P; Carzaniga, Thomas; Zangrossi, Sandro; Briani, Federica; Garcia-Tirado, Esther; Dehò, Gianni; Alonso, Juan C

    2011-11-01

    Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3' → 5' polarity in the presence of Mn(2+) and low inorganic phosphate (Pi) concentration, or to extend a 3'-OH end in the presence dNDP · Mn(2+). Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3'-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ.

  10. A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.

    Science.gov (United States)

    Shen, Yulong; Tang, Xiao-Feng; Yokoyama, Hideshi; Matsui, Eriko; Matsui, Ikuo

    2004-01-01

    Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain. We show that D363, H365 and H454 are the essential residues, while D407, N453, H500, H563 and H565 are critical residues for the activity. We provide experimental evidence demonstrating that manganese, rather than magnesium, is the preferable metal ion for the nuclease activity of PolD. We also show that DP1 alone is insufficient to perform full catalysis, which additionally requires the formation of the PolD complex and manganese ion. We found that a 21 amino acid, subunit-interacting peptide of the sequence from cysteine cluster II of the large subunit (DP2) stimulates the exonuclease activity of DP1 and the internal deletion mutants of PolD lacking the 21-aa sequence. This indicates that the putative zinc finger motif of the cysteine cluster II is deeply involved in the nucleolytic catalysis.

  11. Binding Affinities among DNA Helicase-Primase, DNA Polymerase, and Replication Intermediates in the Replisome of Bacteriophage T7.

    Science.gov (United States)

    Zhang, Huidong; Tang, Yong; Lee, Seung-Joo; Wei, Zeliang; Cao, Jia; Richardson, Charles C

    2016-01-15

    The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging-DNA strands and provides a mechanism for recycling of the lagging-strand DNA polymerase. As an Okazaki fragment is completed, the loop is released, and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase, and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7, the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strand DNA from polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymerase · DNA complex are much faster at a nick DNA region than the release from a ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging-strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNA polymerase at the replication fork for the synthesis of next Okazaki fragment.

  12. The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

    Science.gov (United States)

    Leem, S H; Ropp, P A; Sugino, A

    1994-08-11

    We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

  13. Proficient Replication of the Yeast Genome by a Viral DNA Polymerase.

    Science.gov (United States)

    Stodola, Joseph L; Stith, Carrie M; Burgers, Peter M

    2016-05-27

    DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication.

  14. Extrachromosomal recombination in vaccinia-infected cells requires a functional DNA polymerase participating at a level other than DNA replication.

    Science.gov (United States)

    Colinas, R J; Condit, R C; Paoletti, E

    1990-12-01

    Homologous recombination was measured in vaccinia-infected cells cotransfected with two plasmid recombination substrates. One plasmid contains a vaccinia protein lacZ coding region bearing a 1.1 kb 3' terminal deletion while the other plasmid contains a non-promoted lacZ coding region bearing a 1.1 kb 5' terminal deletion. Homologous recombination occurring between the 825 bp of lacZ common to both plasmids regenerates a functional lacZ gene from which B-galactosidase expression was measured. The entire 3 kb lacZ gene was used as a positive control. A panel of thermosensitive mutants was screened in cells either transfected with the positive control plasmid or cotransfected with the recombination substrates. A DNA - mutant, ts42, known to map to the viral DNA polymerase gene was found to be defective in recombination. Significantly, other DNA - mutants, ts17 or ts25, or other DNA polymerase mutants did not exhibit a defect in recombination similar to ts42. Inhibitors of viral DNA synthesis did not uniformly affect recombination. Cytosine arabinoside and aphidicolin inhibited B-galactosidase expression from the recombination substrates but not from the positive control plasmid, whereas hydroxyurea enhanced expression from both. Marker rescue with the cloned wildtype DNA polymerase gene repaired the defect in ts42. Southern and western analyses demonstrated that B-galactosidase activity was consistent with a recombined lacZ gene and unit size 116 kDa protein. Measurement of plasmid and viral DNA replication in cells infected with the different DNA - mutants indicated that recombination was independent of plasmid and viral DNA replication. Together these results suggest that the vaccinia DNA polymerase participates in homologous recombination at a level other than that of DNA replication.

  15. Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

    Science.gov (United States)

    Furutani, Shunsuke; Naruishi, Nahoko; Hagihara, Yoshihisa; Nagai, Hidenori

    2016-08-01

    On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

  16. Versatility of the carboxy-terminal domain of the alpha subunit of RNA polymerase in transcriptional activation: use of the DNA contact site as a protein contact site for MarA.

    Science.gov (United States)

    Dangi, Bindi; Gronenborn, Angela M; Rosner, Judah L; Martin, Robert G

    2004-10-01

    The transcriptional activator, MarA, interacts with RNA polymerase (RNAP) to activate promoters of the mar regulon. Here, we identify the interacting surfaces of MarA and of the carboxy-terminal domain of the alpha subunit of RNAP (alpha-CTD) by NMR-based chemical shift mapping. Spectral changes were monitored for a MarA-DNA complex upon titration with alpha-CTD, and for alpha-CTD upon titration with MarA-DNA. The mapping results were confirmed by mutational studies and retention chromatography. A model of the ternary complex shows that alpha-CTD uses a '265-like determinant' to contact MarA at a surface distant from the DNA. This is unlike the interaction of alpha-CTD with the CRP or Fis activators where the '265 determinant' contacts DNA while another surface of the same alpha-CTD molecule contacts the activator. These results reveal a new versatility for alpha-CTD in transcriptional activation.

  17. DNA Polymerases λ and β: The Double-Edged Swords of DNA Repair

    Directory of Open Access Journals (Sweden)

    Elisa Mentegari

    2016-08-01

    Full Text Available DNA is constantly exposed to both endogenous and exogenous damages. More than 10,000 DNA modifications are induced every day in each cell’s genome. Maintenance of the integrity of the genome is accomplished by several DNA repair systems. The core enzymes for these pathways are the DNA polymerases. Out of 17 DNA polymerases present in a mammalian cell, at least 13 are specifically devoted to DNA repair and are often acting in different pathways. DNA polymerases β and λ are involved in base excision repair of modified DNA bases and translesion synthesis past DNA lesions. Polymerase λ also participates in non-homologous end joining of DNA double-strand breaks. However, recent data have revealed that, depending on their relative levels, the cell cycle phase, the ratio between deoxy- and ribo-nucleotide pools and the interaction with particular auxiliary proteins, the repair reactions carried out by these enzymes can be an important source of genetic instability, owing to repair mistakes. This review summarizes the most recent results on the ambivalent properties of these enzymes in limiting or promoting genetic instability in mammalian cells, as well as their potential use as targets for anticancer chemotherapy.

  18. Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering

    Directory of Open Access Journals (Sweden)

    Takeshi eYamagami

    2014-09-01

    Full Text Available DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, DNA labeling, mutagenesis, and other experiments. Thermostable DNA polymerases are especially useful and became quite valuable after the development of PCR technology. A DNA polymerase from Thermus aquaticus (Taq polymerase is the most famous DNA polymerase as a PCR enzyme, and has been widely used all over the world. In this study, the gene fragments of the family A DNA polymerases were amplified by PCR from the DNAs from microorganisms within environmental soil samples, using a primer set for the two conserved regions. The corresponding region of the pol gene for Taq polymerase was substituted with the amplified gene fragments, and various chimeric DNA polymerases were prepared. Based on the properties of these chimeric enzymes and their sequences, two residues, E742 and A743, in Taq polymerase were found to be critical for its elongation ability. Taq polymerases with mutations at 742 and 743 actually showed higher DNA affinity and faster primer extension ability. These factors also affected the PCR performance of the DNA polymerase, and improved PCR results were observed with the mutant Taq polymerase.

  19. Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA.

    Science.gov (United States)

    Furukohri, Asako; Nishikawa, Yoshito; Akiyama, Masahiro Tatsumi; Maki, Hisaji

    2012-07-01

    DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.

  20. Real-time DNA sequencing from single polymerase molecules.

    Science.gov (United States)

    Eid, John; Fehr, Adrian; Gray, Jeremy; Luong, Khai; Lyle, John; Otto, Geoff; Peluso, Paul; Rank, David; Baybayan, Primo; Bettman, Brad; Bibillo, Arkadiusz; Bjornson, Keith; Chaudhuri, Bidhan; Christians, Frederick; Cicero, Ronald; Clark, Sonya; Dalal, Ravindra; Dewinter, Alex; Dixon, John; Foquet, Mathieu; Gaertner, Alfred; Hardenbol, Paul; Heiner, Cheryl; Hester, Kevin; Holden, David; Kearns, Gregory; Kong, Xiangxu; Kuse, Ronald; Lacroix, Yves; Lin, Steven; Lundquist, Paul; Ma, Congcong; Marks, Patrick; Maxham, Mark; Murphy, Devon; Park, Insil; Pham, Thang; Phillips, Michael; Roy, Joy; Sebra, Robert; Shen, Gene; Sorenson, Jon; Tomaney, Austin; Travers, Kevin; Trulson, Mark; Vieceli, John; Wegener, Jeffrey; Wu, Dawn; Yang, Alicia; Zaccarin, Denis; Zhao, Peter; Zhong, Frank; Korlach, Jonas; Turner, Stephen

    2009-01-02

    We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

  1. An unusual polyanion from Physarum polycephalum that inhibits homologous DNA polymerase. alpha. in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, H.; Erdmann, S.; Holler, E. (Universitaet Regensburg (West Germany))

    1989-06-13

    From extracts of microplasmodia of Physarum polycephalum and their culture medium, an unusual substance was isolated which inhibited homologous DNA polymerase {alpha} of this slime mold but not {beta}-like DNA polymerase and not heterologous DNA polymerases. Analysis, especially NMR spectroscopy, revealed the major component to be an anionic polyester of L-malic acid and the inhibition to be due to poly(L-malate) in binding reversibly to DNA polymerase {alpha}. The mode of inhibition is competitive with substrate DNA and follows an inhibition constant K{sub i} = 10 ng/mL. Inhibition is reversed in the presence of spermine, spermidine, poly(ethylene imine), and calf thymus histone H1. According to its ester nature, the inhibitor is slightly labile at neutral and instable at acid and alkaline conditions. Its largest size corresponds to a molecular mass of 40-50 kDa, but the bulk of the material after purification has lower molecular masses. The inhibitory activity depends on the polymer size and has a minimal size requirement.

  2. Archaeal DNA Polymerase-B as a DNA Template Guardian: Links between Polymerases and Base/Alternative Excision Repair Enzymes in Handling the Deaminated Bases Uracil and Hypoxanthine

    Directory of Open Access Journals (Sweden)

    Javier Abellón-Ruiz

    2016-01-01

    Full Text Available In Archaea repair of uracil and hypoxanthine, which arise by deamination of cytosine and adenine, respectively, is initiated by three enzymes: Uracil-DNA-glycosylase (UDG, which recognises uracil; Endonuclease V (EndoV, which recognises hypoxanthine; and Endonuclease Q (EndoQ, (which recognises both uracil and hypoxanthine. Two archaeal DNA polymerases, Pol-B and Pol-D, are inhibited by deaminated bases in template strands, a feature unique to this domain. Thus the three repair enzymes and the two polymerases show overlapping specificity for uracil and hypoxanthine. Here it is demonstrated that binding of Pol-D to primer-templates containing deaminated bases inhibits the activity of UDG, EndoV, and EndoQ. Similarly Pol-B almost completely turns off EndoQ, extending earlier work that demonstrated that Pol-B reduces catalysis by UDG and EndoV. Pol-B was observed to be a more potent inhibitor of the enzymes compared to Pol-D. Although Pol-D is directly inhibited by template strand uracil, the presence of Pol-B further suppresses any residual activity of Pol-D, to near-zero levels. The results are compatible with Pol-D acting as the replicative polymerase and Pol-B functioning primarily as a guardian preventing deaminated base-induced DNA mutations.

  3. Role of swi7H4 mutant allele of DNA polymerase α in the DNA damage checkpoint response.

    Science.gov (United States)

    Khan, Saman; Ahmed, Shakil

    2015-01-01

    Besides being a mediator of initiation of DNA replication, DNA polymerase α plays a key role in chromosome maintenance. Swi7H4, a novel temperature sensitive mutant of DNA polymerase α was shown to be defective in transcriptional silencing at the mating type centromere and telomere loci. It is also required for the establishment of chromatin state that can recruit the components of the heterochromatin machinery at these regions. Recently the role of DNA polymerase α in the S-phase alkylation damage response in S. pombe has also been studied. Here we investigate whether defects generated by swi7H4, a mutant allele of DNA polymerase α can activate a checkpoint response. We show that swi7H4 exhibit conditional synthetic lethality with chk1 null mutant and the double mutant of swi7H4 with chk1 deletion aggravate the chromosome segregation defects. More importantly swi7H4 mutant cells delay the mitotic progression at non permissive temperature that is mediated by checkpoint protein kinase Chk1. In addition we show that, in the swi7H4 mutant background, cells accumulate DNA damage at non permissive temperature activating the checkpoint kinase protein Chk1. Further, we observed synthetic lethality between swi7H4 and a number of genes involved in DNA repair pathway at semi permissive temperature. We summarize that defects in swi7H4 mutant results in DNA damage that delay mitosis in a Chk1 dependent manner that also require the damage repair pathway for proper recovery.

  4. Role of swi7H4 mutant allele of DNA polymerase α in the DNA damage checkpoint response.

    Directory of Open Access Journals (Sweden)

    Saman Khan

    Full Text Available Besides being a mediator of initiation of DNA replication, DNA polymerase α plays a key role in chromosome maintenance. Swi7H4, a novel temperature sensitive mutant of DNA polymerase α was shown to be defective in transcriptional silencing at the mating type centromere and telomere loci. It is also required for the establishment of chromatin state that can recruit the components of the heterochromatin machinery at these regions. Recently the role of DNA polymerase α in the S-phase alkylation damage response in S. pombe has also been studied. Here we investigate whether defects generated by swi7H4, a mutant allele of DNA polymerase α can activate a checkpoint response. We show that swi7H4 exhibit conditional synthetic lethality with chk1 null mutant and the double mutant of swi7H4 with chk1 deletion aggravate the chromosome segregation defects. More importantly swi7H4 mutant cells delay the mitotic progression at non permissive temperature that is mediated by checkpoint protein kinase Chk1. In addition we show that, in the swi7H4 mutant background, cells accumulate DNA damage at non permissive temperature activating the checkpoint kinase protein Chk1. Further, we observed synthetic lethality between swi7H4 and a number of genes involved in DNA repair pathway at semi permissive temperature. We summarize that defects in swi7H4 mutant results in DNA damage that delay mitosis in a Chk1 dependent manner that also require the damage repair pathway for proper recovery.

  5. Molecular Basis for DNA Double-Strand Break Annealing and Primer Extension by an NHEJ DNA Polymerase

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    Nigel C. Brissett

    2013-11-01

    Full Text Available Nonhomologous end-joining (NHEJ is one of the major DNA double-strand break (DSB repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5′-3′ direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3′ overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3′ overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3′ ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

  6. Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

    Science.gov (United States)

    Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

    1999-01-01

    Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

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

    Science.gov (United States)

    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.

  8. Bending the Rules of Transcriptional Repression: Tightly Looped DNA Directly Represses T7 RNA Polymerase

    OpenAIRE

    Lionberger, Troy A.; Meyhöfer, Edgar

    2010-01-01

    From supercoiled DNA to the tight loops of DNA formed by some gene repressors, DNA in cells is often highly bent. Despite evidence that transcription by RNA polymerase (RNAP) is affected in systems where DNA is deformed significantly, the mechanistic details underlying the relationship between polymerase function and mechanically stressed DNA remain unclear. Seeking to gain additional insight into the regulatory consequences of highly bent DNA, we hypothesize that tightly looping DNA is alone...

  9. Measuring cation dependent DNA polymerase fidelity landscapes by deep sequencing.

    Directory of Open Access Journals (Sweden)

    Bradley Michael Zamft

    Full Text Available High-throughput recording of signals embedded within inaccessible micro-environments is a technological challenge. The ideal recording device would be a nanoscale machine capable of quantitatively transducing a wide range of variables into a molecular recording medium suitable for long-term storage and facile readout in the form of digital data. We have recently proposed such a device, in which cation concentrations modulate the misincorporation rate of a DNA polymerase (DNAP on a known template, allowing DNA sequences to encode information about the local cation concentration. In this work we quantify the cation sensitivity of DNAP misincorporation rates, making possible the indirect readout of cation concentration by DNA sequencing. Using multiplexed deep sequencing, we quantify the misincorporation properties of two DNA polymerases--Dpo4 and Klenow exo(---obtaining the probability and base selectivity of misincorporation at all positions within the template. We find that Dpo4 acts as a DNA recording device for Mn(2+ with a misincorporation rate gain of ∼2%/mM. This modulation of misincorporation rate is selective to the template base: the probability of misincorporation on template T by Dpo4 increases >50-fold over the range tested, while the other template bases are affected less strongly. Furthermore, cation concentrations act as scaling factors for misincorporation: on a given template base, Mn(2+ and Mg(2+ change the overall misincorporation rate but do not alter the relative frequencies of incoming misincorporated nucleotides. Characterization of the ion dependence of DNAP misincorporation serves as the first step towards repurposing it as a molecular recording device.

  10. Human REV3 DNA Polymerase Zeta Localizes to Mitochondria and Protects the Mitochondrial Genome.

    Science.gov (United States)

    Singh, Bhupendra; Li, Xiurong; Owens, Kjerstin M; Vanniarajan, Ayyasamy; Liang, Ping; Singh, Keshav K

    2015-01-01

    To date, mitochondrial DNA polymerase γ (POLG) is the only polymerase known to be present in mammalian mitochondria. A dogma in the mitochondria field is that there is no other polymerase present in the mitochondria of mammalian cells. Here we demonstrate localization of REV3 DNA polymerase in the mammalian mitochondria. We demonstrate localization of REV3 in the mitochondria of mammalian tissue as well as cell lines. REV3 associates with POLG and mitochondrial DNA and protects the mitochondrial genome from DNA damage. Inactivation of Rev3 leads to reduced mitochondrial membrane potential, reduced OXPHOS activity, and increased glucose consumption. Conversely, inhibition of the OXPHOS increases expression of Rev3. Rev3 expression is increased in human primary breast tumors and breast cancer cell lines. Inactivation of Rev3 decreases cell migration and invasion, and localization of Rev3 in mitochondria increases survival and the invasive potential of cancer cells. Taken together, we demonstrate that REV3 functions in mammalian mitochondria and that mitochondrial REV3 is associated with the tumorigenic potential of cells.

  11. Characterization of Family D DNA polymerase from Thermococcus sp. 9°N

    OpenAIRE

    Greenough, Lucia; Menin, Julie F.; Desai, Nirav S.; Kelman, Zvi; Gardner, Andrew F.

    2014-01-01

    Accurate DNA replication is essential for maintenance of every genome. All archaeal genomes except Crenarchaea, encode for a member of Family B (polB) and Family D (polD) DNA polymerases. Gene deletion studies in Thermococcus kodakaraensis and Methanococcus maripaludis show that polD is the only essential DNA polymerase in these organisms. Thus, polD may be the primary replicative DNA polymerase for both leading and lagging strand synthesis. To understand this unique archaeal enzyme, we repor...

  12. Characterization of Family D DNA polymerase from Thermococcus sp. 9°N

    OpenAIRE

    Greenough, Lucia; Menin, Julie F.; Desai, Nirav S.; Kelman, Zvi; Gardner, Andrew F.

    2014-01-01

    Accurate DNA replication is essential for maintenance of every genome. All archaeal genomes except Crenarchaea, encode for a member of Family B (polB) and Family D (polD) DNA polymerases. Gene deletion studies in Thermococcus kodakaraensis and Methanococcus maripaludis show that polD is the only essential DNA polymerase in these organisms. Thus, polD may be the primary replicative DNA polymerase for both leading and lagging strand synthesis. To understand this unique archaeal enzyme, we repor...

  13. Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase γ.

    Science.gov (United States)

    Kasiviswanathan, Rajesh; Minko, Irina G; Lloyd, R Stephen; Copeland, William C

    2013-05-17

    Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N(6)-propano-2'-deoxyadenosine (γ-HOPdA). The bulky γ-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized translesion DNA synthesis polymerases to tolerate these lesions. Thus, it is important to understand how pol γ, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of pol γ to bypass the minor groove γ-HOPdG and major groove γ-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of pol γ-catalyzed bypass of γ-HOPdG was low, and surprisingly, pol γ preferred to incorporate purine nucleotides opposite the adduct. Pol γ also exhibited ∼2-fold lower rates of excision of the misincorporated purine nucleotides opposite γ-HOPdG compared with the corresponding nucleotides opposite dG. Extension of primers from the termini opposite γ-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, pol γ preferentially incorporated dT opposite the γ-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that γ-HOPdA is probably nonmutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but, in the case of the minor groove γ-HOPdG adduct, at the cost of unprecedented high mutation rates.

  14. Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus

    Science.gov (United States)

    Grúz, Petr; Shimizu, Masatomi; Pisani, Francesca M.; Felice, Mariarita De; Kanke, Yusuke; Nohmi, Takehiko

    2003-01-01

    Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80°C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3–4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP. PMID:12853619

  15. Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi.

    Science.gov (United States)

    Castillo-Lizardo, Melissa; Henneke, Ghislaine; Viguera, Enrique

    2014-01-01

    Replication slippage or slipped-strand mispairing involves the misalignment of DNA strands during the replication of repeated DNA sequences, and can lead to genetic rearrangements such as microsatellite instability. Here, we show that PolB and PolD replicative DNA polymerases from the archaeal model Pyrococcus abyssi (Pab) slip in vitro during replication of a single-stranded DNA template carrying a hairpin structure and short direct repeats. We find that this occurs in both their wild-type (exo+) and exonuclease deficient (exo-) forms. The slippage behavior of PabPolB and PabPolD, probably due to limited strand displacement activity, resembles that observed for the high fidelity P. furiosus (Pfu) DNA polymerase. The presence of PabPCNA inhibited PabPolB and PabPolD slippage. We propose a model whereby PabPCNA stimulates strand displacement activity and polymerase progression through the hairpin, thus permitting the error-free replication of repetitive sequences.

  16. Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi

    Directory of Open Access Journals (Sweden)

    Melissa G. eCastillo-Lizardo

    2014-08-01

    Full Text Available Replication slippage or slipped-strand mispairing involves the misalignment of DNA strands during the replication of repeated DNA sequences, and can lead to genetic rearrangements such as microsatellite instability. Here, we show that PolB and PolD replicative DNA polymerases from the archaeal model Pyrococcus abyssi (Pab slip in vitro during replication of a single-stranded DNA template carrying a hairpin structure and short direct repeats. We find that this occurs in both their wild-type (exo+ and exonuclease deficient (exo- forms. The slippage behavior of PabPolB and PabPolD, probably due to limited strand displacement activity, resembles that observed for the high fidelity Pyrococcus furiosus (Pfu DNA polymerase. The presence of PabPCNA inhibited PabPolB and PabPolD slippage. We propose a model whereby PabPCNA stimulates strand displacement activity and polymerase progression through the hairpin, thus permitting the error-free replication of repetitive sequences.

  17. DNA polymerase zeta (polζ) in higher eukaryotes

    Institute of Scientific and Technical Information of China (English)

    Gregory N Gan; John P Wittschieben; Birgitte φ Wittschieben; Richard D Wood

    2008-01-01

    Most current knowledge about DNA polymerase zeta (pol ζ) comes from studies of the enzyme in the budding yeast Saccharomyces cerevisiae, where polζ consists of a complex of the catalytic subunit Rev3 with Rev7, which associates with Rev1. Most spontaneous and induced mutagenesis in yeast is dependent on these gene products, and yeast pol can mediate translesion DNA synthesis past some adducts in DNA templates. Study of the homologous gene products in higher eukaryotes is in a relatively early stage, but additional functions for the eukaryotic proteins are already appar-ent. Suppression of vertebrate REV3L function not only reduces induced point mutagenesis but also causes larger-scale genuine instability by raising the frequency of spontaneous chromosome translocations. Disruption of Rev3L function is tolerated in Drosophila, Arabidopsis, and in vertebrate cell lines under some conditions, but is incompatible with mouse embryonic development. Functions for REV3L and REV7(MAD2B) in higher eukaryotes have been suggested not only in translesion DNA synthesis but also in some forms of homologous recombination, repair ofinterstrand DNA erosslinks, somatic hypermutation of immunoglobulin genes and cell-cycle control. This review discusses recent devel-opments in these areas.

  18. Pyrovanadolysis: a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate MN2plus and DNA Polymerase of Bacteriophage T7

    Energy Technology Data Exchange (ETDEWEB)

    B Akabayov; A Kulczyk; S Akabayov; C Thiele; L McLaughlin; B Beauchamp; C Richardson

    2011-12-31

    DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PP{sub i}). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry of the oxygen connecting the two phosphorus atoms of PP{sub i}, a variety of compounds was examined for their ability to carry out a reaction similar to pyrophosphorolysis. We describe a manganese-mediated pyrophosphorolysis-like activity using pyrovanadate (VV) catalyzed by the DNA polymerase of bacteriophage T7. We designate this reaction pyrovanadolysis. X-ray absorption spectroscopy reveals a shorter Mn-V distance of the polymerase-VV complex than the Mn-P distance of the polymerase-PP{sub i} complex. This structural arrangement at the active site accounts for the enzymatic activation by Mn-VV. We propose that the Mn{sup 2+}, larger than Mg{sup 2+}, fits the polymerase active site to mediate binding of VV into the active site of the polymerase. Our results may be the first documentation that vanadium can substitute for phosphorus in biological processes.

  19. XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

    Institute of Scientific and Technical Information of China (English)

    Julie K Horton; Mary Watson; Donna F Stefanick; Daniel T Shaughnessy; Jack A Taylor; Samuel H Wilson

    2008-01-01

    Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision re-pair (BER). SSBs generally have blocked termini lacking the conventional 5'-phosphate and 3'-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β (polβ) is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1-/-, and to a lesser extent in polβ-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type andpolβ-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induccd cytotoxicity. XRCC1-/- cellsare also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role inmodulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.

  20. DNA structure in human RNA polymerase II promoters

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Chauvin, Yves

    1998-01-01

    the high-bendability regions position nucleosomes at the downstream end of the transcriptional start point, and consider the possibility of interaction between histone-like TAFs and this area. We also propose the use of this structural signature in computational promoter-finding algorithms.......The fact that DNA three-dimensional structure is important for transcriptional regulation begs the question of whether eukaryotic promoters contain general structural features independently of what genes they control. We present an analysis of a large set of human RNA polymerase II promoters...... with a very low level of sequence similarity. The sequences, which include both TATA-containing and TATA-less promoters, are aligned by hidden Markov models. Using three different models of sequence-derived DNA bendability, the aligned promoters display a common structural profile with bendability being low...

  1. Study on the Interaction of Colloidal Gold with Taq DNA Polymerase

    Institute of Scientific and Technical Information of China (English)

    ZHU,Hong-Ping; MI,Li-Juan; CHEN,Shi-Mou; WANG,Wen-Feng; YAO,Si-De

    2007-01-01

    The interaction of colloidal gold with Taq DNA polymerase (Taq) was investigated in this study. Taq-gold conjugate was formed by adding the enzyme to the colloidal gold solution, as evidenced by UV-Vis spectroscopy,X-ray photoelectron spectroscopy, and photon cross correlation spectroscopy measurements. The conjugate was further characterized by transmission electron microscopy. It was found that the Taq-gold conjugate particles were still spherical and well-dispersed. The influence of gold nanoparticles on the bioactivity of Taq was studied by analyzing the yield of the polymerase chain reaction amplification. Results indicated that the enzymatic activity of Taq decreased after interaction with the colloidal gold.

  2. Increased learning and brain long-term potentiation in aged mice lacking DNA polymerase μ.

    Directory of Open Access Journals (Sweden)

    Daniel Lucas

    Full Text Available A definitive consequence of the aging process is the progressive deterioration of higher cognitive functions. Defects in DNA repair mechanisms mostly result in accelerated aging and reduced brain function. DNA polymerase µ is a novel accessory partner for the non-homologous end-joining DNA repair pathway for double-strand breaks, and its deficiency causes reduced DNA repair. Using associative learning and long-term potentiation experiments, we demonstrate that Polµ(-/- mice, however, maintain the ability to learn at ages when wild-type mice do not. Expression and biochemical analyses suggest that brain aging is delayed in Polµ(-/- mice, being associated with a reduced error-prone DNA oxidative repair activity and a more efficient mitochondrial function. This is the first example in which the genetic ablation of a DNA-repair function results in a substantially better maintenance of learning abilities, together with fewer signs of brain aging, in old mice.

  3. DNAPKcs-dependent arrest of RNA polymerase II transcription in the presence of DNA breaks.

    Science.gov (United States)

    Pankotai, Tibor; Bonhomme, Céline; Chen, David; Soutoglou, Evi

    2012-02-12

    DNA double-strand break (DSB) repair interferes with ongoing cellular processes, including replication and transcription. Although the process of replication stalling upon collision of replication forks with damaged DNA has been extensively studied, the fate of elongating RNA polymerase II (RNAPII) that encounters a DSB is not well understood. We show that the occurrence of a single DSB at a human RNAPII-transcribed gene leads to inhibition of transcription elongation and reinitiation. Upon inhibition of DNA protein kinase (DNAPK), RNAPII bypasses the break and continues transcription elongation, suggesting that it is not the break per se that inhibits the processivity of RNAPII, but the activity of DNAPK. We also show that the mechanism of DNAPK-mediated transcription inhibition involves the proteasome-dependent pathway. The results point to the pivotal role of DNAPK activity in the eviction of RNAPII from DNA upon encountering a DNA lesion.

  4. DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair.

    OpenAIRE

    Mossi, R; Ferrari, E; Hübscher, U

    1998-01-01

    The joining of single-stranded breaks in double-stranded DNA is an essential step in many important processes such as DNA replication, DNA repair, and genetic recombination. Several data implicate a role for DNA ligase I in DNA replication, probably coordinated by the action of other enzymes and proteins. Since both DNA polymerases delta and epsilon show multiple functions in different DNA transactions, we investigated the effect of DNA ligase I on various DNA synthesis events catalyzed by th...

  5. Role of Human DNA Polymerase kappa in Extension Opposite from a cis-syn Thymine Dimer

    Energy Technology Data Exchange (ETDEWEB)

    R Vasquez-Del Carpio; T Silverstein; S Lone; R Johnson; L Prakash; S Prakash; A Aggarwal

    2011-12-31

    Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase {Kappa} (Pol{Kappa}), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Pol{Kappa} in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure reveals a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Pol{Kappa} in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo.

  6. DNA polymerase ε and δ exonuclease domain mutations in endometrial cancer.

    Science.gov (United States)

    Church, David N; Briggs, Sarah E W; Palles, Claire; Domingo, Enric; Kearsey, Stephen J; Grimes, Jonathon M; Gorman, Maggie; Martin, Lynn; Howarth, Kimberley M; Hodgson, Shirley V; Kaur, Kulvinder; Taylor, Jenny; Tomlinson, Ian P M

    2013-07-15

    Accurate duplication of DNA prior to cell division is essential to suppress mutagenesis and tumour development. The high fidelity of eukaryotic DNA replication is due to a combination of accurate incorporation of nucleotides into the nascent DNA strand by DNA polymerases, the recognition and removal of mispaired nucleotides (proofreading) by the exonuclease activity of DNA polymerases δ and ε, and post-replication surveillance and repair of newly synthesized DNA by the mismatch repair (MMR) apparatus. While the contribution of defective MMR to neoplasia is well recognized, evidence that faulty DNA polymerase activity is important in cancer development has been limited. We have recently shown that germline POLE and POLD1 exonuclease domain mutations (EDMs) predispose to colorectal cancer (CRC) and, in the latter case, to endometrial cancer (EC). Somatic POLE mutations also occur in 5-10% of sporadic CRCs and underlie a hypermutator, microsatellite-stable molecular phenotype. We hypothesized that sporadic ECs might also acquire somatic POLE and/or POLD1 mutations. Here, we have found that missense POLE EDMs with good evidence of pathogenic effects are present in 7% of a set of 173 endometrial cancers, although POLD1 EDMs are uncommon. The POLE mutations localized to highly conserved residues and were strongly predicted to affect proofreading. Consistent with this, POLE-mutant tumours were hypermutated, with a high frequency of base substitutions, and an especially large relative excess of G:C>T:A transversions. All POLE EDM tumours were microsatellite stable, suggesting that defects in either DNA proofreading or MMR provide alternative mechanisms to achieve genomic instability and tumourigenesis.

  7. DNA polymerase ɛ and δ exonuclease domain mutations in endometrial cancer

    Science.gov (United States)

    Church, David N.; Briggs, Sarah E.W.; Palles, Claire; Domingo, Enric; Kearsey, Stephen J.; Grimes, Jonathon M.; Gorman, Maggie; Martin, Lynn; Howarth, Kimberley M.; Hodgson, Shirley V.; Kaur, Kulvinder; Taylor, Jenny; Tomlinson, Ian P.M.

    2013-01-01

    Accurate duplication of DNA prior to cell division is essential to suppress mutagenesis and tumour development. The high fidelity of eukaryotic DNA replication is due to a combination of accurate incorporation of nucleotides into the nascent DNA strand by DNA polymerases, the recognition and removal of mispaired nucleotides (proofreading) by the exonuclease activity of DNA polymerases δ and ɛ, and post-replication surveillance and repair of newly synthesized DNA by the mismatch repair (MMR) apparatus. While the contribution of defective MMR to neoplasia is well recognized, evidence that faulty DNA polymerase activity is important in cancer development has been limited. We have recently shown that germline POLE and POLD1 exonuclease domain mutations (EDMs) predispose to colorectal cancer (CRC) and, in the latter case, to endometrial cancer (EC). Somatic POLE mutations also occur in 5–10% of sporadic CRCs and underlie a hypermutator, microsatellite-stable molecular phenotype. We hypothesized that sporadic ECs might also acquire somatic POLE and/or POLD1 mutations. Here, we have found that missense POLE EDMs with good evidence of pathogenic effects are present in 7% of a set of 173 endometrial cancers, although POLD1 EDMs are uncommon. The POLE mutations localized to highly conserved residues and were strongly predicted to affect proofreading. Consistent with this, POLE-mutant tumours were hypermutated, with a high frequency of base substitutions, and an especially large relative excess of G:C>T:A transversions. All POLE EDM tumours were microsatellite stable, suggesting that defects in either DNA proofreading or MMR provide alternative mechanisms to achieve genomic instability and tumourigenesis. PMID:23528559

  8. Structural Basis of Transcription Initiation: An RNA Polymerase Holoenzyme-DNA Complex

    Science.gov (United States)

    Murakami, Katsuhiko S.; Masuda, Shoko; Campbell, Elizabeth A.; Muzzin, Oriana; Darst, Seth A.

    2002-05-01

    The crystal structure of Thermus aquaticus RNA polymerase holoenzyme (α2ββ'ωσA) complexed with a fork-junction promoter DNA fragment has been determined by fitting high-resolution x-ray structures of individual components into a 6.5-angstrom resolution map. The DNA lies across one face of the holoenzyme, completely outside the RNA polymerase active site channel. All sequence-specific contacts with core promoter elements are mediated by the σ subunit. A universally conserved tryptophan is ideally positioned to stack on the exposed face of the base pair at the upstream edge of the transcription bubble. Universally conserved basic residues of the σ subunit provide critical contacts with the DNA phosphate backbone and play a role in directing the melted DNA template strand into the RNA polymerase active site. The structure explains how holoenzyme recognizes promoters containing variably spaced -10 and -35 elements and provides the basis for models of the closed and open promoter complexes.

  9. Solution structures of 2 : 1 and 1 : 1 DNA polymerase-DNA complexes probed by ultracentrifugation and small-angle X-ray scattering

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Kuo-Hsiang; Niebuhr, Marc; Aulabaugh, Ann; Tsai, Ming-Daw [OSU; (Wyeth); (SSRL)

    2008-03-25

    We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase β (Pol β) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol β-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol β-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5'-dRP lyase domain of the second Pol β molecule with the active site of the 1 : 1 Pol β-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5'-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5'-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol β-DNA complex enhances the function of Pol β.

  10. Solution Structures of 2 : 1 And 1 : 1 DNA Polymerase - DNA Complexes Probed By Ultracentrifugation And Small-Angle X-Ray Scattering

    Energy Technology Data Exchange (ETDEWEB)

    Tang, K.H.; /Ohio State U.; Niebuhr, M.; /SLAC, SSRL; Aulabaugh, A.; /Wyeth Res. Biophys., Pearl River; Tsai, M.D.; /Ohio State U. /SLAC, SSRL

    2009-04-30

    We report small-angle X-ray scattering (SAXS) and sedimentation velocity (SV) studies on the enzyme-DNA complexes of rat DNA polymerase {beta} (Pol {beta}) and African swine fever virus DNA polymerase X (ASFV Pol X) with one-nucleotide gapped DNA. The results indicated formation of a 2 : 1 Pol {beta}-DNA complex, whereas only 1 : 1 Pol X-DNA complex was observed. Three-dimensional structural models for the 2 : 1 Pol {beta}-DNA and 1 : 1 Pol X-DNA complexes were generated from the SAXS experimental data to correlate with the functions of the DNA polymerases. The former indicates interactions of the 8 kDa 5{prime}-dRP lyase domain of the second Pol {beta} molecule with the active site of the 1 : 1 Pol {beta}-DNA complex, while the latter demonstrates how ASFV Pol X binds DNA in the absence of DNA-binding motif(s). As ASFV Pol X has no 5{prime}-dRP lyase domain, it is reasonable not to form a 2 : 1 complex. Based on the enhanced activities of the 2 : 1 complex and the observation that the 8 kDa domain is not in an optimal configuration for the 5{prime}-dRP lyase reaction in the crystal structures of the closed ternary enzyme-DNA-dNTP complexes, we propose that the asymmetric 2 : 1 Pol {beta}-DNA complex enhances the function of Pol {beta}.

  11. Proteomic Profiling Reveals a Specific Role for Translesion DNA Polymerase η in the Alternative Lengthening of Telomeres

    Directory of Open Access Journals (Sweden)

    Laura Garcia-Exposito

    2016-11-01

    Full Text Available Cancer cells rely on the activation of telomerase or the alternative lengthening of telomeres (ALT pathways for telomere maintenance and survival. ALT involves homologous recombination (HR-dependent exchange and/or HR-associated synthesis of telomeric DNA. Utilizing proximity-dependent biotinylation (BioID, we sought to determine the proteome of telomeres in cancer cells that employ these distinct telomere elongation mechanisms. Our analysis reveals that multiple DNA repair networks converge at ALT telomeres. These include the specialized translesion DNA synthesis (TLS proteins FANCJ-RAD18-PCNA and, most notably, DNA polymerase eta (Polη. We observe that the depletion of Polη leads to increased ALT activity and late DNA polymerase δ (Polδ-dependent synthesis of telomeric DNA in mitosis. We propose that Polη fulfills an important role in managing replicative stress at ALT telomeres, maintaining telomere recombination at tolerable levels and stimulating DNA synthesis by Polδ.

  12. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

    Science.gov (United States)

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-05-31

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells.

  13. Cloning and sequence analysis of novel DNA polymerases from thermophilic Geobacillus species isolated from hot springs in Turkey: characterization of a DNA polymerase I from Geobacillus kaue strain NB.

    Science.gov (United States)

    Çağlayan, Melike; Bilgin, Neş'e

    2011-11-01

    The complete coding sequences of the polA genes from seven thermophilic Geobacillus species, isolated from hot springs of Gönen and Hisaralan in Turkey, were cloned and sequenced. The polA genes of these Geobacillus species contain a long open reading frame of 2,637 bp encoding DNA polymerase I with a calculated molecular mass of 99 kDa. Amino acid sequences of these Geobacillus DNA polymerases are closely related. The multiple sequence alignments show all include the conserved amino acids in the polymerase and 5'-3' exonuclease domains, but the catalytic residues varied in 3'-5' exonuclease domain of these Geobacillus DNA polymerases. One of them, DNA polymerase I from Geobacillus kaue strain NB (Gkaue polI) is purified to homogeneity and biochemically characterized in vitro. The optimum temperature for enzymatic activity of Gkaue polI is 70 °C at pH 7.5-8.5 in the presence of 8 mM Mg(2+) and 80-100 mM of monovalent ions. The addition of polyamines stimulates the polymerization activity of the enzyme. Three-dimensional structure of Gkaue polI predicted using homology modeling confirmed the conservation of all the functionally important regions in the polymerase active site.

  14. Rapid Detection and Identification of a Pathogen's DNA Using Phi29 DNA Polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Y.; Dunn, J.; Gao, S.; Bruno, J. F.; Luft, B. J.

    2008-10-31

    Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments.

  15. Mechanistic Studies with DNA Polymerases Reveal Complex Outcomes following Bypass of DNA Damage

    Directory of Open Access Journals (Sweden)

    Robert L. Eoff

    2010-01-01

    Full Text Available DNA is a chemically reactive molecule that is subject to many different covalent modifications from sources that are both endogenous and exogenous in origin. The inherent instability of DNA is a major obstacle to genomic maintenance and contributes in varying degrees to cellular dysfunction and disease in multi-cellular organisms. Investigations into the chemical and biological aspects of DNA damage have identified multi-tiered and overlapping cellular systems that have evolved as a means of stabilizing the genome. One of these pathways supports DNA replication events by in a sense adopting the mantra that one must “make the best of a bad situation” and tolerating covalent modification to DNA through less accurate copying of the damaged region. Part of this so-called DNA damage tolerance pathway involves the recruitment of specialized DNA polymerases to sites of stalled or collapsed replication forks. These enzymes have unique structural and functional attributes that often allow bypass of adducted template DNA and successful completion of genomic replication. What follows is a selective description of the salient structural features and bypass properties of specialized DNA polymerases with an emphasis on Y-family members.

  16. Evolutionary connection between the catalytic subunits of DNA-dependent RNA polymerases and eukaryotic RNA-dependent RNA polymerases and the origin of RNA polymerases

    Directory of Open Access Journals (Sweden)

    Aravind L

    2003-01-01

    evolution of RNA polymerases. In addition to the β' subunit, β subunit of DDRP also contains a DPBB domain, which is, however, distorted by large inserts and does not harbor a counterpart of the DbDGD motif. The DPBB domains of the two DDRP subunits together form the catalytic cleft, with the domain from the β' subunit supplying the metal-coordinating DbDGD motif and the one from the β subunit providing two lysine residues involved in catalysis. Given that the two DPBB domains of DDRP contribute completely different sets of active residues to the catalytic center, it is hypothesized that the ultimate ancestor of RNA polymerases functioned as a homodimer of a generic, RNA-binding DPBB domain. This ancestral protein probably did not have catalytic activity and served as a cofactor for a ribozyme RNA polymerase. Subsequent evolution of DDRP and RDRP involved accretion of distinct sets of additional domains. In the DDRPs, these included a RNA-binding Zn-ribbon, an AT-hook-like module and a sandwich-barrel hybrid motif (SBHM domain. Further, lineage-specific accretion of SBHM domains and other, DDRP-specific domains is observed in bacterial DDRPs. In contrast, the orthologs of the β' subunit in archaea and eukaryotes contains a four-stranded α + β domain that is shared with the α-subunit of bacterial DDRP, eukaryotic DDRP subunit RBP11, translation factor eIF1 and type II topoisomerases. The additional domains of the RDRPs remain to be characterized. Conclusions Eukaryotic RNA-dependent RNA polymerases share the catalytic double-psi β-barrel domain, containing a signature metal-coordinating motif, with the universally conserved β' subunit of DNA-dependent RNA polymerases. Beyond this core catalytic domain, the two classes of RNA polymerases do not have common domains, suggesting early divergence from a common ancestor, with subsequent independent domain accretion. The β-subunit of DDRP contains another, highly diverged DPBB domain. The presence of two distinct DPBB

  17. Improving Polymerase Activity with Unnatural Substrates by Sampling Mutations in Homologous Protein Architectures.

    Science.gov (United States)

    Dunn, Matthew R; Otto, Carine; Fenton, Kathryn E; Chaput, John C

    2016-05-20

    The ability to synthesize and propagate genetic information encoded in the framework of xeno-nucleic acid (XNA) polymers would inform a wide range of topics from the origins of life to synthetic biology. While directed evolution has produced examples of engineered polymerases that can accept XNA substrates, these enzymes function with reduced activity relative to their natural counterparts. Here, we describe a biochemical strategy that enables the discovery of engineered polymerases with improved activity for a given unnatural polymerase function. Our approach involves identifying specificity determining residues (SDRs) that control polymerase activity, screening mutations at SDR positions in a model polymerase scaffold, and assaying key gain-of-function mutations in orthologous protein architectures. By transferring beneficial mutations between homologous protein structures, we show that new polymerases can be identified that function with superior activity relative to their starting donor scaffold. This concept, which we call scaffold sampling, was used to generate engineered DNA polymerases that can faithfully synthesize RNA and TNA (threose nucleic acid), respectively, on a DNA template with high primer-extension efficiency and low template sequence bias. We suggest that the ability to combine phenotypes from different donor and recipient scaffolds provides a new paradigm in polymerase engineering where natural structural diversity can be used to refine the catalytic activity of synthetic enzymes.

  18. Structural basis of transcription: nucleotide selection by rotation in the RNA polymerase II active center.

    Science.gov (United States)

    Westover, Kenneth D; Bushnell, David A; Kornberg, Roger D

    2004-11-12

    Binding of a ribonucleoside triphosphate to an RNA polymerase II transcribing complex, with base pairing to the template DNA, was revealed by X-ray crystallography. Binding of a mismatched nucleoside triphosphate was also detected, but in an adjacent site, inverted with respect to the correctly paired nucleotide. The results are consistent with a two-step mechanism of nucleotide selection, with initial binding to an entry (E) site beneath the active center in an inverted orientation, followed by rotation into the nucleotide addition (A) site for pairing with the template DNA. This mechanism is unrelated to that of single subunit RNA polymerases and so defines a new paradigm for the large, multisubunit enzymes. Additional findings from these studies include a third nucleotide binding site that may define the length of backtracked RNA; DNA double helix unwinding in advance of the polymerase active center; and extension of the diffraction limit of RNA polymerase II crystals to 2.3 A.

  19. Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase {eta} from Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Santiago, Maria Jesus [Departamento de Genetica, Facultad de Ciencias, Edificio Gregor Mendel, Campus Rabanales, Universidad de Cordoba (Spain); Alejandre-Duran, Encarna [Departamento de Genetica, Facultad de Ciencias, Edificio Gregor Mendel, Campus Rabanales, Universidad de Cordoba (Spain); Ruiz-Rubio, Manuel [Departamento de Genetica, Facultad de Ciencias, Edificio Gregor Mendel, Campus Rabanales, Universidad de Cordoba (Spain)]. E-mail: ge1rurum@uco.es

    2006-10-10

    DNA polymerase {eta} belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Pol{eta} homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Pol{eta} activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates.

  20. Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase eta from Arabidopsis thaliana.

    Science.gov (United States)

    Santiago, María Jesús; Alejandre-Durán, Encarna; Ruiz-Rubio, Manuel

    2006-10-10

    DNA polymerase eta belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Pol eta homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Pol eta activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates.

  1. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    Science.gov (United States)

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations.

  2. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

    Science.gov (United States)

    Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye

    2015-01-01

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. PMID:26627003

  3. Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ.

    Science.gov (United States)

    Copeland, William C; Kasiviswanathan, Rajesh; Longley, Matthew J

    2016-01-01

    Mitochondrial DNA is replicated by the nuclear-encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand cross-links from chemotherapy agents. Although many of these lesions block DNA replication, pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis.

  4. Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ

    Science.gov (United States)

    Copeland, William C.; Kasiviswanathan, Rajesh; Longley, Matthew J.

    2016-01-01

    Summary Mitochondrial DNA is replicated by the nuclear encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand crosslinks from chemotherapy agents. Although many of these lesions block DNA replication, Pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by Pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis. PMID:26530671

  5. T-DNA integration in plants results from polymerase-θ-mediated DNA repair.

    Science.gov (United States)

    van Kregten, Maartje; de Pater, Sylvia; Romeijn, Ron; van Schendel, Robin; Hooykaas, Paul J J; Tijsterman, Marcel

    2016-10-31

    Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown(1). Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA-plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3' end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer-template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.

  6. Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.

    Science.gov (United States)

    Zheng, Wenjun; Wang, Qingsong; Bi, Qun

    2016-04-01

    Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni(2+) chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future.

  7. Potentiation of temozolomide cytotoxicity by inhibition of DNA polymerase beta is accentuated by BRCA2 mutation.

    Science.gov (United States)

    Stachelek, Gregory C; Dalal, Shibani; Donigan, Katherine A; Campisi Hegan, Denise; Sweasy, Joann B; Glazer, Peter M

    2010-01-01

    Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly used in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid (LCA), an inhibitor of the key BER enzyme DNA polymerase beta (pol beta), in cells deficient in expression of the homology-dependent repair factor BRCA2. In vitro studies show that LCA suppresses the DNA polymerase and 5'-deoxyribose phosphate lyase activities of DNA pol beta by preventing the formation of a stable pol beta-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that LCA exhibits synergism with the alkylating agent temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both LCA and temozolomide individually. The potentiation of temozolomide cytotoxicity by LCA owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by gammaH2AX immunofluorescence. Death seems to be induced in cotreated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.

  8. New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III.

    Science.gov (United States)

    Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

    2014-01-01

    Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells.

  9. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

    Institute of Scientific and Technical Information of China (English)

    GU Fu; LI YuYang; L(U) Hong; YOU Chun; LIU JianPing; CHEN Ao; YU Yao; WANG Xiang; WAN DaFang; GU JianRen; YUAN HanYing

    2007-01-01

    DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recombination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepatocellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepatocarcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E.coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chromatography in an FPLC system. The analysis using isotope α-32p-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

  10. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recom-bination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepato-cellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepato-carcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chro-matography in an FPLC system. The analysis using isotope α-32P-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

  11. Translesion DNA polymerases Pol , Pol , Pol , Pol and Rev1 are not essential for repeat-induced point mutation in Neurospora crassa

    Indian Academy of Sciences (India)

    Ranjan Tamuli; C Ravindran; Durgadas P Kasbekar

    2006-12-01

    Pol , Pol , Pol , Pol and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) in Neurospora crassa. Additionally, we demonstrate that the upr-1 gene, which encodes the catalytic subunit of Pol , is a highly polymorphic locus in Neurospora.

  12. Role of the LEXE Motif of Protein-primed DNA Polymerases in the Interaction with the Incoming Nucleotide*

    Science.gov (United States)

    Santos, Eugenia; Lázaro, José M.; Pérez-Arnaiz, Patricia; Salas, Margarita; de Vega, Miguel

    2014-01-01

    The LEXE motif, conserved in eukaryotic type DNA polymerases, is placed close to the polymerization active site. Previous studies suggested that the second Glu was involved in binding a third noncatalytic ion in bacteriophage RB69 DNA polymerase. In the protein-primed DNA polymerase subgroup, the LEXE motif lacks the first Glu in most cases, but it has a conserved Phe/Trp and a Gly preceding that position. To ascertain the role of those residues, we have analyzed the behavior of mutants at the corresponding φ29 DNA polymerase residues Gly-481, Trp-483, Ala-484, and Glu-486. We show that mutations at Gly-481 and Trp-483 hamper insertion of the incoming dNTP in the presence of Mg2+ ions, a reaction highly improved when Mn2+ was used as metal activator. These results, together with previous crystallographic resolution of φ29 DNA polymerase ternary complex, allow us to infer that Gly-481 and Trp-483 could form a pocket that orients Val-250 to interact with the dNTP. Mutants at Glu-486 are also defective in polymerization and, as mutants at Gly-481 and Trp-483, in the pyrophosphorolytic activity with Mg2+. Recovery of both reactions with Mn2+ supports a role for Glu-486 in the interaction with the pyrophosphate moiety of the dNTP. PMID:24324256

  13. α,β-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

    Directory of Open Access Journals (Sweden)

    Olivier Martínez

    Full Text Available (S(C5', R(P α,β-D- Constrained Nucleic Acids (CNA are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

  14. α,β-D-Constrained Nucleic Acids Are Strong Terminators of Thermostable DNA Polymerases in Polymerase Chain Reaction

    Science.gov (United States)

    Mahéo, Sabrina; Gross, Grégori; Bodin, Pierre; Teissié, Justin; Escudier, Jean-Marc; Paquereau, Laurent

    2011-01-01

    (SC5′, RP) α,β-D- Constrained Nucleic Acids (CNA) are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5′C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases. PMID:21991314

  15. An in-frame deletion at the polymerase active site of POLD1 causes a multisystem disorder with lipodystrophy

    OpenAIRE

    Weedon, Michael N; Ellard, Sian; Prindle, Marc J.; Caswell, Richard; Allen, Hana Lango; Oram, Richard; Godbole, Koumudi; Yajnik, Chittaranjan S.; Sbraccia, Paolo; Novelli, Giuseppe; Turnpenny, Peter; McCann, Emma; Goh, Kim Jee; Wang, Yukai; Fulford, Jonathan

    2013-01-01

    DNA polymerase delta, whose catalytic subunit is encoded by POLD1, is responsible for lagging strand DNA synthesis during DNA replication 1 . It achieves this with high fidelity due to its intrinsic 3′ to 5′ exonuclease activity, which confers proofreading ability. Missense mutations in the exonuclease domain of POLD1 have recently been shown to predispose to colorectal and endometrial cancer 2 . Here we report a recurring heterozygous single amino acid deletion at the polymerase active site ...

  16. Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions.

    Directory of Open Access Journals (Sweden)

    Sung-Kun Kim

    Full Text Available Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2',4'-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2',4'-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2',4'-bridged thymidine operates. With a Ki value of 9.7 ± 1.1 μM, the 2',4'-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2',4'-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2',4'-bridged thymidine. Molecular dynamics (MD simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2',4'-bridged thymidine suggests the potential of bridged nucleosides as drug candidates.

  17. Cell cycle phase dependent role of DNA polymerase beta in DNA repair and survival after ionizing radiation.

    NARCIS (Netherlands)

    Vermeulen, C.; Verwijs-Janssen, M.; Begg, A.C.; Vens, C.

    2008-01-01

    PURPOSE: The purpose of the present study was to determine the role of DNA polymerase beta in repair and response after ionizing radiation in different phases of the cell cycle. METHODS AND MATERIALS: Synchronized cells deficient and proficient in DNA polymerase beta were irradiated in different pha

  18. Polymerase chain reaction and conventional DNA tests in detection of HPV DNA in cytologically normal and abnormal cervical scrapes

    DEFF Research Database (Denmark)

    Kalia, A.; Jalava, T.; Nieminen, P.

    1992-01-01

    Med.mikrobiologi, polymerase chain reaction, DNA tests, human papillomavirus (HPV), cervical smear, hybridisation, cytologi, affiProbe HPV test, ViraType test......Med.mikrobiologi, polymerase chain reaction, DNA tests, human papillomavirus (HPV), cervical smear, hybridisation, cytologi, affiProbe HPV test, ViraType test...

  19. The discovery of error-prone DNA polymerase V and its unique regulation by RecA and ATP.

    Science.gov (United States)

    Goodman, Myron F

    2014-09-26

    My career pathway has taken a circuitous route, beginning with a Ph.D. degree in electrical engineering from The Johns Hopkins University, followed by five postdoctoral years in biology at Hopkins and culminating in a faculty position in biological sciences at the University of Southern California. My startup package in 1973 consisted of $2,500, not to be spent all at once, plus an ancient Packard scintillation counter that had a series of rapidly flashing light bulbs to indicate a radioactive readout in counts/minute. My research pathway has been similarly circuitous. The discovery of Escherichia coli DNA polymerase V (pol V) began with an attempt to identify the mutagenic DNA polymerase responsible for copying damaged DNA as part of the well known SOS regulon. Although we succeeded in identifying a DNA polymerase, one that was induced as part of the SOS response, we actually rediscovered DNA polymerase II, albeit in a new role. A decade later, we discovered a new polymerase, pol V, whose activity turned out to be regulated by bound molecules of RecA protein and ATP. This Reflections article describes our research trajectory, includes a review of key features of DNA damage-induced SOS mutagenesis leading us to pol V, and reflects on some of the principal researchers who have made indispensable contributions to our efforts.

  20. DNA polymerase-associated lectin (DPAL) and its binding to the galactose-containing glycoconjugate of the replication complex.

    Science.gov (United States)

    Kelley, T J; St Amand, T; Groll, J M; Ray, S; Basu, S

    1999-10-01

    The highly purified DNA Pol-alpha from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488-1492]. Binding of ConA and RCA to human recombinant DNA polymerase-alpha showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231-6237]. The catalytic polypeptide, DNA polymerase-alpha of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-alpha from embryonic chicken brain (ECB) contains an alpha-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-alpha reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567-573] by the treatment with methyl-alpha-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an alpha-galactose binding, 56kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-alpha as determined by immunostaining with the polymerase-alpha-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-alpha and a complete separation of polymerase complex and

  1. Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth.

    Science.gov (United States)

    Rahn, Jennifer J; Bestman, Jennifer E; Stackley, Krista D; Chan, Sherine S L

    2015-12-02

    DNA polymerase gamma (POLG) is essential for replication and repair of mitochondrial DNA (mtDNA). Mutations in POLG cause mtDNA instability and a diverse range of poorly understood human diseases. Here, we created a unique Polg animal model, by modifying polg within the critical and highly conserved polymerase domain in zebrafish. polg(+/-) offspring were indistinguishable from WT siblings in multiple phenotypic and biochemical measures. However, polg(-/-) mutants developed severe mtDNA depletion by one week post-fertilization (wpf), developed slowly and had regenerative defects, yet surprisingly survived up to 4 wpf. An in vivo mtDNA polymerase activity assay utilizing ethidium bromide (EtBr) to deplete mtDNA, showed that polg(+/-) and WT zebrafish fully recover mtDNA content two weeks post-EtBr removal. EtBr further reduced already low levels of mtDNA in polg(-/-) animals, but mtDNA content did not recover following release from EtBr. Despite significantly decreased respiration that corresponded with tissue-specific levels of mtDNA, polg(-/-) animals had WT levels of ATP and no increase in lactate. This zebrafish model of mitochondrial disease now provides unique opportunities for studying mtDNA instability from multiple angles, as polg(-/-) mutants can survive to juvenile stage, rather than lose viability in embryogenesis as seen in Polg mutant mice.

  2. Analysis of unassisted translesion replication by the DNA polymerase III holoenzyme.

    Science.gov (United States)

    Tomer, G; Livneh, Z

    1999-05-01

    DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotides, as model DNA substrates for translesion replication. Using these substrates, we show that the DNA polymerase III holoenzyme from Escherichia coli can bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of the polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when standing-start and running-start replication were compared. A comparison of translesion replication by DNA polymerase I, DNA polymerase II, the DNA polymerase III core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by adding the gamma complex and beta subunit, did not increase bypass. These results support the model that SOS regulation was imposed on a fundamentally constitutive translesion replication reaction to achieve tight control of mutagenesis.

  3. Crystal Structure of a Replicative DNA Polymerase Bound to the Oxidized Guanine Lesion Guanidinohydantoin

    Energy Technology Data Exchange (ETDEWEB)

    Aller, Pierre; Ye, Yu; Wallace, Susan S.; Burrows, Cynthia J.; Doubli, Sylvie (Vermont); (Utah)

    2010-04-12

    The oxidation of guanine generates one of the most common DNA lesions, 8-oxo-7,8-dihydroguanine (8-oxoG). The further oxidation of 8-oxoG can produce either guanidinohydantoin (Gh) in duplex DNA or spiroiminodihydantoin (Sp) in nucleosides and ssDNA. Although Gh can be a strong block for replicative DNA polymerases such as RB69 DNA polymerase, this lesion is also mutagenic: DNA polymerases bypass Gh by preferentially incorporating a purine with a slight preference for adenine, which results in G {center_dot} C {yields} T {center_dot} A or G {center_dot} C {yields} C {center_dot} G transversions. The 2.15 {angstrom} crystal structure of the replicative RB69 DNA polymerase in complex with DNA containing Gh reveals that Gh is extrahelical and rotated toward the major groove. In this conformation Gh is no longer in position to serve as a templating base for the incorporation of an incoming nucleotide. This work also constitutes the first crystallographic structure of Gh, which is stabilized in the R configuration in the two polymerase/DNA complexes present in the crystal asymmetric unit. In contrast to 8-oxoG, Gh is found in a high syn conformation in the DNA duplex and therefore presents the same hydrogen bond donor and acceptor pattern as thymine, which explains the propensity of DNA polymerases to incorporate a purine opposite Gh when bypass occurs.

  4. N-Aroyl Indole Thiobarbituric Acids as Inhibitors of DNA Repair and Replication Stress Response Polymerases

    Science.gov (United States)

    Coggins, Grace E.; Maddukuri, Leena; Penthala, Narsima R.; Hartman, Jessica H.; Eddy, Sarah; Ketkar, Amit; Crooks, Peter A.; Eoff, Robert L.

    2013-01-01

    Using a robust and quantitative assay, we have identified a novel class of DNA polymerase inhibitors that exhibits some specificity against an enzyme involved in resistance to anti-cancer drugs, namely human DNA polymerase eta (hpol η). In our initial screen, we identified the indole thiobarbituric acid (ITBA) derivative 5-((1-(2-bromobenzoyl)-5-chloro-1H-indol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (ITBA-12) as an inhibitor of the Y-family DNA member hpol η, an enzyme that has been associated with increased resistance to cisplatin and doxorubicin treatments. An additional seven DNA polymerases from different sub-families were tested for inhibition by ITBA-12. Hpol η was the most potently inhibited enzyme (30 ± 3 μM), with hpol β, hpol γ and hpol κ exhibiting comparable but higher IC50 values of 41 ± 24 μM, 49 ± 6 μM and 59 ± 11 μM, respectively. The other polymerases tested had IC50 values closer to 80 μM. Steady-state kinetic analysis was used to investigate the mechanism of polymerase inhibition by ITBA-12. Based on changes in the Michaelis constant, it was determined that ITBA-12 acts as an allosteric (or partial) competitive inhibitor of dNTP binding. The parent ITBA scaffold was modified to produce 20 derivatives and establish structure-activity relationships by testing for inhibition of hpol η. Two compounds with N-naphthoyl Ar-substituents, ITBA-16 and ITBA-19, were both found to have improved potency against hpol η with IC50 values of 16 ± 3 μM and 17 ± 3 μM, respectively. Moreover, the specificity of ITBA-16 was improved relative to ITBA-12. The presence of a chloro substituent at position 5 on the indole ring appears to be crucial for effective inhibition of hpol η, with the indole N-1-naphthoyl and N-2-naphthoyl analogs being the most potent inhibitors of hpol η. These results provide a framework from which second-generation ITBA derivatives may be developed against specialized polymerases that are involved in

  5. Gastrointestinal hyperplasia with altered expression of DNA polymerase beta.

    Directory of Open Access Journals (Sweden)

    Katsuhiko Yoshizawa

    Full Text Available BACKGROUND: Altered expression of DNA polymerase beta (Pol beta has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol beta over-expression has not yet been evaluated in a mouse model. METHODOLOGY/PRINCIPAL FINDINGS: We have recently developed a novel transgenic mouse model that over-expresses Pol beta. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner's gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol beta over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol beta expression. We observed elevated expression of Pol beta in stomach adenomas and thyroid follicular carcinomas, but reduced Pol beta expression in esophageal adenocarcinomas and squamous carcinomas. CONCLUSIONS/SIGNIFICANCE: These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation.

  6. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases. delta. and. beta. are involved in DNA repair synthesis induced by N-methyl-N prime -nitro-N-nitrosoguanidine

    Energy Technology Data Exchange (ETDEWEB)

    Hammond, R.A.; Miller, M.R. (West Virginia Univ. Health Sciences Center, Morgantown (USA)); McClung, J.K. (Samuel Roberts Noble Foundation, Inc., East Ardmore, OK (USA))

    1990-01-09

    The involvement of DNA polymerases {alpha}, {beta}, and {delta} in DNA repair synthesis induced by N-methyl-N{prime}-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase {alpha}) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of ({sup 3}H)thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 {mu}g of aphidicolin/mL, 6% by 10 {mu}M BuPdGTP, 13% by anti-(DNA polymerse {alpha}) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 {mu}g of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase {alpha}) antibodies into HF nuclei. These results indicate that both DNA polymerase {delta} and {beta} are involved in repairing DNA damage caused by MNNG.

  7. The beta subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme.

    Science.gov (United States)

    Tomer, G; Reuven, N B; Livneh, Z

    1998-11-24

    The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD' and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the beta subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the beta subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the beta subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly -1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD', UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the beta subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.

  8. Conformational Selection and Induced Fit for RNA Polymerase and RNA/DNA Hybrid Backtracked Recognition

    Directory of Open Access Journals (Sweden)

    Haifeng eChen

    2015-11-01

    Full Text Available RNA polymerase catalyzes transcription with a high fidelity. If DNA/RNA mismatch or DNA damage occurs downstream, a backtracked RNA polymerase can proofread this situation. However, the backtracked mechanism is still poorly understood. Here we have performed multiple explicit-solvent molecular dynamics (MD simulations on bound and apo DNA/RNA hybrid to study backtracked recognition. MD simulations at room temperature suggest that specific electrostatic interactions play key roles in the backtracked recognition between the polymerase and DNA/RNA hybrid. Kinetics analysis at high temperature shows that bound and apo DNA/RNA hybrid unfold via a two-state process. Both kinetics and free energy landscape analyses indicate that bound DNA/RNA hybrid folds in the order of DNA/RNA contracting, the tertiary folding and polymerase binding. The predicted Φ-values suggest that C7, G9, dC12, dC15 and dT16 are key bases for the backtracked recognition of DNA/RNA hybrid. The average RMSD values between the bound structures and the corresponding apo ones and Kolmogorov-Smirnov (KS P test analyses indicate that the recognition between DNA/RNA hybrid and polymerase might follow an induced fit mechanism for DNA/RNA hybrid and conformation selection for polymerase. Furthermore, this method could be used to relative studies of specific recognition between nucleic acid and protein.

  9. L-Homoserylaminoethanol, a novel dipeptide alcohol inhibitor of eukaryotic DNA polymerase from a plant cultured cells, Nicotina tabacum L.

    Science.gov (United States)

    Kuriyama, Isoko; Asano, Naoki; Kato, Ikuo; Oshige, Masahiko; Sugino, Akio; Kadota, Yasuhiro; Kuchitsu, Kazuyuki; Yoshida, Hiromi; Sakaguchi, Kengo; Mizushina, Yoshiyuki

    2004-03-01

    We found a novel inhibitor specific to eukaryotic DNA polymerase epsilon(pol epsilon) from plant cultured cells, Nicotina tabacum L. The compound (compound 1) was a dipeptide alcohol, L-homoserylaminoethanol. The 50% inhibition of pol epsilon activity by the compound was 43.6 microg/mL, and it had almost no effect on the activities of the other eukaryotic DNA polymerases such as alpha, beta, gamma and delta, prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human telomerase, human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, human DNA topoisomerase I and II, T4 polynucleotide kinase and bovine deoxyribonuclease I. Kinetic studies showed that inhibition of pol epsilon by the compound was non-competitive with respect to both template-primer DNA and nucleotide substrate. We succeeded in chemically synthesizing the stereoisomers, L-homoserylaminoethanol and D-homoserylaminoethanol, and found both were effective to the same extent. The IC(50) values of L- and D-homoserylaminoethanols for pol epsilon were 42.0 and 41.5 microg/mL, respectively. This represents the second discovery of a pol epsilon-specific inhibitor, and the first report on a water-soluble peptide-like compound as the inhibitor, which is required in biochemical studies of pol epsilon.

  10. Bacillus subtilis DNA polymerases, PolC and DnaE, are required for both leading and lagging strand synthesis in SPP1 origin-dependent DNA replication.

    Science.gov (United States)

    Seco, Elena M; Ayora, Silvia

    2017-08-21

    Firmicutes have two distinct replicative DNA polymerases, the PolC leading strand polymerase, and PolC and DnaE synthesizing the lagging strand. We have reconstituted in vitro Bacillus subtilis bacteriophage SPP1 θ-type DNA replication, which initiates unidirectionally at oriL. With this system we show that DnaE is not only restricted to lagging strand synthesis as previously suggested. DnaG primase and DnaE polymerase are required for initiation of DNA replication on both strands. DnaE and DnaG synthesize in concert a hybrid RNA/DNA 'initiation primer' on both leading and lagging strands at the SPP1 oriL region, as it does the eukaryotic Pol α complex. DnaE, as a RNA-primed DNA polymerase, extends this initial primer in a reaction modulated by DnaG and one single-strand binding protein (SSB, SsbA or G36P), and hands off the initiation primer to PolC, a DNA-primed DNA polymerase. Then, PolC, stimulated by DnaG and the SSBs, performs the bulk of DNA chain elongation at both leading and lagging strands. Overall, these modulations by the SSBs and DnaG may contribute to the mechanism of polymerase switch at Firmicutes replisomes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Steady-State Kinetic Analysis of DNA Polymerase Single-Nucleotide Incorporation Products

    Science.gov (United States)

    O'Flaherty, Derek K.

    2014-01-01

    This unit describes the experimental procedures for the steady-state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady-state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases. PMID:25501593

  12. Biochemical analysis of six genetic variants of error-prone human DNA polymerase ι involved in translesion DNA synthesis.

    Science.gov (United States)

    Kim, Jinsook; Song, Insil; Jo, Ara; Shin, Joo-Ho; Cho, Hana; Eoff, Robert L; Guengerich, F Peter; Choi, Jeong-Yun

    2014-10-20

    DNA polymerase (pol) ι is the most error-prone among the Y-family polymerases that participate in translesion synthesis (TLS). Pol ι can bypass various DNA lesions, e.g., N(2)-ethyl(Et)G, O(6)-methyl(Me)G, 8-oxo-7,8-dihydroguanine (8-oxoG), and an abasic site, though frequently with low fidelity. We assessed the biochemical effects of six reported genetic variations of human pol ι on its TLS properties, using the recombinant pol ι (residues 1-445) proteins and DNA templates containing a G, N(2)-EtG, O(6)-MeG, 8-oxoG, or abasic site. The Δ1-25 variant, which is the N-terminal truncation of 25 residues resulting from an initiation codon variant (c.3G > A) and also is the formerly misassigned wild-type, exhibited considerably higher polymerase activity than wild-type with Mg(2+) (but not with Mn(2+)), coinciding with its steady-state kinetic data showing a ∼10-fold increase in kcat/Km for nucleotide incorporation opposite templates (only with Mg(2+)). The R96G variant, which lacks a R96 residue known to interact with the incoming nucleotide, lost much of its polymerase activity, consistent with the kinetic data displaying 5- to 72-fold decreases in kcat/Km for nucleotide incorporation opposite templates either with Mg(2+) or Mn(2+), except for that opposite N(2)-EtG with Mn(2+) (showing a 9-fold increase for dCTP incorporation). The Δ1-25 variant bound DNA 20- to 29-fold more tightly than wild-type (with Mg(2+)), but the R96G variant bound DNA 2-fold less tightly than wild-type. The DNA-binding affinity of wild-type, but not of the Δ1-25 variant, was ∼7-fold stronger with 0.15 mM Mn(2+) than with Mg(2+). The results indicate that the R96G variation severely impairs most of the Mg(2+)- and Mn(2+)-dependent TLS abilities of pol ι, whereas the Δ1-25 variation selectively and substantially enhances the Mg(2+)-dependent TLS capability of pol ι, emphasizing the potential translational importance of these pol ι genetic variations, e.g., individual differences

  13. RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template.

    Science.gov (United States)

    Pai, Dave A; Kaplan, Craig D; Kweon, Hye Kyong; Murakami, Kenji; Andrews, Philip C; Engelke, David R

    2014-05-01

    Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

  14. Stability of the human polymerase δ holoenzyme and its implications in lagging strand DNA synthesis.

    Science.gov (United States)

    Hedglin, Mark; Pandey, Binod; Benkovic, Stephen J

    2016-03-29

    In eukaryotes, DNA polymerase δ (pol δ) is responsible for replicating the lagging strand template and anchors to the proliferating cell nuclear antigen (PCNA) sliding clamp to form a holoenzyme. The stability of this complex is integral to every aspect of lagging strand replication. Most of our understanding comes from Saccharomyces cerevisae where the extreme stability of the pol δ holoenzyme ensures that every nucleobase within an Okazaki fragment is faithfully duplicated before dissociation but also necessitates an active displacement mechanism for polymerase recycling and exchange. However, the stability of the human pol δ holoenzyme is unknown. We designed unique kinetic assays to analyze the processivity and stability of the pol δ holoenzyme. Surprisingly, the results indicate that human pol δ maintains a loose association with PCNA while replicating DNA. Such behavior has profound implications on Okazaki fragment synthesis in humans as it limits the processivity of pol δ on undamaged DNA and promotes the rapid dissociation of pol δ from PCNA on stalling at a DNA lesion.

  15. Role of a GAG hinge in the nucleotide-induced conformational change governing nucleotide specificity by T7 DNA polymerase.

    Science.gov (United States)

    Jin, Zhinan; Johnson, Kenneth A

    2011-01-14

    A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches.

  16. Maintenance of Genome Integrity: How Mammalian Cells Orchestrate Genome Duplication by Coordinating Replicative and Specialized DNA Polymerases

    OpenAIRE

    Barnes, Ryan; Eckert, Kristin

    2017-01-01

    Precise duplication of the human genome is challenging due to both its size and sequence complexity. DNA polymerase errors made during replication, repair or recombination are central to creating mutations that drive cancer and aging. Here, we address the regulation of human DNA polymerases, specifically how human cells orchestrate DNA polymerases in the face of stress to complete replication and maintain genome stability. DNA polymerases of the B-family are uniquely adept at accurate genome ...

  17. AraUTP-Affi-Gel 10: a novel affinity absorbent for the specific purification of DNA polymerase alpha-primase.

    Science.gov (United States)

    Izuta, S; Saneyoshi, M

    1988-10-01

    For the specific purification of eukaryotic DNA-dependent DNA polymerase alpha, we prepared two novel affinity resins bearing 5-(E)-(4-aminostyryl) araUTP as a ligand. One of them was araUTP-Sepharose 4B which was coupled directly with the ligand and the other was araUTP-Affi-Gel 10 which was coupled with the ligand through a spacer. No DNA polymerase alpha-primase activity from cherry salmon (Oncorhynchus masou) testes was bound on the araUTP-Sepharose 4B in all cases examined. On the other hand, the araUTP-Affi-Gel 10 retains this enzyme activity when poly(dA) or poly(dA)-oligo(dT)12-18 is present. The retained enzyme activity was sharply eluted around 100-mM KCl concentrations as a single peak, and this fraction showed a specific activity of about 170,000 units/mg as alpha-polymerase activity. The highly purified DNA polymerase alpha-primase isolated using the araUTP-Affi-Gel 10 contained only three polypeptides, which showed Mr values of 120,000, 62,000, and 58,000, respectively, as judged using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

  18. Proteinase-Polymerase Precursor as the Active Form of Feline Calicivirus RNA-Dependent RNA Polymerase

    OpenAIRE

    Wei, Lai; Huhn, Jason S.; Mory, Aaron; Pathak, Harsh B.; Sosnovtsev, Stanislav V.; Green, Kim Y.; Cameron, Craig E.

    2001-01-01

    The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a three...

  19. Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana

    Science.gov (United States)

    Kashiwagi, Sayo; Kuraoka, Isao; Fujiwara, Yoshie; Hitomi, Kenichi; Cheng, Quen J.; Fuss, Jill O.; Shin, David S.; Masutani, Chikahide; Tainer, John A.; Hanaoka, Fumio; Iwai, Shigenori

    2010-01-01

    Human DNA polymerase η (HsPolη) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolη from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolη shares sequence homology with HsPolη and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolη is more thermostable than HsPolη, as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPolη provides a robust, human-like Polη that is more active after exposure to high temperatures and organic solvents. PMID:20936172

  20. Mitochondrial DNA polymerase gamma variants in idiopathic sporadic Parkinson disease.

    Science.gov (United States)

    Luoma, P T; Eerola, J; Ahola, S; Hakonen, A H; Hellström, O; Kivistö, K T; Tienari, P J; Suomalainen, A

    2007-09-11

    Dysfunction of mitochondrial DNA polymerase gamma (POLG) has been recently recognized as an important cause of inherited neurodegenerative diseases. We have reported dominant and recessive inheritance of parkinsonism, mitochondrial myopathy, and premature amenorrhea in five ethnically distinct families with POLG1 mutations. This prompted us to carry out a detailed analysis of the coding region and intron-exon boundaries of POLG1 in Finnish patients with idiopathic sporadic Parkinson disease (PD) and in nonparkinsonian controls. The coding region of POLG1 was analyzed in 140 Finnish patients with PD and their 127 spouses as age- and ethnically matched controls. Further, we analyzed the intragenic CAG-repeat region of POLG1 in 126 additional patients with nonparkinsonian neurologic disorders and in 516 Finnish population controls. We found clustering of rare variants of the POLG1 CAG-repeat, encoding a polyglutamine tract, in Finnish patients with idiopathic PD as compared to their spouses (p = 0.003; OR 3.01, 95% CI 1.35 to 6.71), population controls (p = 0.001; OR 2.45, 95% CI 1.45 to 4.14), and patients with nonparkinsonian neurologic disorders (p = 0.05, OR 1.98, 95% CI 0.97 to 4.05). We found several amino acid substitutions, none of them associating with PD. These included a previously parkinsonism-associated POLG variant Y831C, found in one patient with PD, but also in five controls, suggesting that it is a neutral amino acid polymorphism. Our results suggest that POLG polyglutamine tract variants should be considered as a predisposing genetic factor in idiopathic sporadic Parkinson disease.

  1. Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

    Science.gov (United States)

    Gardner, Shea N.; Mariella, Jr., Raymond P.; Christian, Allen T.; Young, Jennifer A.; Clague, David S.

    2011-01-18

    A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

  2. Possible Role of DNA Polymerase beta in Protecting Human Bronchial Epithelial Cells Against Cytotoxicity of Hydroquinone

    Institute of Scientific and Technical Information of China (English)

    DA-LIN HU; JIAN-PING YANG; DAO-KUI FANG; YAN SHA; XIAO-ZHI TU; ZHI-XIONG ZHUANG; HUAN-WEN TANG; HAI-RONG LIANG; DONG-SHENG TANG; YI-MING LIU; WEI-DONG JI; JIAN-HUI YUAN; YUN HE; ZHENG-YU ZHU

    2007-01-01

    Objective To explore the toxicological mechanism of hydroquinone in human bronchial epithelial cells and to investigate whether DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone. Methods DNA polymerase beta knock-down cell line was established via RNA interference as an experimental group. Normal human bronchial epithelial cells and cells transfected with the empty vector of pEGFP-Cl were used as controls. Cells were treated with different concentrations of hydroquinone (ranged from 10 μmol/L to 120 μmol/L) for 4 hours. MTT assay and Comet assay [single-cell gel electrophoresis (SCGE)] were performed respectively to detect the toxicity of hydroquinone. Results MTT assay showed that DNA polymerase beta knock-down cells treated with different concentrations of hydroquinone had a lower absorbance value at 490 nm than the control cells in a dose-dependant manner. Comet assay revealed that different concentrations of hydroquinone caused more severe DNA damage in DNA polymerase beta knock-down cell line than in control cells and there was no significant difference in the two control groups. Conclusions Hydroquinone has significant toxicity to human bronchial epithelial cells and causes DNA damage. DNA polymerase beta knock-down cell line appears more sensitive to hydroquinone than the control cells. The results suggest that DNA polymerase beta is involved in protecting cells from damage caused by hydroquinone.

  3. Dynamic Conformational Change Regulates the Protein-DNA Recognition: An Investigation on Binding of a Y-Family Polymerase to Its Target DNA

    Science.gov (United States)

    Chu, Xiakun; Liu, Fei; Maxwell, Brian A.; Wang, Yong; Suo, Zucai; Wang, Haijun; Han, Wei; Wang, Jin

    2014-01-01

    Protein-DNA recognition is a central biological process that governs the life of cells. A protein will often undergo a conformational transition to form the functional complex with its target DNA. The protein conformational dynamics are expected to contribute to the stability and specificity of DNA recognition and therefore may control the functional activity of the protein-DNA complex. Understanding how the conformational dynamics influences the protein-DNA recognition is still challenging. Here, we developed a two-basin structure-based model to explore functional dynamics in Sulfolobus solfataricus DNA Y-family polymerase IV (DPO4) during its binding to DNA. With explicit consideration of non-specific and specific interactions between DPO4 and DNA, we found that DPO4-DNA recognition is comprised of first 3D diffusion, then a short-range adjustment sliding on DNA and finally specific binding. Interestingly, we found that DPO4 is under a conformational equilibrium between multiple states during the binding process and the distributions of the conformations vary at different binding stages. By modulating the strength of the electrostatic interactions, the flexibility of the linker, and the conformational dynamics in DPO4, we drew a clear picture on how DPO4 dynamically regulates the DNA recognition. We argue that the unique features of flexibility and conformational dynamics in DPO4-DNA recognition have direct implications for low-fidelity translesion DNA synthesis, most of which is found to be accomplished by the Y-family DNA polymerases. Our results help complete the description of the DNA synthesis process for the Y-family polymerases. Furthermore, the methods developed here can be widely applied for future investigations on how various proteins recognize and bind specific DNA substrates. PMID:25188490

  4. DNA Polymerase θ: Duct Tape and Zip Ties for a Fragile Genome.

    Science.gov (United States)

    Willis, Nicholas A; Scully, Ralph

    2016-08-18

    Using a combination of genetics and cellular DNA rejoining assays, in this issue of Molecular Cell, Wyatt et al. (2016) demonstrate a critical role for mammalian DNA polymerase θ in the rejoining of DNA ends that are poor substrates for classical non-homologous end joining.

  5. Polymerase chain reaction-mediated DNA fingerprinting for epidemiological studies on Campylobacter spp

    NARCIS (Netherlands)

    Giesendorf, B A; Goossens, H; Niesters, H G; Van Belkum, A; Koeken, A; Endtz, H P; Stegeman, H; Quint, W G

    1994-01-01

    The applicability of polymerase chain reaction (PCR)-mediated DNA typing, with primers complementary to dispersed repetitive DNA sequences and arbitrarily chosen DNA motifs, to study the epidemiology of campylobacter infection was evaluated. With a single PCR reaction and simple gel electrophoresis,

  6. Polymerase chain reaction-mediated DNA fingerprinting for epidemiological studies on Campylobacter spp

    NARCIS (Netherlands)

    Giesendorf, B A; Goossens, H; Niesters, H G; Van Belkum, A; Koeken, A; Endtz, H P; Stegeman, H; Quint, W G

    The applicability of polymerase chain reaction (PCR)-mediated DNA typing, with primers complementary to dispersed repetitive DNA sequences and arbitrarily chosen DNA motifs, to study the epidemiology of campylobacter infection was evaluated. With a single PCR reaction and simple gel electrophoresis,

  7. Polymerase study: Improved detection of Salmonella and Campylobacter through the optimized use of DNA polymerases in diagnostic real-time PCR

    DEFF Research Database (Denmark)

    Søndergaard, Mette Sofie Rousing; Löfström, Charlotta; Al-Habib, Zahra Fares Sayer;

    commercially available polymerases and four master mixes in two validated PCR assays, for Campylobacter and Salmonella, respectively, to develop more sensitive, robust and cost effective assays. The polymerases were screened on purified DNA and the five best performing, for each PCR assay, were then applied...... and robustness of a PCR assay, as some polymerases are more resistant to inhibitors, and thus be a simple strategy for assay optimization. Identifying an optimal polymerase can even render costly and time-consuming sample preparation unnecessary. The aim of this study was to evaluate the performance of 16...... different DNA extraction methods for Campylobacter. Results show that VeriQuest qPCR master mix have the best general performance, while the AmpliTaq Gold and HotMasterTaq DNA polymerases performed well with meat samples and poorly with fecal samples. Tth DNA polymerase performed well only with the purest...

  8. Hybrid Methods Reveal Multiple Flexibly Linked DNA Polymerases within the Bacteriophage T7 Replisome.

    Science.gov (United States)

    Wallen, Jamie R; Zhang, Hao; Weis, Caroline; Cui, Weidong; Foster, Brittni M; Ho, Chris M W; Hammel, Michal; Tainer, John A; Gross, Michael L; Ellenberger, Tom

    2017-01-03

    The physical organization of DNA enzymes at a replication fork enables efficient copying of two antiparallel DNA strands, yet dynamic protein interactions within the replication complex complicate replisome structural studies. We employed a combination of crystallographic, native mass spectrometry and small-angle X-ray scattering experiments to capture alternative structures of a model replication system encoded by bacteriophage T7. Two molecules of DNA polymerase bind the ring-shaped primase-helicase in a conserved orientation and provide structural insight into how the acidic C-terminal tail of the primase-helicase contacts the DNA polymerase to facilitate loading of the polymerase onto DNA. A third DNA polymerase binds the ring in an offset manner that may enable polymerase exchange during replication. Alternative polymerase binding modes are also detected by small-angle X-ray scattering with DNA substrates present. Our collective results unveil complex motions within T7 replisome higher-order structures that are underpinned by multivalent protein-protein interactions with functional implications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Computational Evaluation of Nucleotide Insertion Opposite Expanded and Widened DNA by the Translesion Synthesis Polymerase Dpo4.

    Science.gov (United States)

    Albrecht, Laura; Wilson, Katie A; Wetmore, Stacey D

    2016-06-23

    Expanded (x) and widened (y) deoxyribose nucleic acids (DNA) have an extra benzene ring incorporated either horizontally (xDNA) or vertically (yDNA) between a natural pyrimidine base and the deoxyribose, or between the 5- and 6-membered rings of a natural purine. Far-reaching applications for (x,y)DNA include nucleic acid probes and extending the natural genetic code. Since modified nucleobases must encode information that can be passed to the next generation in order to be a useful extension of the genetic code, the ability of translesion (bypass) polymerases to replicate modified bases is an active area of research. The common model bypass polymerase DNA polymerase IV (Dpo4) has been previously shown to successfully replicate and extend past a single modified nucleobase on a template DNA strand. In the current study, molecular dynamics (MD) simulations are used to evaluate the accommodation of expanded/widened nucleobases in the Dpo4 active site, providing the first structural information on the replication of (x,y)DNA. Our results indicate that the Dpo4 catalytic (palm) domain is not significantly impacted by the (x,y)DNA bases. Instead, the template strand is displaced to accommodate the increased C1'-C1' base-pair distance. The structural insights unveiled in the present work not only increase our fundamental understanding of Dpo4 replication, but also reveal the process by which Dpo4 replicates (x,y)DNA, and thereby will contribute to the optimization of high fidelity and efficient polymerases for the replication of modified nucleobases.

  10. DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate.

    Science.gov (United States)

    Rajão, M A; Passos-Silva, D G; DaRocha, W D; Franco, G R; Macedo, A M; Pena, S D J; Teixeira, S M; Machado, C R

    2009-01-01

    DNA polymerase kappa (Pol kappa) is a low-fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y-family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Pol kappa from the protozoan Trypanosoma cruzi (TcPol kappa). The role of this TcPol kappa copy was investigated by analysing its subcellular localization, its activities in vitro, and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N-terminal extension which is present only in eukaryotic DinB members, but its C-terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C(2)HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPol kappa efficiently bypasses 8-oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPol kappa increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.

  11. Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

    OpenAIRE

    1993-01-01

    A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected inse...

  12. DNA's Liaison with RNA Polymerase Physical Consequences of a Twisted Relationship

    Science.gov (United States)

    Kulic, Igor; Nelson, Phil

    2006-03-01

    RNA polymerase is the molecular motor that performs the fundamental process of transcription. Besides being the key- protagonist of gene regulation it is one of the most powerful nano-mechanical force generators known inside the cell. The fact that polymerase strictly tracks only one of DNA's strands together with DNA's helical geometry induces a force-to-torque transmission, with several important biological consequences like the ``twin supercoil domain'' effect and remote torsional interaction of genes. In the first part of the talk we theoretically explore the mechanisms of non-equilibrium transport of twist generated by a moving polymerase. We show that these equations are intrinsically non-linear in the crowded cellular environment and lead to peculiar effects like self-confinement of torsional strain by generation of alternative DNA structures like cruciforms. We demonstrate how the asymmetric conformational properties of DNA lead to a ``torsional diode'' effect, i.e. a rectification of polymerase-generated twist currents of different signs. In the second part we explore the possibility of exploiting the polymerase as a powerful workhorse for nanomechanical devices. We propose simple and easy to assemble arrangements of DNA templates interconnected by strand-hybridization that when transcribed by the polymerase linearly contract by tenfold. We show that the typical forces generated by such ``DNA stress fibers'' are in the piconewton range. We discuss their kinetics of contraction and relaxation and draw parallels to natural muscle fiber design.

  13. Development of an efficient process intensification strategy for enhancing Pfu DNA polymerase production in recombinant Escherichia coli.

    Science.gov (United States)

    Hu, Jian-Hua; Wang, Feng; Liu, Chun-Zhao

    2015-04-01

    An efficient induction strategy that consisted of multiple additions of small doses of isopropyl-β-D-thiogalactopyranoside (IPTG) in the early cell growth phase was developed for enhancing Pfu DNA polymerase production in Escherichia coli. In comparison to the most commonly used method of a single induction of 1 mM IPTG, the promising induction strategy resulted in an increase in the Pfu activity of 13.5% in shake flasks, while simultaneously decreasing the dose of IPTG by nearly half. An analysis of the intracellular IPTG concentrations indicated that the cells need to maintain an optimum intracellular IPTG concentration after 6 h for efficient Pfu DNA polymerase production. A significant increase in the Pfu DNA polymerase activity of 31.5% under the controlled dissolved oxygen concentration of 30% in a 5 L fermentor was achieved using the multiple IPTG induction strategy in comparison with the single IPTG induction. The induction strategy using multiple inputs of IPTG also avoided over accumulation of IPTG and reduced the cost of Pfu DNA polymerase production.

  14. Roles of the Y-family DNA polymerase Dbh in accurate replication of the Sulfolobus genome at high temperature.

    Science.gov (United States)

    Sakofsky, Cynthia J; Foster, Patricia L; Grogan, Dennis W

    2012-04-01

    The intrinsically thermostable Y-family DNA polymerases of Sulfolobus spp. have revealed detailed three-dimensional structure and catalytic mechanisms of trans-lesion DNA polymerases, yet their functions in maintaining their native genomes remain largely unexplored. To identify functions of the Y-family DNA polymerase Dbh in replicating the Sulfolobus genome under extreme conditions, we disrupted the dbh gene in Sulfolobus acidocaldarius and characterized the resulting mutant strains phenotypically. Disruption of dbh did not cause any obvious growth defect, sensitivity to any of several DNA-damaging agents, or change in overall rate of spontaneous mutation at a well-characterized target gene. Loss of dbh did, however, cause significant changes in the spectrum of spontaneous forward mutation in each of two orthologous target genes of different sequence. Relative to wild-type strains, dbh(-) constructs exhibited fewer frame-shift and other small insertion-deletion mutations, but exhibited more base-pair substitutions that converted G:C base pairs to T:A base pairs. These changes, which were confirmed to be statistically significant, indicate two distinct activities of the Dbh polymerase in Sulfolobus cells growing under nearly optimal culture conditions (78-80°C and pH 3). The first activity promotes slipped-strand events within simple repetitive motifs, such as mononucleotide runs or triplet repeats, and the second promotes insertion of C opposite a potentially miscoding form of G, thereby avoiding G:C to T:A transversions.

  15. A comprehensive strategy to discover inhibitors of the translesion synthesis DNA polymerase κ.

    Directory of Open Access Journals (Sweden)

    Kinrin Yamanaka

    Full Text Available Human DNA polymerase kappa (pol κ is a translesion synthesis (TLS polymerase that catalyzes TLS past various minor groove lesions including N(2-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N(2-dG DNA-DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen ∼16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.

  16. RNA polymerase activity of Ustilago maydis virus

    Energy Technology Data Exchange (ETDEWEB)

    Yie, S.W.

    1986-01-01

    Ustilago maydis virus has an RNA polymerase enzyme which is associated with virion capsids. In the presence of Mg/sup 2 +/ ion and ribonucleotide triphosphate, the enzyme catalyzes the in vitro synthesis of mRNA by using dsRNA as a template. The products of the UmV RNA polymerase were both ssRNA and dsRNA. The dsRNA was determined by characteristic mobilities in gel electrophoresis, lack of sensitivity to RNase, and specific hybridization tests. The ssRNAs were identified by elution from a CF-11 column and by their RNase sensitivity. On the basis of the size of ssRNAs, it was concluded that partial transcripts were produced from H dsRNA segments, and full length transcripts were produced from M and L dsRNA segments. The following observations indicates that transcription occurs by strand displacement; (1) Only the positive strand of M2 dsRNA was labeled by the in vitro reaction. (2) The M2 dsRNA which had been labeled with /sup 32/''P-UTP in vitro could be chased from dsRNA with unlabeled UTP. The transcription products of three UmV strains were compared, and the overall pattern of transcription was very similar among them.

  17. Investigation of Influenza Virus Polymerase Activity in Pig Cells

    Science.gov (United States)

    Moncorgé, Olivier; Long, Jason S.; Cauldwell, Anna V.; Zhou, Hongbo; Lycett, Samantha J.

    2013-01-01

    Reassortant influenza viruses with combinations of avian, human, and/or swine genomic segments have been detected frequently in pigs. As a consequence, pigs have been accused of being a “mixing vessel” for influenza viruses. This implies that pig cells support transcription and replication of avian influenza viruses, in contrast to human cells, in which most avian influenza virus polymerases display limited activity. Although influenza virus polymerase activity has been studied in human and avian cells for many years by use of a minigenome assay, similar investigations in pig cells have not been reported. We developed the first minigenome assay for pig cells and compared the activities of polymerases of avian or human influenza virus origin in pig, human, and avian cells. We also investigated in pig cells the consequences of some known mammalian host range determinants that enhance influenza virus polymerase activity in human cells, such as PB2 mutations E627K, D701N, G590S/Q591R, and T271A. The two typical avian influenza virus polymerases used in this study were poorly active in pig cells, similar to what is seen in human cells, and mutations that adapt the avian influenza virus polymerase for human cells also increased activity in pig cells. In contrast, a different pattern was observed in avian cells. Finally, highly pathogenic avian influenza virus H5N1 polymerase activity was tested because this subtype has been reported to replicate only poorly in pigs. H5N1 polymerase was active in swine cells, suggesting that other barriers restrict these viruses from becoming endemic in pigs. PMID:23077313

  18. RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

    Directory of Open Access Journals (Sweden)

    Isabel X. Wang

    2014-03-01

    Full Text Available RNA sequences are expected to be identical to their corresponding DNA sequences. Here, we found all 12 types of RNA-DNA sequence differences (RDDs in nascent RNA. Our results show that RDDs begin to occur in RNA chains ∼55 nt from the RNA polymerase II (Pol II active site. These RDDs occur so soon after transcription that they are incompatible with known deaminase-mediated RNA-editing mechanisms. Moreover, the 55 nt delay in appearance indicates that they do not arise during RNA synthesis by Pol II or as a direct consequence of modified base incorporation. Preliminary data suggest that RDD and R-loop formations may be coupled. These findings identify sequence substitution as an early step in cotranscriptional RNA processing.

  19. Requirement of Rad5 for DNA Polymerase ζ-Dependent Translesion Synthesis in Saccharomyces cerevisiae

    Science.gov (United States)

    Pagès, Vincent; Bresson, Anne; Acharya, Narottam; Prakash, Satya; Fuchs, Robert P.; Prakash, Louise

    2008-01-01

    In yeast, Rad6–Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases η or ζ or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities—a ubiquitin ligase that promotes Mms2–Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polζ. Using duplex plasmids carrying different site-specific DNA lesions—an abasic site, a cis–syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct—we show that Rad5 is needed for Polζ-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS. PMID:18757916

  20. Kinetic selection vs. free energy of DNA base pairing in control of polymerase fidelity.

    Science.gov (United States)

    Oertell, Keriann; Harcourt, Emily M; Mohsen, Michael G; Petruska, John; Kool, Eric T; Goodman, Myron F

    2016-04-19

    What is the free energy source enabling high-fidelity DNA polymerases (pols) to favor incorporation of correct over incorrect base pairs by 10(3)- to 10(4)-fold, corresponding to free energy differences of ΔΔGinc∼ 5.5-7 kcal/mol? Standard ΔΔG° values (∼0.3 kcal/mol) calculated from melting temperature measurements comparing matched vs. mismatched base pairs at duplex DNA termini are far too low to explain pol accuracy. Earlier analyses suggested that pol active-site steric constraints can amplify DNA free energy differences at the transition state (kinetic selection). A recent paper [Olson et al. (2013)J Am Chem Soc135:1205-1208] used Vent pol to catalyze incorporations in the presence of inorganic pyrophosphate intended to equilibrate forward (polymerization) and backward (pyrophosphorolysis) reactions. A steady-state leveling off of incorporation profiles at long reaction times was interpreted as reaching equilibrium between polymerization and pyrophosphorolysis, yielding apparent ΔG° = -RTlnKeq, indicating ΔΔG° of 3.5-7 kcal/mol, sufficient to account for pol accuracy without need of kinetic selection. Here we perform experiments to measure and account for pyrophosphorolysis explicitly. We show that forward and reverse reactions attain steady states far from equilibrium for wrong incorporations such as G opposite T. Therefore,[Formula: see text]values obtained from such steady-state evaluations ofKeqare not dependent on DNA properties alone, but depend largely on constraints imposed on right and wrong substrates in the polymerase active site.

  1. Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii.

    Science.gov (United States)

    Tang, Xiao-Feng; Shen, Yulong; Matsui, Eriko; Matsui, Ikuo

    2004-09-21

    Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea. Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays. A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200). Specific interaction between the remaining part of the small subunit, DP1(201-622), and the N-terminus of the large subunit, DP2(1-300), was detected by the Y2H assay. The SPR assay also indicated the intrasubunit interaction within the N-terminus, DP2(1-100), and the C-terminus, DP2(792-1163), of the large subunit. A synthetic 21 amino acid peptide corresponding to the sequence from cysteine cluster II, DP2(1290-1310), tightly interacted (a dissociation constant K(D) = 4.3 nM) with the N-terminus of the small subunit, DP1(1-200). Since the peptide could increase the 3'-5' exonuclease activity of DP1 [Shen et al. (2004) Nucleic Acids Res. 32, 158], the short region DP2(1290-1310) seems to play dual roles to form the PhoPolD complex and to regulate the 3'-5' exonuclease activity of DP1 through interaction with DP1(1-200). Furthermore, DP2(792-1163) containing the catalytic residues for DNA polymerization, Asp1122 and Asp1124, interacted with the intrasubunit domain, DP2(1-100), and the intersubunit domain, DP1(1-200). DP2(792-1163) probably forms the most important domain deeply involved in both the catalysis of DNA polymerization and stabilization of the PhoPolD complex through these multiple interactions.

  2. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.

    2010-05-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  3. Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

    Science.gov (United States)

    Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

    2015-09-01

    5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion.

  4. Sequence and transcription analysis of the human cytomegalovirus DNA polymerase gene

    Energy Technology Data Exchange (ETDEWEB)

    Kouzarides, T.; Bankier, A.T.; Satchwell, S.C.; Weston, K.; Tomlinson, P.; Barrell, B.G.

    1987-01-01

    DNA sequence analysis has revealed that the gene coding for the human cytomegalovirus (HCMV) DNA polymerase is present within the long unique region of the virus genome. Identification is based on extensive amino acid homology between the predicted HCMV open reading frame HFLF2 and the DNA polymerase of herpes simplex virus type 1. The authors present here a 5280 base-pair DNA sequence containing the HCMV pol gene, along with the analysis of transcripts encoded within this region. Since HCMV pol also shows homology to the predicted Epstein-Barr virus pol, they were able to analyze the extent of homology between the DNA polymerases of three distantly related herpes viruses, HCMV, Epstein-Barr virus, and herpes simplex virus. The comparison shows that these DNA polymerases exhibit considerable amino acid homology and highlights a number of highly conserved regions; two such regions show homology to sequences within the adenovirus type 2 DNA polymerase. The HCMV pol gene is flanked by open reading frames with homology to those of other herpes viruses; upstream, there is a reading frame homologous to the glycoprotein B gene of herpes simplex virus type I and Epstein-Barr virus, and downstream there is a reading frame homologous to BFLF2 of Epstein-Barr virus.

  5. ExpandplusCrystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA

    Energy Technology Data Exchange (ETDEWEB)

    M Langelier; J Planck; S Roy; J Pascal

    2011-12-31

    Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNA interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.

  6. Urinary tract infection drives genome instability in uropathogenic Escherichia coli and necessitates translesion synthesis DNA polymerase IV for virulence.

    Science.gov (United States)

    Gawel, Damian; Seed, Patrick C

    2011-01-01

    Uropathogenic Escherichia coli (UPEC) produces ~80% of community-acquired UTI, the second most common infection in humans. During UTI, UPEC has a complex life cycle, replicating and persisting in intracellular and extracellular niches. Host and environmental stresses may affect the integrity of the UPEC genome and threaten its viability. We determined how the host inflammatory response during UTI drives UPEC genome instability and evaluated the role of multiple factors of genome replication and repair for their roles in the maintenance of genome integrity and thus virulence during UTI. The urinary tract environment enhanced the mutation frequency of UPEC ~100-fold relative to in vitro levels. Abrogation of inflammation through a host TLR4-signaling defect significantly reduced the mutation frequency, demonstrating in the importance of the host response as a driver of UPEC genome instability. Inflammation induces the bacterial SOS response, leading to the hypothesis that the UPEC SOS-inducible translesion synthesis (TLS) DNA polymerases would be key factors in UPEC genome instability during UTI. However, while the TLS DNA polymerases enhanced in vitro, they did not increase in vivo mutagenesis. Although it is not a source of enhanced mutagenesis in vivo, the TLS DNA polymerase IV was critical for the survival of UPEC during UTI during an active inflammatory assault. Overall, this study provides the first evidence of a TLS DNA polymerase being critical for UPEC survival during urinary tract infection and points to independent mechanisms for genome instability and the maintenance of genome replication of UPEC under host inflammatory stress.

  7. Preparation of Taq DNA Polymerase by Thermal Purification%Taq DNA聚合酶的热纯化制备

    Institute of Scientific and Technical Information of China (English)

    丁燕华; 刘树涛; 齐庆远

    2011-01-01

    [Objective]The paper was to improve the preparation efficacy of Taq DNA polymerase.[Method]Ni column was used to purify Taq DNA polymerase carrying with 6xHis tag, and recombined vector.Using the thermal-resistant characteristics of Taq DNA polymerase, the crude extract was treated at 75 ℃ for 1 h, and the activity of prepared enzyme solution was verified by PCR test.[Result]The recombinant pET-32A-Taq could highly express in BL21 (DE3) host bacteria and remove hybrid protein by thermal denaturation.The enzyme preparation with the activity further higher than purchased Taq DNA polymerase was obtained.[Conclusion]Taq DNA polymerase prepared by thermal purification method is simple with low cost, and can meet the needs of a large number of conventional PCR amplification.%[目的]提高Taq DNA聚合酶的制备效率.[方法]利用Ni柱亲和色谱纯化载有6xHis标记的Taq DNA聚合酶,并重组载体,利用Taq DNA聚合酶的耐热特性,对粗提液75℃处理1h,之后通过PCR试验验证制备酶液的活力.[结果]所获重组的pET-32A-Taq能够在BL21(DE3)宿主菌中高效表达并可通过热变性去除杂蛋白,获得了活力远高于购买的Taq DNA聚合酶的酶制剂.[结论]使用热纯化法制备的Taq DNA聚合酶工艺简单,成本较低,能满足常规大量PCR实验要求.

  8. Preparation of Taq DNA Polymerase by Thermal Purification%Taq DNA聚合酶的热纯化制备

    Institute of Scientific and Technical Information of China (English)

    丁燕华; 刘树涛; 齐庆远

    2011-01-01

    [ Objective ] The paper was to improve the preparation efficacy of Taq DNA polymerase. [ Method ] Ni column was used to purify Taq DNA polymerase carrying with 6xHis tag, and recombined vector. Using the thermal -resistant characteristics of TaqDNA polymerase, the crude extract was treated at 75 ℃ for 1 h, and the activity of prepared enzyme solution was verified by PCR test. [ Result] The recombinant pET-32A-Taq could highly express in BL21 (DE3) host bacteria and remove hybrid protein by thermal denaturation. The enzyme preparation with the activity further higher than purchased TaqDNA polymerase was obtained. [ Conclusion ] Taq DNA polymerase prepared by thermal purification method is simple with low cost, and can meet the needs of a large number of conventional PCR amplification.%[目的]提高Taq DNA聚合酶的制备效率.[方法]利用Ni柱亲和色谱纯化载有6xHis标记的Taq DNA聚合酶,并重组载体,利用Taq DNA聚合酶的耐热特性,对粗提液75℃处理1h,之后通过PCR试验验证制备酶液的活力.[结果]所获重组的pET-32A-Taq能够在BL21(DE3)宿主菌中高效表达并可通过热变性去除杂蛋白,获得了活力远高于购买的Taq DNA聚合酶的酶制剂.[结论]使用热纯化法制备的Taq DNA聚合酶工艺简单,成本较低,能满足常规大量PCR实验要求.

  9. Role of the first aspartate residue of the "YxDTDS" motif of phi29 DNA polymerase as a metal ligand during both TP-primed and DNA-primed DNA synthesis.

    Science.gov (United States)

    Saturno, J; Lázaro, J M; Blanco, L; Salas, M

    1998-10-30

    Almost all known nucleic acid polymerases require three acidic residues to bind the metal ion during catalysis of nucleotide incorporation. Nevertheless, recent crystallographic data on bacteriophage RB69 DNA polymerase indicate that the first aspartate residue belonging to the conserved motif "YxDTDS" could have a merely structural role. To address this question, a mutant protein at the homologous aspartate residue (Asp456) in phi29 DNA polymerase was made 3'-5' exonuclease deficient. This allowed us to analyse the functional importance of this residue in different metal-dependent reactions that can be performed using either terminal protein (TP) or DNA primers. When Mg2+ was used as the metal activator, the synthetic activities of the mutant phi29 DNA polymerase, TP-primed initiation and DNA-primed polymerisation, were about 50-fold less efficient than those of the wild-type enzyme. Interestingly, the use of Mn2+ as the metal activator partially restored the wild-type phenotype. When polymerisation required an efficient translocation along the template, mutation of Asp456 strongly affected the catalytic efficiency of phi29 DNA polymerase. The results presented here indicate that Asp456 has a catalytic role as a metal-activator ligand, but also contributes to enzyme translocation along the DNA, required during consecutive nucleotide incorporation cycles. Moreover, Asp456 appears to be critical to remodel the active site during transition from TP priming to DNA priming. The results are discussed in the light of structural information corresponding to distantly related polymerases. Copyright 1998 Academic Press.

  10. Absence of RNase H allows replication of pBR322 in Escherichia coli mutants lacking DNA polymerase I.

    Science.gov (United States)

    Kogoma, T

    1984-12-01

    rnh (formerly termed sdrA) mutants of Escherichia coli K-12, capable of continuous DNA replication in the absence of protein synthesis (stable DNA replication), are devoid of ribonuclease H (RNase H, EC 3.1.26.4) activity. Plasmid pBR322 was found to replicate in rnh mutants in the absence of DNA polymerase I, the polA gene product, which is normally required for replication of this plasmid. The plasmid copy number in polA rnh double mutants was as high as in the wild-type strains. When a chimeric construct between pBR322 and pSC101 was introduced into a polA rnh double mutant, the replication of the plasmid via the pBR322 replicon was inhibited if the plasmid also carried an rnh+ gene or if the host harbored an F' plasmid carrying an rnh+ gene. Thus, DNA polymerase I-independent replication of pBR322 requires the absence of RNase H activity. This alternative mechanism requiring neither DNA polymerase I nor RNase H appears to involve a transcriptional event in the region of the normal origin of replication.

  11. Kynurenine signaling increases DNA polymerase kappa expression and promotes genomic instability in glioblastoma cells

    Science.gov (United States)

    Bostian, April C.L.; Maddukuri, Leena; Reed, Megan R.; Savenka, Tatsiana; Hartman, Jessica H.; Davis, Lauren; Pouncey, Dakota L.; Miller, Grover P.; Eoff, Robert L.

    2015-01-01

    Over-expression of the translesion synthesis polymerase (TLS pol) hpol κ in glioblastomas has been linked to a poor patient prognosis; however, the mechanism promoting higher expression in these tumors remains unknown. We determined that activation of the aryl hydrocarbon receptor (AhR) pathway in glioblastoma cells leads to increased hpol κ mRNA and protein levels. We blocked nuclear translocation and DNA binding by the AhR in glioblastoma cells using a small-molecule and observed decreased hpol κ expression. Pharmacological inhibition of tryptophan-2,3-dioxygenase (TDO), the enzyme largely responsible for activating the AhR in glioblastomas, led to a decrease in the endogenous AhR agonist kynurenine (Kyn) and a corresponding decrease in hpol κ protein levels. Importantly, we discovered that inhibiting TDO activity, AhR signaling, or suppressing hpol κ expression with RNA interference led to decreased chromosomal damage in glioblastoma cells. Epistasis assays further supported the idea that TDO activity, activation of AhR signaling and the resulting over-expression of hpol κ function primarily in the same pathway to increase endogenous DNA damage. These findings indicate that up-regulation of hpol κ through glioblastoma-specific TDO activity and activation of AhR signaling likely contributes to the high levels of replication stress and genomic instability observed in these tumors. PMID:26651356

  12. Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution.

    Science.gov (United States)

    Christian, Thomas D; Romano, Louis J; Rueda, David

    2009-12-15

    The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule Förster resonance energy transfer intensity drops in discrete steps to values consistent with single-nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer/template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change after the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the preinsertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.

  13. Variations of Human DNA Polymerase Genes as Biomarkers of Prostate Cancer Progression

    Science.gov (United States)

    2013-07-01

    obtain the percentage of product formed. Time courses were linear for the chosen enzyme concentration and time interval. 2.8. Data analysis The...1] E.C. Friedberg DNA damage and repair, Nature 421 (2003) 436-440. [2] W.A. Beard, S.H. Wilson Structure and mechanism of DNA polymerase β, Chem

  14. Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET

    NARCIS (Netherlands)

    Hohlbein, J.; Kapanidis, A.N.

    2016-01-01

    Monitoring conformational changes in DNA polymerases using single-molecule Förster resonance energy transfer (smFRET) has provided new tools for studying fidelity-related mechanisms that promote the rejection of incorrect nucleotides before DNA synthesis. In addition to the previously known open

  15. A Model of Sequence Dependent Rna-Polymerase Diffusion Along Dna

    CERN Document Server

    Barbi, M; Popkov, V; Salerno, M; Barbi, Maria; Place, Christophe; Popkov, Vladislav; Salerno, Mario

    2001-01-01

    We introduce a probabilistic model for the RNA-polymerase sliding motion along DNA during the promoter search. The model accounts for possible effects due to sequence-dependent interactions between the nonspecific DNA and the enzyme. We focus on T7 RNA-polymerase and exploit the available information about its interaction at the promoter site in order to investigate the influence of bacteriophage T7 DNA sequence on the dynamics of the sliding process. Hydrogen bonds in the major groove are used as the main sequence-dependent interaction between the RNA-polymerase and the DNA. The resulting dynamical properties and the possibility of an experimental validation are discussed in details. We show that, while at large times the process reaches a pure diffusive regime, it initially displays a sub-diffusive behavior. The crossover from anomalous to normal diffusion may occur at times large enough to be of biological interest.

  16. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response.

    Science.gov (United States)

    Yoshimura, Akari; Kobayashi, Yume; Tada, Shusuke; Seki, Masayuki; Enomoto, Takemi

    2014-09-12

    WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH(-/-)) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

  17. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA methylation

    Science.gov (United States)

    Varshney, Dhaval; Vavrova-Anderson, Jana; Oler, Andrew J.; Cowling, Victoria H.; Cairns, Bradley R.; White, Robert J.

    2015-01-01

    Short interspersed nuclear elements (SINEs), such as Alu, spread by retrotransposition, which requires their transcripts to be copied into DNA and then inserted into new chromosomal sites. This can lead to genetic damage through insertional mutagenesis and chromosomal rearrangements between non-allelic SINEs at distinct loci. SINE DNA is heavily methylated and this was thought to suppress its accessibility and transcription, thereby protecting against retrotransposition. Here we provide several lines of evidence that methylated SINE DNA is occupied by RNA polymerase III, including the use of high-throughput bisulphite sequencing of ChIP DNA. We find that loss of DNA methylation has little effect on accessibility of SINEs to transcription machinery or their expression in vivo. In contrast, a histone methyltransferase inhibitor selectively promotes SINE expression and occupancy by RNA polymerase III. The data suggest that methylation of histones rather than DNA plays a dominant role in suppressing SINE transcription. PMID:25798578

  18. Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota.

    Science.gov (United States)

    Faili, Ahmad; Aoufouchi, Said; Flatter, Eric; Guéranger, Quentin; Reynaud, Claude-Agnès; Weill, Jean-Claude

    2002-10-31

    Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases--pol eta, pol iota, pol kappa and rev1--are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitt's lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.

  19. Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases.

    Science.gov (United States)

    Su, Yan; Peter Guengerich, F

    2016-06-01

    Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. © 2016 by John Wiley & Sons, Inc.

  20. SOS-induced DNA polymerases enhance long-term survival and evolutionary fitness

    Science.gov (United States)

    Yeiser, Bethany; Pepper, Evan D.; Goodman, Myron F.; Finkel, Steven E.

    2002-01-01

    Escherichia coli encodes three SOS-induced DNA polymerases: pol II, pol IV, and pol V. We show here that each of these polymerases confers a competitive fitness advantage during the stationary phase of the bacterial life cycle, in the absence of external DNA-damaging agents known to induce the SOS response. When grown individually, wild-type and SOS pol mutants exhibit indistinguishable temporal growth and death patterns. In contrast, when grown in competition with wild-type E. coli, mutants lacking one or more SOS polymerase suffer a severe reduction in fitness. These mutants also fail to express the “growth advantage in stationary phase” phenotype as do wild-type strains, instead expressing two additional new types of “growth advantage in stationary phase” phenotype. These polymerases contribute to survival by providing essential functions to ensure replication of the chromosome and by generating genetic diversity. PMID:12060704

  1. Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

    Energy Technology Data Exchange (ETDEWEB)

    Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita [Department of Genetics, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu (Estonia); Kivisaar, Maia, E-mail: maiak@ebc.ee [Department of Genetics, Institute of Molecular and Cell Biology, Tartu University and Estonian Biocentre, 23 Riia Street, 51010 Tartu (Estonia)

    2011-09-01

    The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif{sup r} mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

  2. MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

    Directory of Open Access Journals (Sweden)

    Jeffrey D Stumpf

    2014-10-01

    Full Text Available Mitochondrial DNA (mtDNA encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS, would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA

  3. RNA-Dependent RNA Polymerase Activity in Influenza Virions

    Science.gov (United States)

    Penhoet, Edward; Miller, Henry; Doyle, Michael; Blatti, Stanley

    1971-01-01

    An RNA-dependent RNA polymerase activity has been detected in purified preparations of influenza virus. In contrast to the replicase activity induced in influenza-infected cells, the virion-associated enzyme has an absolute requirement for Mn++. Most of the RNA synthesized in vitro is complementary to virion RNA. PMID:5288388

  4. Comparison of proteases in DNA extraction via quantitative polymerase chain reaction.

    Science.gov (United States)

    Eychner, Alison M; Lebo, Roberta J; Elkins, Kelly M

    2015-06-01

    We compared four proteases in the QIAamp DNA Investigator Kit (Qiagen) to extract DNA for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate alternate proteases for improved DNA recovery as compared with proteinase K for forensic, biochemical research, genetic paternity and immigration, and molecular diagnostic purposes. The Quantifiler Kit TaqMan quantitative PCR assay was used to measure the recovery of DNA from human blood, semen, buccal cells, breastmilk, and earwax in addition to low-template samples, including diluted samples, computer keyboard swabs, chewing gum, and cigarette butts. All methods yielded amplifiable DNA from all samples.

  5. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  6. DNA Polymerases BI and D from the Hyperthermophilic Archaeon Pyrococcus furiosus Both Bind to Proliferating Cell Nuclear Antigen with Their C-Terminal PIP-Box Motifs▿

    Science.gov (United States)

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-01-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3′-5′ exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex. PMID:17496095

  7. DNA polymerases BI and D from the hyperthermophilic archaeon Pyrococcus furiosus both bind to proliferating cell nuclear antigen with their C-terminal PIP-box motifs.

    Science.gov (United States)

    Tori, Kazuo; Kimizu, Megumi; Ishino, Sonoko; Ishino, Yoshizumi

    2007-08-01

    Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3'-5' exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.

  8. Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Wei-Ping Qian; Li-Ka Shing; Yue-Qiu Tan; Ying Chen; Ying Peng; Zhi Li; Guang-Xiu Lu; Marie C. Lin; Hsiang-Fu Kung; Ming-Ling He

    2005-01-01

    AIM: To examine the sensitivity and accuracy of real-time polymerase chain reaction (PCR) for the quantification of hepatitis B virus (HBV) DNA in semen.METHODS: Hepatitis B viral DNA was isolated from HBV carriers' semen and sera using phenol extraction method and QTAamp DNA blood mini kit (Qiagen, Germany). HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR (quantitative polymerase chain reaction (qPCR)). The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction.RESULTS: Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×107 and 1.67×107 copies of HBV DNA per mL in two HBV infected patients' sera, while 2.L4×105 and 3.02×105 copies of HBV DNA per mL in the semen.CONCLUSION: Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

  9. Apoptotic DNA Fragmentation May Be a Cooperative Activity between Caspase-activated Deoxyribonuclease and the Poly(ADP-ribose) Polymerase-regulated DNAS1L3, an Endoplasmic Reticulum-localized Endonuclease That Translocates to the Nucleus during Apoptosis*

    Science.gov (United States)

    Errami, Youssef; Naura, Amarjit S.; Kim, Hogyoung; Ju, Jihang; Suzuki, Yasuhiro; El-Bahrawy, Ali H.; Ghonim, Mohamed A.; Hemeida, Ramadan A.; Mansy, Moselhy S.; Zhang, Jianhua; Xu, Ming; Smulson, Mark E.; Brim, Hassan; Boulares, A. Hamid

    2013-01-01

    Caspase-activated DNase (CAD) is the most favorable candidate for chromatin degradation during apoptosis. Ca2+-dependent endonucleases are equally important in internucleosomal DNA fragmentation (INDF), including the PARP-1-regulated DNAS1L3. Despite the elaborate work on these endonucleases, the question of whether these enzymes cooperate during INDF was not addressed. Here, we show a lack of correlation between INDF and CAD expression levels and inactivation by cleavage of its inhibitor (ICAD) during apoptosis. The cells that failed to induce INDF accumulated large amounts of 50-kb breaks, which is suggestive of incomplete chromatin processing. Similarly, INDF was blocked by Ca2+ chelation without a block in ICAD cleavage or caspase-3 activation, which is consistent with the involvement of CAD in 50-kb DNA fragmentation and its Ca2+ independence. However, DNAS1L3 expression in INDF-deficient cells promoted INDF during apoptosis and was blocked by Ca2+ chelation. Interestingly, expression of DNAS1L3 in ICAD-deficient cells failed to promote tumor necrosis factor α-induced INDF but required the coexpression of ICAD. These results suggest a cooperative activity between CAD and DNAS1L3 to accomplish INDF. In HT-29 cells, endogenous DNAS1L3 localized to the endoplasmic reticulum (ER) and translocated to the nucleus upon apoptosis induction but prior to INDF manifestation, making it the first reported Ca2+-dependent endonuclease to migrate from the ER to the nucleus. The nuclear accumulation of DNAS1L3, but not its exit out of the ER, required the activity of cysteine and serine proteases. Interestingly, the endonuclease accumulated in the cytosol upon inhibition of serine, but not cysteine, proteases. These results exemplify the complexity of chromatin degradation during apoptosis. PMID:23229555

  10. Structural Insight into Processive Human Mitochondrial DNA Synthesis and Disease-Related Polymerase Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Young-Sam; Kennedy, W. Dexter; Yin, Y. Whitney; (Texas)

    2010-09-07

    Human mitochondrial DNA polymerase (Pol {gamma}) is the sole replicase in mitochondria. Pol {gamma} is vulnerable to nonselective antiretroviral drugs and is increasingly associated with mutations found in patients with mitochondriopathies. We determined crystal structures of the human heterotrimeric Pol {gamma} holoenzyme and, separately, a variant of its processivity factor, Pol {gamma}B. The holoenzyme structure reveals an unexpected assembly of the mitochondrial DNA replicase where the catalytic subunit Pol {gamma}A interacts with its processivity factor primarily via a domain that is absent in all other DNA polymerases. This domain provides a structural module for supporting both the intrinsic processivity of the catalytic subunit alone and the enhanced processivity of holoenzyme. The Pol {gamma} structure also provides a context for interpreting the phenotypes of disease-related mutations in the polymerase and establishes a foundation for understanding the molecular basis of toxicity of anti-retroviral drugs targeting HIV reverse transcriptase.

  11. Domain structures and inter-domain interactions defining the holoenzyme architecture of archaeal d-family DNA polymerase.

    Science.gov (United States)

    Matsui, Ikuo; Matsui, Eriko; Yamasaki, Kazuhiko; Yokoyama, Hideshi

    2013-07-05

    Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information.

  12. Domain Structures and Inter-Domain Interactions Defining the Holoenzyme Architecture of Archaeal D-Family DNA Polymerase

    Directory of Open Access Journals (Sweden)

    Hideshi Yokoyama

    2013-07-01

    Full Text Available Archaea-specific D-family DNA polymerase (PolD forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information.

  13. [Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

    Science.gov (United States)

    Nikitenko, N A; Speiseder, T; Chernolovskaya, E L; Zenkova, M A; Dobner, T; Prassolov, V S

    2016-01-01

    Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

  14. Evidence that in xeroderma pigmentosum variant cells, which lack DNA polymerase eta, DNA polymerase iota causes the very high frequency and unique spectrum of UV-induced mutations.

    Science.gov (United States)

    Wang, Yun; Woodgate, Roger; McManus, Terrence P; Mead, Samantha; McCormick, J Justin; Maher, Veronica M

    2007-04-01

    Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlight-induced cancer. They exhibit a 25-fold higher than normal frequency of UV-induced mutations and very unusual kinds (spectrum), mainly transversions. The primary defect in XPV cells is the lack of functional DNA polymerase (Pol) eta, the translesion synthesis DNA polymerase that readily inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XPV cells strongly suggest that, in the absence of Pol eta, an abnormally error-prone polymerase substitutes. In vitro replication studies of Pol iota show that it replicates past 5'T-T3' and 5'T-U3' cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3' nucleotide. To test the hypothesis that Pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XPV cells, we identified an unlimited lifespan XPV cell line expressing two forms of Pol iota, whose frequency of UV-induced mutations is twice that of XPV cells expressing one form. We eliminated expression of one form and compared the parental cells and derivatives for the frequency and kinds of UV-induced mutations. All exhibited similar sensitivity to the cytotoxicity of UV((254 nm)), and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to UV-induced mutations, and ultimately their malignant transformation.

  15. WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

    Energy Technology Data Exchange (ETDEWEB)

    Yoshimura, Akari, E-mail: akari_yo@stu.musashino-u.ac.jp [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Kobayashi, Yume [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan); Tada, Shusuke [Department of Medical Biochemistry, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi-shi, Chiba 274-8510 (Japan); Seki, Masayuki [Department of Biochemistry, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai-shi, Miyagi 981-8558 (Japan); Enomoto, Takemi [Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585 (Japan)

    2014-09-12

    Highlights: • The UV sensitivity of POLH{sup −/−} cells was suppressed by disruption of WRNIP1. • In WRNIP1{sup −/−/−}/POLH{sup −/−} cells, mutation frequencies and SCE after irradiation reduced. • WRNIP1 defect recovered rate of fork progression after irradiation in POLH{sup −/−} cells. • WRNIP1 functions upstream of Polη in the translesion DNA synthesis pathway. - Abstract: WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH{sup −/−}) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

  16. Are there three polynucleotide strands in the catalytic centre of DNA polymerases?

    Science.gov (United States)

    Lestienne, Patrick P

    2009-01-01

    Mitochondrial DNA may undergo large-scale rearrangements, thus leading to diseases. The mechanisms of these rearrangements are still the matter of debates. Several lines of evidence indicate that breakpoints are characterized by direct repeats (DR), one of them being eliminated from the normal genome. Analysis of DR showed their skewed nucleotide content compatible with the formation of known triple helices. Here, I propose a novel mechanism involving the formation of triplex structures that result from the dissociation of the [synthesized repeat-DNA polymerase] complex. Upon binding to the homologous sequence, replication is initiated from the primer bound in a triple helix manner. This feature implies the initiation of replication on the double-stranded DNA from the triple helix primer. Hereby, I review evidences supporting this model. Indeed, all short d(G)-rich primers 10 nucleotides long can be elongated on double-stranded DNA by phage, bacterial, reverse transcriptases and eukaryotic DNA polymerases. Mismatches may be tolerated between the primer and its double-stranded binding site. In contrast to previous studies, evidences for the parallel binding of the triple helix to its homologous strand are provided. This suggest the displacement of the non-template strand by the triple helix primer upon binding within the DNA polymerase catalytic centre. Computer modelling indicates that the triple helix primer lies within the major groove of the double helix, with its 3' hydroxyl end nearby the catalytic amino acids. Taken together, I bring new concepts on DNA rearrangements, and novel features of triple helices and DNA polymerases that can bind three polynucleotide strands similar to RNA polymerases.

  17. Secondary Interaction Interfaces with PCNA Control Conformational Switching of DNA Polymerase PolB from Polymerization to Editing.

    Science.gov (United States)

    Xu, Xiaojun; Yan, Chunli; Kossmann, Bradley R; Ivanov, Ivaylo

    2016-08-25

    Replicative DNA polymerases (Pols) frequently possess two distinct DNA processing activities: DNA synthesis (polymerization) and proofreading (3'-5' exonuclease activity). The polymerase and exonuclease reactions are performed alternately and are spatially separated in different protein domains. Thus, the growing DNA primer terminus has to undergo dynamic conformational switching between two distinct functional sites on the polymerase. Furthermore, the transition from polymerization (pol) mode to exonuclease (exo) mode must occur in the context of a DNA Pol holoenzyme, wherein the polymerase is physically associated with processivity factor proliferating cell nuclear antigen (PCNA) and primer-template DNA. The mechanism of this conformational switching and the role that PCNA plays in it have remained obscure, largely due to the dynamic nature of ternary Pol/PCNA/DNA assemblies. Here, we present computational models of ternary assemblies for archaeal polymerase PolB. We have combined all available structural information for the binary complexes with electron microscopy data and have refined atomistic models for ternary PolB/PCNA/DNA assemblies in pol and exo modes using molecular dynamics simulations. In addition to the canonical PIP-box/interdomain connector loop (IDCL) interface of PolB with PCNA, contact analysis of the simulation trajectories revealed new secondary binding interfaces, distinct between the pol and exo states. Using targeted molecular dynamics, we explored the conformational transition from pol to exo mode. We identified a hinge region between the thumb and palm domains of PolB that is critical for conformational switching. With the thumb domain anchored onto the PCNA surface, the neighboring palm domain executed rotational motion around the hinge, bringing the core of PolB down toward PCNA to form a new interface with the clamp. A helix from PolB containing a patch of arginine residues was involved in the binding, locking the complex in the exo

  18. RNA Polymerase Collision versus DNA Structural Distortion: Twists and Turns Can Cause Break Failure.

    Science.gov (United States)

    Pannunzio, Nicholas R; Lieber, Michael R

    2016-05-05

    The twisting of DNA due to the movement of RNA polymerases is the basis of numerous classic experiments in molecular biology. Recent mouse genetic models indicate that chromosomal breakage is common at sites of transcriptional turbulence. Two key studies on this point mapped breakpoints to sites of either convergent or divergent transcription but arrived at different conclusions as to which is more detrimental and why. The issue hinges on whether DNA strand separation is the basis for the chromosomal instability or collision of RNA polymerases.

  19. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

    KAUST Repository

    Yamashita, Mami

    2017-05-08

    The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

  20. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    Science.gov (United States)

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-06-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

  1. The Pseudorabies Virus DNA Polymerase Accessory Subunit UL42 Directs Nuclear Transport of the Holoenzyme.

    Science.gov (United States)

    Wang, Yi-Ping; Du, Wen-Juan; Huang, Li-Ping; Wei, Yan-Wu; Wu, Hong-Li; Feng, Li; Liu, Chang-Ming

    2016-01-01

    Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354-370 and that K(354), R(355), and K(367) are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression

  2. Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

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    Watson Spencer K

    2006-12-01

    Full Text Available Abstract Background Formalin-fixed paraffin-embedded (FFPE tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold. Results We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb. When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar. Conclusion Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.

  3. [Features of interaction of Escherichia coli DNA polymerase I and its Klenow fragment with dTTP gamma-p-azidoanilide].

    Science.gov (United States)

    Kudriashova, N V; Shamanina, M Iu; Godovikova, T S; Anan'ko, E A; Akhmadieva, F F; Romashchenko, A G

    1993-02-01

    gamma-p-Azidoanilidate of dTTP was used to study the photoaffinity modification of DNA polymerase I and Klenow fragment. The analog was found to be a mixed-type inhibitor with respect to dTTP of the polymerization reaction catalyzed by DNA polymerase I and Klenow fragment. In the absence of the reagent both UV-irradiated enzymes were rapidly inactivated. Substrates (dNTP and template-primer) protected the enzymes from inactivation by UV-light with different efficiency. In the presence of the template-primer UV-irradiation induced activation of DNA polymerase I. The effect of the analog on both enzyme forms under irradiation is different. At concentration of 10(-5)M gamma-p-anilidate of dTTP accelerated the activation of DNA polymerase I initiated by UV-irradiation and at 10(-4)M concentration it inactivated the enzyme by 20-25%. Under such conditions one enzyme molecule covalently bound two molecules of the analog. While the template-complementary substrate (dTTP) protected DNA polymerase I both from inactivation and modification, the non-complementary one (dCTP) worked only against modification. In contrast to DNA polymerase I Klenow fragment was not inactivated when exposed to UV-irradiation and gamma-p-anilidate of dTTP neither modified the protein nor exerted any significant effect on its polymerization activity. The data accumulated suggest the presence on the DNA polymerase I molecule of a regulatory region providing additional dNTP binding sites.

  4. DNA聚合酶Polι的研究进展%Progress in DNA polymerase iota

    Institute of Scientific and Technical Information of China (English)

    周虎传; 杨劲

    2011-01-01

    Y-family DNA polymerases are one kind of polymerases which replicating damaged template.Y-family DNA polymerases are widely distributed among the three kingdoms of life.Human cells contain at least four:i.e, Revl Polκ Pol(ι) and Polη , and Pol(ι) is different from the other three DNA polymerases of bypassing damaged template for the rate of mismatching when it replicates DNA is very high.DNA polymerase iota has the lowest fidelity in all of DNA polymerases so far.The high rate of mismatching result in high rate of mutation, Even mismatching was reported to be related to the occurrence of cancer Therefore the DNA polymerase iota were researched worldwide, for its polymerase from different properties, and gain a series of outcomes.In addition, the prospect of future research is addressed.%Y家族DNA聚合酶是一种跨损伤复制酶,即能以损伤的DNA为模板进行复制.Y家族DNA聚合酶广泛分布生物界,人类细胞中Y家族DNA聚合酶至少包括Revl、Polκ、Polι、Polη四种,Polι在以DNA为模板进行复制时错配率很高而不同于其他跨损伤DNA聚合酶,Polι是目前发现的所有DNA聚合酶中保真性最低的DNA聚合酶.很高的错配率导致很高的突变率,最后基因的突变导致癌症的发生,因此Polι在各个国家被广泛的研究,并且对Polι的各个不同的特性进行了研究,取得了一系列成果,现对Polι的研究进展予以综述,并展望了未来的研究趋势.

  5. DNA polymerase β as a novel target for chemotherapeutic intervention of colorectal cancer.

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    Aruna S Jaiswal

    Full Text Available Chemoprevention presents a major strategy for the medical management of colorectal cancer. Most drugs used for colorectal cancer therapy induce DNA-alkylation damage, which is primarily repaired by the base excision repair (BER pathway. Thus, blockade of BER pathway is an attractive option to inhibit the spread of colorectal cancer. Using an in silico approach, we performed a structure-based screen by docking small-molecules onto DNA polymerase β (Pol-β and identified a potent anti-Pol-β compound, NSC-124854. Our goal was to examine whether NSC-124854 could enhance the therapeutic efficacy of DNA-alkylating agent, Temozolomide (TMZ, by blocking BER. First, we determined the specificity of NSC-124854 for Pol-β by examining in vitro activities of APE1, Fen1, DNA ligase I, and Pol-β-directed single nucleotide (SN- and long-patch (LP-BER. Second, we investigated the effect of NSC-124854 on the efficacy of TMZ to inhibit the growth of mismatch repair (MMR-deficient and MMR-proficient colon cancer cell lines using in vitro clonogenic assays. Third, we explored the effect of NSC-124854 on TMZ-induced in vivo tumor growth inhibition of MMR-deficient and MMR-proficient colonic xenografts implanted in female homozygous SCID mice. Our data showed that NSC-124854 has high specificity to Pol-β and blocked Pol-β-directed SN- and LP-BER activities in in vitro reconstituted system. Furthermore, NSC-124854 effectively induced the sensitivity of TMZ to MMR-deficient and MMR-proficient colon cancer cells both in vitro cell culture and in vivo xenograft models. Our findings suggest a potential novel strategy for the development of highly specific structure-based inhibitor for the prevention of colonic tumor progression.

  6. Reevaluation of the role of DNA polymerase theta in somatic hypermutation of immunoglobulin genes.

    Science.gov (United States)

    Martomo, Stella A; Saribasak, Huseyin; Yokoi, Masayuki; Hanaoka, Fumio; Gearhart, Patricia J

    2008-09-01

    DNA polymerase theta has been implicated in the process of somatic hypermutation in immunoglobulin variable genes based on several reports of alterations in the frequency and spectra of mutations from Polq(-/-) mice. However, these studies have contrasting results on mutation frequencies and the types of nucleotide substitutions, which question the role of polymerase theta in hypermutation. DNA polymerase eta has a dominant effect on mutation and may substitute in the absence of polymerase theta to affect the pattern. Therefore, we have examined mutation in mice deficient for both polymerases theta and eta. The mutation frequencies in rearranged variable genes from Peyer's patches were similar in wild type, Polq(-/-), Polh(-/-), and Polq(-/-)Polh(-/-) mice. The types of substitutions were also similar between wild type and Polq(-/-) clones, and between Polh(-/-) and Polq(-/-)Polh(-/-) clones. Furthermore, there was no difference in heavy chain class switching in splenic B cells from the four groups of mice. These results indicate that polymerase theta does not play a significant role in the generation of somatic mutation in immunoglobulin genes.

  7. DNA polymerase [gamma] and disease: what we have learned from yeast

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    Tiziana eLodi

    2015-03-01

    Full Text Available Mip1 is the Saccharomyces cerevisiae DNA polymerase [gamma] (Pol [gamma], which is responsible for the replication of mitochondrial DNA (mtDNA. It belongs to the family A of the DNA polymerases and it is orthologous to human POLGA. In humans, mutations in POLG(1 cause many mitochondrial pathologies, such as PEO, Alpers’ syndrome and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in i validating the pathological mutations found in human POLG and modeled in MIP1, ii determining the molecular defects caused by these mutations and iii finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol [gamma] holoenzyme is expressed in yeast and complements the loss of Mip1.

  8. Active RNA polymerases: mobile or immobile molecular machines?

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    Argyris Papantonis

    Full Text Available It is widely assumed that active RNA polymerases track along their templates to produce a transcript. We test this using chromosome conformation capture and human genes switched on rapidly and synchronously by tumour necrosis factor alpha (TNFalpha; one is 221 kbp SAMD4A, which a polymerase takes more than 1 h to transcribe. Ten minutes after stimulation, the SAMD4A promoter comes together with other TNFalpha-responsive promoters. Subsequently, these contacts are lost as new downstream ones appear; contacts are invariably between sequences being transcribed. Super-resolution microscopy confirms that nascent transcripts (detected by RNA fluorescence in situ hybridization co-localize at relevant times. Results are consistent with an alternative view of transcription: polymerases fixed in factories reel in their respective templates, so different parts of the templates transiently lie together.

  9. Bending the rules of transcriptional repression: tightly looped DNA directly represses T7 RNA polymerase.

    Science.gov (United States)

    Lionberger, Troy A; Meyhöfer, Edgar

    2010-08-09

    From supercoiled DNA to the tight loops of DNA formed by some gene repressors, DNA in cells is often highly bent. Despite evidence that transcription by RNA polymerase (RNAP) is affected in systems where DNA is deformed significantly, the mechanistic details underlying the relationship between polymerase function and mechanically stressed DNA remain unclear. Seeking to gain additional insight into the regulatory consequences of highly bent DNA, we hypothesize that tightly looping DNA is alone sufficient to repress transcription. To test this hypothesis, we have developed an assay to quantify transcription elongation by bacteriophage T7 RNAP on small, circular DNA templates approximately 100 bp in size. From these highly bent transcription templates, we observe that the elongation velocity and processivity can be repressed by at least two orders of magnitude. Further, we show that minicircle templates sustaining variable levels of twist yield only moderate differences in repression efficiency. We therefore conclude that the bending mechanics within the minicircle templates dominate the observed repression. Our results support a model in which RNAP function is highly dependent on the bending mechanics of DNA and are suggestive of a direct, regulatory role played by the template itself in regulatory systems where DNA is known to be highly bent. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms.

    Science.gov (United States)

    Evans, Geraint W; Hohlbein, Johannes; Craggs, Timothy; Aigrain, Louise; Kapanidis, Achillefs N

    2015-07-13

    DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

  11. A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP

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    Yogesh eChander

    2014-08-01

    Full Text Available Meeting the goal of providing point of care (POC tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol is a thermostable viral enzyme that enables true POC use in clinics or in field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations . Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular

  12. Liquid Chromatography-Mass Spectrometry Analysis of DNA Polymerase Reaction Products

    Science.gov (United States)

    Chowdhury, Goutam; Guengerich, F. Peter

    2013-01-01

    This unit describes experimental and analytical procedures for characterizing the efficiency and fidelity of translesion DNA synthesis across various DNA damages by DNA polymerases in vitro. This procedure utilizes primer extension assays followed by LC-MS and LC-MS/MS analysis of the extension products. Detailed explanations for the analysis of the LC-MS/MS data for deciphering the nucleotide sequences of the DNA fragments are also presented. This approach provides a significant improvement over conventional methods, as it allows detection of misincorporation, as well as frameshift products. PMID:22147421

  13. Thermostable DNA polymerase from a viral metagenome is a potent RT-PCR enzyme.

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    Michael J Moser

    Full Text Available Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR. Wild-type 3173 Pol contains a proofreading 3'-5' exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth and two-enzyme (MMLV RT/Taq RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics.

  14. Cloning of Thermostable DNA Polymerase Gene from a Thermophilic Brevibacillus sp. Isolated from Sikidang Crater, Dieng Plateu, Central Java

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    Lucia Dhiantika Witasari

    2015-11-01

    Full Text Available Thermostable DNA polymerase has an important role for amplifying small amount of DNA through polymerase chain reaction (PCR. Thermophillic bacteria Brevibacillus sp. was isolated from Sikidang Crater, Dieng Plateu, Central Java. Previous study showed that crude protein of the isolate could be used in PCR. Unfortunately, like most native thermostable enzymes, the thermostable DNA polymerase of the isolate is synthesized in a very low level and therefore is cumbersome to purify. The purpose of this research is to clone thermostable DNA polymerase gene of the isolate. The DNA polymerase gene was amplified by means of PCR using spesific primers. The amplified fragment was then isolated, purified, and ligated into the pGEM-T cloning vector. The recombinant plasmid was then transformed to competent E. coli JM109 cells using heat shock method. The cloned thermostable DNA polymerase gene from the thermophilic isolate was then characterized for its nucleotide base sequence. The result showed that the DNA Pol I gene was successfully be amplified from the isolate DNA genom, resulting in ± 2,7 kb DNA fragment in length. Sequence analysis of segment of targeted gene showed high similarity to that of thermostable DNA polymerase genes from other Bacillus.Key words : Thermostable DNA Pol I, Brevibacillus sp., PCR, cloning

  15. Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

    Science.gov (United States)

    Fujita, Keisuke; Iwaki, Mitsuhiro; Yanagida, Toshio

    2016-12-01

    Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an in vitro single-molecule measurement system to analyse the kinetics of transcriptional bursting. The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting.

  16. Sensitivitas dan Spesifisitas Nested Polymerase Chain Reaction untuk Mendeteksi DNA Coxiella burnetii (SENSITIVITY AND SPECIFICITY OF NESTED POLYMERASE CHAIN REACTION FOR DETECTION OF COXIELLA BURNETII DNA

    Directory of Open Access Journals (Sweden)

    Trioso Purnawarman

    2014-04-01

    Full Text Available Sensitivity and specificity of nested polymerase chain reaction (nested PCR to detect Coxiella burnetii(C. burnetii DNA were studied. The primer system which consists of external primers (OMP1 and OMP2and internal primers (OMP3 and OMP4, was designed from the nucleotide sequence of the com I geneencoding for 27 kDa outer membrane protein and used to specifically amplify a 501 bp and 438 bp fragment.This nested PCR assay was 50 fold more sensitive than that of using PCR external primer only. TheNested PCR has a detection limit as low as 300 pg/?l. Specificity studies showed that nested PCR onlydetected C. burnetii DNA and did not happened Brucella abortus, Escherichia coli, Pseudomonas aeruginosaand Campylobacter Jejuni DNA. Nested PCR has high senstively and specificaly diagnostic method of C.burnetii as agent of Q fever disease.

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

    Science.gov (United States)

    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.

  18. On-Chip integration of sample pretreatment and Multiplex polymerase chain reaction (PCR) for DNA analysis

    DEFF Research Database (Denmark)

    Brivio, Monica; Snakenborg, Detlef; Søgaard, E.;

    2008-01-01

    In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a custom-made thermocycler (Figure 1) and includes a simple...

  19. Specific Inhibition of Herpes Simplex Virus DNA Polymerase by Helical Peptides Corresponding to the Subunit Interface

    Science.gov (United States)

    Digard, Paul; Williams, Kevin P.; Hensley, Preston; Brooks, Ian S.; Dahl, Charles E.; Coen, Donald M.

    1995-02-01

    The herpes simplex virus DNA polymerase consists of two subunits-a catalytic subunit and an accessory subunit, UL42, that increases processivity. Mutations affecting the extreme C terminus of the catalytic subunit specifically disrupt subunit interactions and ablate virus replication, suggesting that new antiviral drugs could be rationally designed to interfere with polymerase heterodimerization. To aid design, we performed circular dichroism (CD) spectroscopy and analytical ultracentrifugation studies, which revealed that a 36-residue peptide corresponding to the C terminus of the catalytic subunit folds into a monomeric structure with partial α-helical character. CD studies of shorter peptides were consistent with a model where two separate regions of α-helix interact to form a hairpin-like structure. The 36-residue peptide and a shorter peptide corresponding to the C-terminal 18 residues blocked UL42-dependent long-chain DNA synthesis at concentrations that had no effect on synthesis by the catalytic subunit alone or by calf thymus DNA polymerase δ and its processivity factor. These peptides, therefore, represent a class of specific inhibitors of herpes simplex virus DNA polymerase that act by blocking accessory-subunit-dependent synthesis. These peptides or their structures may form the basis for the synthesis of clinically effective drugs.

  20. Localized Cerebral Energy Failure in DNA Polymerase Gamma-Associated Encephalopathy Syndromes

    Science.gov (United States)

    Tzoulis, Charalampos; Neckelmann, Gesche; Mork, Sverre J.; Engelsen, Bernt E.; Viscomi, Carlo; Moen, Gunnar; Ersland, Lars; Zeviani, Massimo; Bindoff, Laurence A.

    2010-01-01

    Mutations in the catalytic subunit of the mitochondrial DNA-polymerase gamma cause a wide spectrum of clinical disease ranging from infantile hepato-encephalopathy to juvenile/adult-onset spinocerebellar ataxia and late onset progressive external ophthalmoplegia. Several of these syndromes are associated with an encephalopathy that…

  1. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Alkhatib, H.M.; Chen, D.; Cherney, B.; Bhatia, K.; Notario, V.; Giri, C.; Stein, G.; Slattery, E.; Roeder, R.G.; Smulson, M.E.

    1987-03-01

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambdagt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambdagt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

  2. MUSCLE-SPECIFIC OVEREXPRESSION OF THE CATALYTIC SUBUNIT OF DNA POLYMERASE γ INDUCES PUPAL LETHALITY IN Drosophila melanogaster

    Science.gov (United States)

    Martínez-Azorín, Francisco; Calleja, Manuel; Hernández-Sierra, Rosana; Farr, Carol L.; Kaguni, Laurie S.; Garesse, Rafael

    2015-01-01

    We show the physiological effects and molecular characterization of overexpression of the catalytic core of mitochondrial DNA (mtDNA) polymerase (pol γ-α) in muscle of Drosophila melanogaster. Muscle-specific overexpression of pol γ-α using the UAS/GAL4 (where UAS is upstream activation sequence) system produced more than 90% of lethality at the end of pupal stage at 25°C, and the survivor adult flies showed a significant reduction in life span. The survivor flies displayed a decreased mtDNA level that is accompanied by a corresponding decrease in the levels of the nucleoid-binding protein mitochondrial transcription factor A (mtTFA). Furthermore, an increase in apoptosis is detected in larvae and adults overexpressing pol γ-α. We suggest that the pupal lethality and reduced life span of survivor adult flies are both caused mainly by massive apoptosis of muscle cells induced by mtDNA depletion. PMID:23729397

  3. DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Elena K Braithwaite

    Full Text Available Base excision repair (BER is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda, was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

  4. Temporal order of evolution of DNA replication systems inferred by comparison of cellular and viral DNA polymerases

    Directory of Open Access Journals (Sweden)

    Koonin Eugene V

    2006-12-01

    Full Text Available Abstract Background The core enzymes of the DNA replication systems show striking diversity among cellular life forms and more so among viruses. In particular, and counter-intuitively, given the central role of DNA in all cells and the mechanistic uniformity of replication, the core enzymes of the replication systems of bacteria and archaea (as well as eukaryotes are unrelated or extremely distantly related. Viruses and plasmids, in addition, possess at least two unique DNA replication systems, namely, the protein-primed and rolling circle modalities of replication. This unexpected diversity makes the origin and evolution of DNA replication systems a particularly challenging and intriguing problem in evolutionary biology. Results I propose a specific succession for the emergence of different DNA replication systems, drawing argument from the differences in their representation among viruses and other selfish replicating elements. In a striking pattern, the DNA replication systems of viruses infecting bacteria and eukaryotes are dominated by the archaeal-type B-family DNA polymerase (PolB whereas the bacterial replicative DNA polymerase (PolC is present only in a handful of bacteriophage genomes. There is no apparent mechanistic impediment to the involvement of the bacterial-type replication machinery in viral DNA replication. Therefore, I hypothesize that the observed, markedly unequal distribution of the replicative DNA polymerases among the known cellular and viral replication systems has a historical explanation. I propose that, among the two types of DNA replication machineries that are found in extant life forms, the archaeal-type, PolB-based system evolved first and had already given rise to a variety of diverse viruses and other selfish elements before the advent of the bacterial, PolC-based machinery. Conceivably, at that stage of evolution, the niches for DNA-viral reproduction have been already filled with viruses replicating with the

  5. DNA polymerase delta, RFC and PCNA are required for repair synthesis of large looped heteroduplexes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Corrette-Bennett, Stephanie E; Borgeson, Claudia; Sommer, Debbie; Burgers, Peter M J; Lahue, Robert S

    2004-01-01

    Small looped mispairs are corrected by DNA mismatch repair (MMR). In addition, a distinct process called large loop repair (LLR) corrects loops up to several hundred nucleotides in extracts of bacteria, yeast or human cells. Although LLR activity can be readily demonstrated, there has been little progress in identifying its protein components. This study identified some of the yeast proteins responsible for DNA repair synthesis during LLR. Polyclonal antisera to either Pol31 or Pol32 subunits of polymerase delta efficiently inhibited LLR in extracts by blocking repair just prior to gap filling. Gap filling was inhibited regardless of whether the loop was retained or removed. These experiments suggest polymerase delta is uniquely required in yeast extracts for LLR-associated synthesis. Similar results were obtained with antisera to the clamp loader proteins Rfc3 and Rfc4, and to PCNA, i.e. LLR was inhibited just prior to gap filling for both loop removal and loop retention. Thus PCNA and RFC seem to act in LLR only during repair synthesis, in contrast to their roles at both pre- and post-excision steps of MMR. These biochemical experiments support the idea that yeast polymerase delta, RFC and PCNA are required for large loop DNA repair synthesis.

  6. RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

    Science.gov (United States)

    Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

    2016-01-01

    Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation. PMID:26392149

  7. DNA from oral bacteria by sodium hydroxide-paper method suitable for polymerase chain reaction.

    Science.gov (United States)

    Lefimil, Claudia; Lozano, Carla; Morales-Bozo, Irene; Plaza, Anita; Maturana, Cristian; Urzúa, Blanca

    2013-02-15

    In the oral cavity, we can find a complex mixture of microorganisms, commensals, and pathogens. The studies of normal oral microbiota, as well as the studies of much oral pathology (e.g., caries, periodontitis), involve the isolation and cultivation of these microorganisms and their molecular analysis. The aim of this study was to validate a quick, easy, efficient, and inexpensive DNA extraction method for the recovery of genomic DNA from gram-positive and gram-negative oral bacteria to be used in polymerase chain reaction amplification. This method worked great with all samples analyzed, providing an approach to extract DNA for different microorganisms. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase.

    Science.gov (United States)

    Lee, Harold; Hanes, Jeremiah; Johnson, Kenneth A

    2003-12-23

    Incorporation of nucleoside analogues by the mitochondrial DNA polymerase has been implicated as the primary cause underlying many of the toxic side effects of these drugs in HIV therapy. Recent success in reconstituting recombinant human enzyme has afforded a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. The toxic side effects of nucleoside analogues are correlated with the kinetics of incorporation by the mitochondrial DNA polymerase, varying over 6 orders of magnitude in the sequence zalcitabine (ddC) > didanosine (ddI metabolized to ddA) > stavudine (d4T) > lamivudine (3TC) > tenofovir (PMPA) > zidovudine (AZT) > abacavir (metabolized to carbovir, CBV). In this review, we summarize our current efforts to examine the mechanistic basis for nucleotide selectivity by the mitochondrial DNA polymerase and its role in mitochondrial toxicity of nucleoside analogues used to treat AIDS and other viral infections. We will also discuss the promise and underlying challenges for the development of new analogues with lower toxicity.

  9. Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance.

    Science.gov (United States)

    Schermerhorn, Kelly M; Gardner, Andrew F

    2015-09-04

    Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, kpol and Kd, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (kpol ∼ 2.5 s(-1)) and especially tight nucleotide binding (Kd (dNTP) ∼ 1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3'-5' exonuclease hydrolysis activity in the presence of Mg(2+) and Mn(2+). Interestingly, substituting Mn(2+) for Mg(2+) accelerated hydrolysis rates > 40-fold (kexo ≥ 110 s(-1) versus ≥ 2.5 s(-1)). Preference for Mn(2+) over Mg(2+) in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance*

    Science.gov (United States)

    Schermerhorn, Kelly M.; Gardner, Andrew F.

    2015-01-01

    Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, kpol and Kd, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (kpol ∼2.5 s−1) and especially tight nucleotide binding (Kd(dNTP) ∼1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg2+ and Mn2+. Interestingly, substituting Mn2+ for Mg2+ accelerated hydrolysis rates >40-fold (kexo ≥110 s−1 versus ≥2.5 s−1). Preference for Mn2+ over Mg2+ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families. PMID:26160179

  11. Distinct co-evolution patterns of genes associated to DNA polymerase III DnaE and PolC

    Directory of Open Access Journals (Sweden)

    Engelen Stefan

    2012-02-01

    Full Text Available Abstract Background Bacterial genomes displaying a strong bias between the leading and the lagging strand of DNA replication encode two DNA polymerases III, DnaE and PolC, rather than a single one. Replication is a highly unsymmetrical process, and the presence of two polymerases is therefore not unexpected. Using comparative genomics, we explored whether other processes have evolved in parallel with each polymerase. Results Extending previous in silico heuristics for the analysis of gene co-evolution, we analyzed the function of genes clustering with dnaE and polC. Clusters were highly informative. DnaE co-evolves with the ribosome, the transcription machinery, the core of intermediary metabolism enzymes. It is also connected to the energy-saving enzyme necessary for RNA degradation, polynucleotide phosphorylase. Most of the proteins of this co-evolving set belong to the persistent set in bacterial proteomes, that is fairly ubiquitously distributed. In contrast, PolC co-evolves with RNA degradation enzymes that are present only in the A+T-rich Firmicutes clade, suggesting at least two origins for the degradosome. Conclusion DNA replication involves two machineries, DnaE and PolC. DnaE co-evolves with the core functions of bacterial life. In contrast PolC co-evolves with a set of RNA degradation enzymes that does not derive from the degradosome identified in gamma-Proteobacteria. This suggests that at least two independent RNA degradation pathways existed in the progenote community at the end of the RNA genome world.

  12. Mitochondrial polymerase gamma dysfunction and aging cause cardiac nuclear DNA methylation changes.

    Science.gov (United States)

    Koczor, Christopher A; Ludlow, Ivan; Fields, Earl; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Lewis, William

    2016-04-01

    Cardiomyopathy (CM) is an intrinsic weakening of myocardium with contractile dysfunction and congestive heart failure (CHF). CHF has been postulated to result from decreased mitochondrial energy production and oxidative stress. Effects of decreased mitochondrial oxygen consumption also can accelerate with aging. We previously showed DNA methylation changes in human hearts with CM. This was associated with mitochondrial DNA depletion, being another molecular marker of CM. We examined the relationship between mitochondrial dysfunction and cardiac epigenetic DNA methylation changes in both young and old mice. We used genetically engineered C57Bl/6 mice transgenic for a cardiac-specific mutant of the mitochondrial polymerase-γ (termed Y955C). Y955C mice undergo left ventricular hypertrophy (LVH) at a young age (∼ 94 days old), and LVH decompensated to CHF at old age (∼ 255 days old). Results found 95 genes differentially expressed as a result of Y955C expression, while 4,452 genes were differentially expressed as a result of aging hearts. Moreover, cardiac DNA methylation patterns differed between Y955C (4,506 peaks with 68.5% hypomethylation) and aged hearts (73,286 peaks with 80.2% hypomethylated). Correlatively, of the 95 Y955C-dependent differentially expressed genes, 30 genes (31.6%) also displayed differential DNA methylation; in the 4,452 age-dependent differentially expressed genes, 342 genes (7.7%) displayed associated DNA methylation changes. Both Y955C and aging demonstrated significant enrichment of CACGTG-associated E-box motifs in differentially methylated regions. Cardiac mitochondrial polymerase dysfunction alters nuclear DNA methylation. Furthermore, aging causes a robust change in cardiac DNA methylation that is partially associated with mitochondrial polymerase dysfunction. Copyright © 2016 the American Physiological Society.

  13. Pyrovanadolysis, a Pyrophosphorolysis-like Reaction Mediated by Pyrovanadate, Mn2+, and DNA Polymerase of Bacteriophage T7

    NARCIS (Netherlands)

    Akabayov, Barak; Kulczyk, Arkadiusz W.; Akabayov, Sabine R.; Theile, Christopher; McLaughlin, Larry W.; Beauchamp, Benjamin; van Oijen, Antoine M.; Richardson, Charles C.

    2011-01-01

    DNA polymerases catalyze the 3'-5'-pyrophosphorolysis of a DNA primer annealed to a DNA template in the presence of pyrophosphate (PPi). In this reversal of the polymerization reaction, deoxynucleotides in DNA are converted to deoxynucleoside 5'-triphosphates. Based on the charge, size, and geometry

  14. Data of self-made Taq DNA polymerase prepared for screening purposes

    Directory of Open Access Journals (Sweden)

    E.V. Konovalova

    2017-04-01

    Full Text Available DNA analysis is a key procedure in genetic engineering. Nowadays the analysis is often done by PCR with Taq DNA polymerase. Although the last enzyme price is quite low, demand for numerous analyses results in much money expenditure which are not affordable for many laboratories. In a meanwhile, many screening tasks do not require the highly purified enzyme. Taking into account the enzyme unique properties it makes possible to marginally simplify its production without resorting to costly or lengthy techniques such as column chromatography and/or dialysis. Here the data of routine usage of Taq DNA polymerase prepared according to the protocol developed in our laboratory is presented. The protocol takes only several hours to realize and does not need qualified personnel or expensive equipment. Yet it gives the enzyme preparation suitable for most screening purposes. The isolated Taq DNA polymerase stock can be stored as ammonium sulfate suspension in a refrigerator for prolonged period, not less than 6 months. The working enzyme solution is prepared from the stock suspension on demand, not more than once in a month and can be stored also in a refrigerator.

  15. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    Science.gov (United States)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  16. (1)H, (13)C, and (15)N backbone resonance assignments of the full-length 40 kDa S. acidocaldarius Y-family DNA polymerase, dinB homolog.

    Science.gov (United States)

    Moro, Sean L; Cocco, Melanie J

    2015-10-01

    The dinB homolog (Dbh) is a member of the Y-family of translesion DNA polymerases, which are specialized to accurately replicate DNA across from a wide variety of lesions in living cells. Lesioned bases block the progression of high-fidelity polymerases and cause detrimental replication fork stalling; Y-family polymerases can bypass these lesions. The active site of the translesion synthesis polymerase is more open than that of a replicative polymerase; consequently Dbh polymerizes with low fidelity. Bypass polymerases also have low processivity. Short extension past the lesion allows the high-fidelity polymerase to switch back onto the site of replication. Dbh and the other Y-family polymerases have been used as structural models to investigate the mechanisms of DNA polymerization and lesion bypass. Many high-resolution crystal structures of Y-family polymerases have been reported. NMR dynamics studies can complement these structures by providing a measure of protein motions. Here we report the (15)N, (1)H, and (13)C backbone resonance assignments at two temperatures (35 and 50 °C) for Sulfolobus acidocaldarius Dbh polymerase. Backbone resonance assignments have been obtained for 86 % of the residues. The polymerase active site is assigned as well as the majority of residues in each of the four domains.

  17. DNA Polymerases and Aminoacyl-tRNA Synthetases: Shared Mechanisms for Ensuring the Fidelity of Gene Expression

    OpenAIRE

    Francklyn, Christopher S.

    2008-01-01

    DNA polymerases and aminoacyl-tRNA synthetases (ARSs) represent large enzyme families with critical roles in the transformation of genetic information from DNA to RNA to protein. DNA polymerases carry out replication and collaborate in the repair of the genome, while ARSs provide aminoacylated tRNA precursors for protein synthesis. Enzymes of both families face the common challenge of selecting their cognate small molecule substrates from a pool of chemically related molecules, achieving high...

  18. Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products.

    Science.gov (United States)

    Debela, Ahmed M; Ortiz, Mayreli; Beni, Valerio; Thorimbert, Serge; Lesage, Denis; Cole, Richard B; O'Sullivan, Ciara K; Hasenknopf, Bernold

    2015-12-01

    The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW11 O39 {Sn(CH2 )2 CO}](8-) and [P2 W17 O61 {Sn(CH2 )2 CO}](6-) have been used to link to a 5'-NH2 terminated 21-mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM-labeled primers was demonstrated through hybridization with a surface-immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.

  19. PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif

    Science.gov (United States)

    Böhlke, Kristina; Pisani, Francesca M.; Vorgias, Constantinos E.; Frey, Bruno; Sobek, Harald; Rossi, Mosè; Antranikian, Garabed

    2000-01-01

    The effect of mutations in the highly conserved Y-GG/A motif of B-type DNA polymerases was studied in the DNA polymerase from the hyperthermophilic euryarchaeon Thermococcus aggregans. This motif plays a critical role in the balance between the synthesis and degradation of the DNA chain. Five different mutations of the tyrosine at position 387 (Tyr387→Phe, Tyr387→Trp, Tyr387→His, Tyr387→Asn and Tyr387→Ser) revealed that an aromatic ring system is crucial for the synthetic activity of the enzyme. Amino acids at this position lacking the ring system (Ser and Asn) led to a significant decrease in polymerase activity and to enhanced exonuclease activity, which resulted in improved enzyme fidelity. Exchange of tyrosine to phenylalanine, tryptophan or histidine led to phenotypes with wild-type-like fidelity but enhanced PCR performance that could be related to a higher velocity of polymerisation. With the help of a modelled structure of T.aggregans DNA polymerase, the biochemical data were interpreted proposing that the conformation of the flexible loop containing the Y-GG/A motif is an important factor for the equilibrium between DNA polymerisation and exonucleolysis. PMID:11024170

  20. Computational study of putative residues involved in DNA synthesis fidelity checking in Thermus aquaticus DNA polymerase I.

    Science.gov (United States)

    Elias, Angela A; Cisneros, G Andrés

    2014-01-01

    A fidelity-checking site for DNA polymerase I has been proposed based on recent single-molecule Förster resonance energy transfer studies. The checking site is believed to ensure proper base pairing of the newly inserted nucleotide. Computational studies have been utilized to predict residues involved in this putative checking site on the Klenow and Bacillus fragments. Here, we employ energy decomposition analysis, electrostatic free energy response, and noncovalent interaction plots to identify the residues involved in the hypothesized checking site in the homologous Klenow fragment from Thermus aquaticus (Klentaq). Our results indicate multiple protein residues that show altered interactions for three mispairs compared to the correctly paired DNA dimer. Many of these residues are also conserved along A family polymerases.

  1. Entecavir for Treatment of Hepatitis B Virus Displays No In Vitro Mitochondrial Toxicity or DNA Polymerase Gamma Inhibition▿

    Science.gov (United States)

    Mazzucco, Charles E.; Hamatake, Robert K.; Colonno, Richard J.; Tenney, Daniel J.

    2008-01-01

    Therapy with nucleoside reverse transcriptase inhibitors (NRTIs) can be associated with mitochondrial toxicity. In vitro studies have been used to predict the predisposition for and characterize the mechanisms causing mitochondrial toxicity. Entecavir (ETV) is an approved deoxyguanosine nucleoside for the treatment of chronic hepatitis B virus (HBV) infection that exhibits potent activity against viral reverse transcriptase. We assessed the potential for mitochondrial toxicity of ETV in long-term cultures of HepG2 hepatoma cells by measuring mitochondrial function (through lactate secretion), levels of mitochondrial DNA (mtDNA), and levels of mitochondrial proteins COX II and COX IV. Furthermore, we tested the activity of ETV-triphosphate (ETV-TP) against mitochondrial DNA polymerase γ (Pol γ) in vitro. ETV concentrations as high as 100 times the maximal clinical exposure (Cmax) did not affect cell proliferation, levels of lactate, mitochondrial DNA, or mitochondrial proteins throughout the 15-day culture. The lack of mitochondrial toxicity was consistent with the finding that ETV-TP was not recognized by mitochondrial DNA Pol γ and failed to be incorporated into DNA or inhibit the polymerase assay at the highest levels tested, 300 μM. Combinations of ETV with each of the other HBV NRTI antivirals, adefovir, tenofovir, and lamivudine at 10 times their respective Cmax levels also failed to result in cellular or mitochondrial toxicity. In summary, cell culture and enzymatic studies yielded no evidence that would predict mitochondrial toxicity of ETV at exposure levels in excess of those expected to be achieved clinically. PMID:18056280

  2. Roles of POLD4, smallest subunit of DNA polymerase {delta}, in nuclear structures and genomic stability of human cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Qin Miao [Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Akashi, Tomohiro [Division of Molecular Mycology and Medicine, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Masuda, Yuji; Kamiya, Kenji [Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Takahashi, Takashi [Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Suzuki, Motoshi, E-mail: msuzuki@med.nagoya-u.ac.jp [Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan)

    2010-01-01

    Mammalian DNA polymerase {delta} (pol {delta}) is essential for DNA replication, though the functions of this smallest subunit of POLD4 have been elusive. We investigated pol {delta} activities in vitro and found that it was less active in the absence of POLD4, irrespective of the presence of the accessory protein PCNA. shRNA-mediated reduction of POLD4 resulted in a marked decrease in colony formation activity by Calu6, ACC-LC-319, and PC-10 cells. We also found that POLD4 reduction was associated with an increased population of karyomere-like cells, which may be an indication of DNA replication stress and/or DNA damage. The karyomere-like cells retained an ability to progress through the cell cycle, suggesting that POLD4 reduction induces modest genomic instability, while allowing cells to grow until DNA damage reaches an intolerant level. Our results indicate that POLD4 is required for the in vitro pol {delta} activity, and that it functions in cell proliferation and maintenance of genomic stability of human cells.

  3. Mutations for Worse or Better: Low-Fidelity DNA Synthesis by SOS DNA Polymerase V Is a Tightly Regulated Double-Edged Sword.

    Science.gov (United States)

    Jaszczur, Malgorzata; Bertram, Jeffrey G; Robinson, Andrew; van Oijen, Antoine M; Woodgate, Roger; Cox, Michael M; Goodman, Myron F

    2016-04-26

    1953, the year of Watson and Crick, bore witness to a less acclaimed yet highly influential discovery. Jean Weigle demonstrated that upon infection of Escherichia coli, λ phage deactivated by UV radiation, and thus unable to form progeny, could be reactivated by irradiation of the bacterial host. Evelyn Witkin and Miroslav Radman later revealed the presence of the SOS regulon. The more than 40 regulon genes are repressed by LexA protein and induced by the coproteolytic cleavage of LexA, catalyzed by RecA protein bound to single-stranded DNA, the RecA* nucleoprotein filament. Several SOS-induced proteins are engaged in repairing both cellular and extracellular damaged DNA. There's no "free lunch", however, because error-free repair is accompanied by error-prone translesion DNA synthesis (TLS), involving E. coli DNA polymerase V (UmuD'2C) and RecA*. This review describes the biochemical mechanisms of pol V-mediated TLS. pol V is active only as a mutasomal complex, pol V Mut = UmuD'2C-RecA-ATP. RecA* donates a single RecA subunit to pol V. We highlight three recent insights. (1) pol V Mut has an intrinsic DNA-dependent ATPase activity that governs polymerase binding and dissociation from DNA. (2) Active and inactive states of pol V Mut are determined at least in part by the distinct interactions between RecA and UmuC. (3) pol V is activated by RecA*, not at a blocked replisome, but at the inner cell membrane.

  4. Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Surajit; Brown, Kyle L.; Egli, Martin; Stone, Michael P. (Vanderbilt)

    2012-07-18

    Aflatoxin B{sub 1} (AFB{sub 1}) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB{sub 1}-N7-dG). The AFB{sub 1}-N7-dG can rearrange to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative. Both AFB{sub 1}-N7-dG and the {beta}-anomer of the AFB{sub 1}-FAPY adduct yield G {yields} T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB{sub 1}-N7-dG in an error-free manner but conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including misinsertion of dATP, consistent with the G {yields} T mutations observed in E. coli. Three ternary (Dpo4-DNA-dNTP) structures with AFB{sub 1}-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB{sub 1}-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O{sup 2} oxygen of dTTP, and between the template T O{sup 4} oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB{sub 1}-N7-dG maintains the 5'-intercalation of the AFB{sub 1} moiety observed in DNA. The bond between N7-dG and C8 of the AFB{sub 1} moiety remains in plane with the alkylated guanine, creating a 16{sup o} inclination of the AFB{sub 1} moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB{sub 1}-FAPY adducted template:primer also maintains 5'-intercalation of the AFB{sub 1} moiety. The {beta}-deoxyribose anomer is observed. Rotation about the FAPY C5-N{sup 5} bond orients the bond between N{sup 5} and C8 of the AFB{sub 1} moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves

  5. Putative DNA-dependent RNA polymerase in Mitochondrial Plasmid of Paramecium caudatum Stock GT704

    Directory of Open Access Journals (Sweden)

    Trina Ekawati Tallei

    2015-10-01

    Full Text Available Mitochondria of Paramecium caudatum stock GT704 has a set of four kinds of linear plasmids with sizes of 8.2, 4.1, 2.8 and 1.4 kb. The plasmids of 8.2 and 2.8 kb exist as dimers consisting of 4.1- and 1.4-kb monomers, respectively. The plasmid 2.8 kb, designated as pGT704-2.8, contains an open reading frame encodes for putative DNA-dependent RNA polymerase (RNAP. This study reveals that this RNAP belongs to superfamily of DNA/RNA polymerase and family of T7/T3 single chain RNA polymerase and those of mitochondrial plasmid of fungi belonging to Basidiomycota and Ascomycota. It is suggested that RNAP of pGT704-2.8 can perform transcription without transcription factor as promoter recognition. Given that only two motifs were found, it could not be ascertained whether this RNAP has a full function independently or integrated with mtDNA in carrying out its function.

  6. Structural basis of transcription: separation of RNA from DNA by RNA polymerase II.

    Science.gov (United States)

    Westover, Kenneth D; Bushnell, David A; Kornberg, Roger D

    2004-02-13

    The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape.

  7. On-Chip integration of sample pretreatment and Multiplex polymerase chain reaction (PCR) for DNA analysis

    DEFF Research Database (Denmark)

    Brivio, Monica; Snakenborg, Detlef; Søgaard, E.

    2008-01-01

    In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a custom-made thermocycler (Figure 1) and includes a simple...... and efficient method for switching the liquid flow between the PT and PCR chamber. Purification of human genomic DNA from EDTA-treated blood and multiplex PCR were successfully carried out on-chip using the developed lab-on-a-chip system....

  8. DNA polymerase zeta is required for proliferation of normal mammalian cells.

    Science.gov (United States)

    Lange, Sabine S; Wittschieben, John P; Wood, Richard D

    2012-05-01

    Unique among translesion synthesis (TLS) DNA polymerases, pol ζ is essential during embryogenesis. To determine whether pol ζ is necessary for proliferation of normal cells, primary mouse fibroblasts were established in which Rev3L could be conditionally inactivated by Cre recombinase. Cells were grown in 2% O(2) to prevent oxidative stress-induced senescence. Cells rapidly became senescent or apoptotic and ceased growth within 3-4 population doublings. Within one population doubling following Rev3L deletion, DNA double-strand breaks and chromatid aberrations were found in 30-50% of cells. These breaks were replication dependent, and found in G1 and G2 phase cells. Double-strand breaks were reduced when cells were treated with the reactive oxygen species scavenger N-acetyl-cysteine, but this did not rescue the cell proliferation defect, indicating that several classes of endogenously formed DNA lesions require Rev3L for tolerance or repair. T-antigen immortalization of cells allowed cell growth. In summary, even in the absence of external challenges to DNA, pol ζ is essential for preventing replication-dependent DNA breaks in every division of normal mammalian cells. Loss of pol ζ in slowly proliferating mouse cells in vivo may allow accumulation of chromosomal aberrations that could lead to tumorigenesis. Pol ζ is unique amongst TLS polymerases for its essential role in cell proliferation.

  9. Characterization of a 7-kilodalton subunit of vaccinia virus DNA-dependent RNA polymerase with structural similarities to the smallest subunit of eukaryotic RNA polymerase II.

    Science.gov (United States)

    Amegadzie, B Y; Ahn, B Y; Moss, B

    1992-05-01

    A previously unrecognized 7-kDa polypeptide copurified with the DNA-dependent RNA polymerase of vaccinia virus virions. Internal amino acid sequences of the small protein matched a viral genomic open reading frame of 63 codons. Antipeptide antiserum was used to confirm the specific and complete association of the 7-kDa protein with RNA polymerase. The amino acid sequence predicted from the viral gene, named rpo7, was 23% identical to that of the smallest subunit of Saccharomyces cerevisiae RNA polymerase II, and a metal-binding motif, Cys-X-X-Cys-Gly, was located at precisely the same location near the N terminus in the two proteins. RNA analyses demonstrated early transcriptional initiation and termination signals in the rpo7 gene sequence. The viral RNA polymerase subunit was synthesized during the early phase of infection and continued to accumulate during the late phase.

  10. DNA polymerase ζ-dependent lesion bypass in Saccharomyces cerevisiae is accompanied by error-prone copying of long stretches of adjacent DNA.

    Directory of Open Access Journals (Sweden)

    Olga V Kochenova

    2015-03-01

    Full Text Available Translesion synthesis (TLS helps cells to accomplish chromosomal replication in the presence of unrepaired DNA lesions. In eukaryotes, the bypass of most lesions involves a nucleotide insertion opposite the lesion by either a replicative or a specialized DNA polymerase, followed by extension of the resulting distorted primer terminus by DNA polymerase ζ (Polζ. The subsequent events leading to disengagement of the error-prone Polζ from the primer terminus and its replacement with an accurate replicative DNA polymerase remain largely unknown. As a first step toward understanding these events, we aimed to determine the length of DNA stretches synthesized in an error-prone manner during the Polζ-dependent lesion bypass. We developed new in vivo assays to identify the products of mutagenic TLS through a plasmid-borne tetrahydrofuran lesion and a UV-induced chromosomal lesion. We then surveyed the region downstream of the lesion site (in respect to the direction of TLS for the presence of mutations indicative of an error-prone polymerase activity. The bypass of both lesions was associated with an approximately 300,000-fold increase in the mutation rate in the adjacent DNA segment, in comparison to the mutation rate during normal replication. The hypermutated tract extended 200 bp from the lesion in the plasmid-based assay and as far as 1 kb from the lesion in the chromosome-based assay. The mutation rate in this region was similar to the rate of errors produced by purified Polζ during copying of undamaged DNA in vitro. Further, no mutations downstream of the lesion were observed in rare TLS products recovered from Polζ-deficient cells. This led us to conclude that error-prone Polζ synthesis continues for several hundred nucleotides after the lesion bypass is completed. These results provide insight into the late steps of TLS and show that error-prone TLS tracts span a substantially larger region than previously appreciated.

  11. Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations

    Directory of Open Access Journals (Sweden)

    Leary Jeffry J

    2002-05-01

    Full Text Available Abstract Background The thymidine kinase (tk mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. Methods A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. Results Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2–4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed signficant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA stucture. Conclusions This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a mutations may be modulated by other viral polypeptides cooperating with Pol, and (b the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

  12. A Crystallographic Study of the Role of Sequence Context in Thymine Glycol Bypass by a Replicative DNA Polymerase Serendipitously Sheds Light on the Exonuclease Complex

    Energy Technology Data Exchange (ETDEWEB)

    Aller, Pierre; Duclos, Stéphanie; Wallace, Susan S.; Doublié, Sylvie (Vermont)

    2012-06-27

    Thymine glycol (Tg) is the most common oxidation product of thymine and is known to be a strong block to replicative DNA polymerases. A previously solved structure of the bacteriophage RB69 DNA polymerase (RB69 gp43) in complex with Tg in the sequence context 5'-G-Tg-G shed light on how Tg blocks primer elongation: The protruding methyl group of the oxidized thymine displaces the adjacent 5'-G, which can no longer serve as a template for primer elongation [Aller, P., Rould, M. A., Hogg, M, Wallace, S. S. and Doublie S. (2007). A structural rationale for stalling of a replicative DNA polymerase at the most common oxidative thymine lesion, thymine glycol. Proc. Natl. Acad. Sci. USA, 104, 814-818.]. Several studies showed that in the sequence context 5'-C-Tg-purine, Tg is more likely to be bypassed by Klenow fragment, an A-family DNA polymerase. We set out to investigate the role of sequence context in Tg bypass in a B-family polymerase and to solve the crystal structures of the bacteriophage RB69 DNA polymerase in complex with Tg-containing DNA in the three remaining sequence contexts: 5'-A-Tg-G, 5'-T-Tg-G, and 5'-C-Tg-G. A combination of several factors - including the associated exonuclease activity, the nature of the 3' and 5' bases surrounding Tg, and the cis-trans interconversion of Tg - influences Tg bypass. We also visualized for the first time the structure of a well-ordered exonuclease complex, allowing us to identify and confirm the role of key residues (Phe123, Met256, and Tyr257) in strand separation and in the stabilization of the primer strand in the exonuclease site.

  13. BORRELIA BURGDORFERI DNA IN BIOLOGICAL SAMPLES FROM PATIENTS WITH SARCOIDOSIS USING THE POLYMERASE CHAIN REACTION TECHNIQUE

    Institute of Scientific and Technical Information of China (English)

    连伟; 罗慰慈

    1995-01-01

    Polymerase chain reaction (PCR) was used to detect the presence of Borretia burgdoferi DNA in biological samples from patients with sarcoidcsis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridlzation with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdor feri genome, even in the presence of a 104-fold excess of human eukaryotic DNA, and was also specific to different B. burgdorferl strains tested. Sera seroiogieally positive to B. burgdorferi (n=26), broncbemlveolar lavage fluid and supematant of BALF (n=26) and peripheral blood (n=9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26, and 0/9, respectively). It was considered that DNA from B. bur gdor feri may be identified in a minority of patients with s,arcoidosis, and it may play a pathogenetic rote in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.

  14. Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

    Directory of Open Access Journals (Sweden)

    Ana Lisa do Vale Gomes

    2006-10-01

    Full Text Available This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA. The efficiency was 0.99 and the correlation coefficient (R² was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83ºC. The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

  15. Contiguous 2,2,4-triamino-5(2H)-oxazolone obstructs DNA synthesis by DNA polymerases α, β, η, ι, κ, REV1 and Klenow Fragment exo-, but not by DNA polymerase ζ.

    Science.gov (United States)

    Suzuki, Masayo; Kino, Katsuhito; Kawada, Taishu; Oyoshi, Takanori; Morikawa, Masayuki; Kobayashi, Takanobu; Miyazawa, Hiroshi

    2016-03-01

    Guanine is the most easily oxidized of the four DNA bases, and contiguous guanines (GG) in a sequence are more readily oxidized than a single guanine in a sequence. Continued oxidation of GGs results in a contiguous oxidized guanine lesion. Two contiguous 2,5-diamino-4H-imidazol-4-ones, an oxidized form of guanine that hydrolyses to 2,2,4-triamino-5(2H)-oxazolone (Oz), are detected following the oxidation of GG. In this study, we analysed translesion synthesis (TLS) across two contiguous Oz molecules (OzOz) using Klenow Fragment exo(-) (KF exo(-)) and DNA polymerases (Pols) α, β, ζ, η, ι, κ and REV1. We found that KF exo(-) and Pols α, β, ι and REV1 inserted one nucleotide opposite the 3' Oz of OzOz and stalled at the subsequent extension, and that Pol κ incorporated no nucleotide. Pol η only inefficiently elongated the primer up to full-length across OzOz; the synthesis of most DNA strands stalled at the 3' or 5' Oz of OzOz. Surprisingly, however, Pol ζ efficiently extended the primer up to full-length across OzOz, unlike the other DNA polymerases, but catalysed error-prone nucleotide incorporation. We therefore believe that Pol ζ is required for efficient TLS of OzOz. These results show that OzOz obstructs DNA synthesis by DNA polymerases except Pol ζ.

  16. Compartmentalized self-replication (CSR) selection of Thermococcus litoralis Sh1B DNA polymerase for diminished uracil binding.

    Science.gov (United States)

    Tubeleviciute, Agne; Skirgaila, Remigijus

    2010-08-01

    The thermostable archaeal DNA polymerase Sh1B from Thermococcus litoralis has a typical uracil-binding pocket, which in nature plays an essential role in preventing the accumulation of mutations caused by cytosine deamination to uracil and subsequent G-C base pair transition to A-T during the genomic DNA replication. The uracil-binding pocket recognizes and binds uracil base in a template strand trapping the polymerase. Since DNA replication stops, the repair systems have a chance to correct the promutagenic event. Archaeal family B DNA polymerases are employed in various PCR applications. Contrary to nature, in PCR the uracil-binding property of archaeal polymerases is disadvantageous and results in decreased DNA amplification yields and lowered sensitivity. Furthermore, in diagnostics qPCR, RT-qPCR and end-point PCR are performed using dNTP mixtures, where dTTP is partially or fully replaced by dUTP. Uracil-DNA glycosylase treatment and subsequent heating of the samples is used to degrade the DNA containing uracil and prevent carryover contamination, which is the main concern in diagnostic laboratories. A thermostable archaeal DNA polymerase with the abolished uracil binding would be a highly desirable and commercially interesting product. An attempt to disable uracil binding in DNA polymerase Sh1B from T. litoralis by generating site-specific mutants did not yield satisfactory results. However, a combination of random mutagenesis of the whole polymerase gene and compartmentalized self-replication was successfully used to select variants of thermostable Sh1B polymerase capable of performing PCR with dUTP instead of dTTP.

  17. Identification of the sequences recognized by phage phi 29 transcriptional activator: possible interaction between the activator and the RNA polymerase.

    Science.gov (United States)

    Nuez, B; Rojo, F; Barthelemy, I; Salas, M

    1991-05-11

    Expression of Bacillus subtilis phage phi 29 late genes requires the transcriptional activator protein p4. This activator binds to a region of the late A3 promoter spanning nucleotides -56 to -102 relative to the transcription start site, generating a strong bending Tin the DNA. In this work the target sequences recognized by protein p4 in the phage phi 29 late A3 promoter have been characterized. The binding of protein p4 to derivatives of the late A3 promoter harbouring deletions in the protein p4 binding site has been studied. When protein p4 recognition sequences were altered, the activator could only bind to the promoter in the presence of RNA polymerase. This strong cooperativity in the binding of protein p4 and RNA polymerase to the promoter suggests the presence of direct protein-protein contacts between them.

  18. Down syndrome as a model of DNA polymerase beta haploinsufficiency and accelerated aging.

    Science.gov (United States)

    Patterson, David; Cabelof, Diane C

    2012-04-01

    Down syndrome is a condition of intellectual disability characterized by accelerated aging. As with other aging syndromes, evidence accumulated over the past several decades points to a DNA repair defect inherent in Down syndrome. This evidence has led us to suggest that Down syndrome results in reduced DNA base excision repair (BER) capacity, and that this contributes to the genomic instability and the aging phenotype of Down syndrome. We propose important roles for microRNA and/or folate metabolism and oxidative stress in the dysregulation of BER in Down syndrome. Further, we suggest these pathways are involved in the leukemogenesis of Down syndrome. We have reviewed the role of BER in the processing of oxidative stress, and the impact of folate depletion on BER capacity. Further, we have reviewed the role that loss of BER, specifically DNA polymerase beta, plays in accelerating the rate of aging. Like that seen in the DNA polymerase beta heterozygous mouse, the aging phenotype of Down syndrome is subtle, unlike the aging phenotypes seen in the classical progeroid syndromes and mouse models of aging. As such, Down syndrome may provide a model for elucidating some of the basic mechanisms of aging.

  19. A bichaperone (Hsp70-Hsp78) system restores mitochondrial DNA synthesis following thermal inactivation of Mip1p polymerase.

    Science.gov (United States)

    Germaniuk, Aleksandra; Liberek, Krzysztof; Marszalek, Jaroslaw

    2002-08-02

    Mitochondrial DNA synthesis is a thermosensitive process in the yeast Saccharomyces cerevisiae. We found that restoration of mtDNA synthesis following heat treatment of cells is dependent on reactivation of the mtDNA polymerase Mip1p through the action of a mitochondrial bichaperone system consisting of the Hsp70 system and the Hsp78 oligomeric protein. mtDNA synthesis was inefficiently restored after heat shock in yeast lacking either functional component of the bichaperone system. Furthermore, the activity of purified Mip1p was also thermosensitive; however, the purified components of the mitochondrial bichaperone system (Ssc1p, Mdj1p, Mge1p, and Hsp78p) were able to protect its activity under moderate heat shock conditions as well as to reactivate thermally inactivated Mip1p. Interestingly, the reactivation of endogenous Mip1p contributed more significantly to the restoration of mtDNA synthesis than did import of newly synthesized Mip1p from the cytosol. These observations suggest an important link between function of mitochondrial chaperones and the propagation of mitochondrial genomes under ever-changing environmental conditions.

  20. Crystal structure of the shrimp proliferating cell nuclear antigen: structural complementarity with WSSV DNA polymerase PIP-box.

    Directory of Open Access Journals (Sweden)

    Jesus S Carrasco-Miranda

    Full Text Available DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA. This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein. The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems.

  1. Rapid identification of genes encoding DNA polymerases by function-based screening of metagenomic libraries derived from glacial ice.

    Science.gov (United States)

    Simon, Carola; Herath, Judith; Rockstroh, Stephanie; Daniel, Rolf

    2009-05-01

    Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.

  2. Rapid Identification of Genes Encoding DNA Polymerases by Function-Based Screening of Metagenomic Libraries Derived from Glacial Ice▿

    OpenAIRE

    2009-01-01

    Small-insert and large-insert metagenomic libraries were constructed from glacial ice of the Northern Schneeferner, which is located on the Zugspitzplatt in Germany. Subsequently, these libraries were screened for the presence of DNA polymerase-encoding genes by complementation of an Escherichia coli polA mutant. Nine novel genes encoding complete DNA polymerase I proteins or domains typical of these proteins were recovered.

  3. Treponema pallidum and Haemophilus ducreyi DNA detection by A Multi-Nested Polymerase Chain Reaction

    Institute of Scientific and Technical Information of China (English)

    郑和平; SylviaBruisten; 何玉山; 黄进梅; 吴兴中

    2004-01-01

    Objectives: To develop a multi-nested polymerase chain reaction in an assay to detect early Treponema pallidum and Haemophilus ducreyi DNA in the swabs of genital ulcers. Methods: Four pairs of outer and inner primers, specific to the basic membrane protein gene of Treponema pallidum and to the 16s rRNA gene of H ducreyi were synthesized. The multi-nested PCR was developed and applied to detect Treponema pallidum and Haemophilus dicreyi in clinical swabs. Result: The two samples of standard strains of Haemophilus ducreyi and one Treponema pallidum were amplified and showed 309-bp rRNA gene of Haemophilus ducreyi and 506-bp DNA of Treponema palidum, respectively. Out of 51 samples of genital ulcer detected, 29 showed Treponemapallidum positive product and noHaemophilus ducreyi DNA was found. Conclusion: The multi-nested PCR for Treponema pallidum and Haemophilus ducreyi could be useful for early detection and distinguishing diagnosis between syphilis and chancroid.

  4. Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration ofa transgene.

    Directory of Open Access Journals (Sweden)

    Tomoyuki eFurukawa

    2015-05-01

    Full Text Available The DNA double-strand break (DSB is a critical type of damage, and can be induced by both endogenous sources (e.g. errors of oxidative metabolism, transposable elements, programmed meiotic breaks, or perturbation of the DNA replication fork and exogenous sources (e.g. ionizing radiation or radiomimetic chemicals. Although higher plants, like mammals, are thought to preferentially repair DSBs via nonhomologous end joining (NHEJ, much remains unclear about plant DSB repair pathways. Our reverse genetic approach suggests that DNA polymerase λ is involved in DSB repair in Arabidopsis. The Arabidopsis T-DNA insertion mutant (atpolλ-1 displayed sensitivity to both gamma-irradiation and treatment with radiomimetic reagents, but not to other DNA damaging treatments. The atpolλ-1 mutant showed a moderate sensitivity to DSBs, while Arabidopsis Ku70 and DNA ligase 4 mutants (atku70-3 and atlig4-2, both of which play critical roles in NHEJ, exhibited a hypersensitivity to these treatments. The atpolλ-1/atlig4-2 double mutant exhibited a higher sensitivity to DSBs than each single mutant, but the atku70/atpolλ-1 showed similar sensitivity to the atku70-3 mutant. We showed that transcription of the DNA ligase 1, DNA ligase 6, and Wee1 genes was quickly induced by BLM in several NHEJ deficient mutants in contrast to wild-type. Finally, the T-DNA transformation efficiency dropped in NHEJ deficient mutants and the lowest transformation efficiency was scored in the atpolλ-1/atlig4-2 double mutant. These results imply that AtPolλ is involved in both DSB repair and DNA damage response pathway.

  5. Differential diagnosis of Taenia saginata and Taenia solium infections: from DNA probes to polymerase chain reaction.

    Science.gov (United States)

    González, Luis Miguel; Montero, Estrella; Sciutto, Edda; Harrison, Leslie J S; Parkhouse, R Michael E; Garate, Teresa

    2002-04-01

    The objective of this work was the rapid and easy differential diagnosis of Taenia saginata and T. solium. First, a T. saginata size-selected genomic deoxyribonucleic acid (gDNA) library was constructed in the vector lambda gt10 using the 2-4 kb fraction from the parasite DNA digested with EcoR1, under 'star' conditions. After differential screening of the library and hybridization analysis with DNA from T. saginata, T. solium, T. taeniaeformis, T. crassiceps, and Echinococcus granulosus (bovine, porcine, and human), 2 recombinant phages were selected. They were designated HDP1 and HDP2. HDP1 reacted specifically with T. saginata DNA, and HDP2 recognized DNA from both T. saginata and T. solium. The 2 DNA probes were then sequenced and further characterized. HDP1 was a repetitive sequence with a 53 bp monomeric unit repeated 24 times in direct tandem along the 1272 bp fragment, while the 3954 bp HDP2 was not a repetitive sequence. Using the sequencing data, oligonucleotides were designed and used in a polymerase chain reaction (PCR). The 2 selected oligonucleotides from probe HDP1 (PTs4F1 and PTs4R1) specifically amplified gDNA from T. saginata, but not T. solium or other related cestodes, with a sensitivity of < 10 pg of T. saginata gDNA, about the quantity of DNA in one taeniid egg. The 3 oligonucleotides selected from the HDP2 sequence (PTs7S35F1, PTs7S35F2, and PTs7S35R1) allowed the differential amplification of gDNA from T. saginata, T. solium and E. granulosus in a multiplex PCR, again with a sensitivity of < 10 pg. These diagnostic tools have immediate application in the differential diagnosis of T. solium and T. saginata in humans and in the diagnosis of dubious cysts in the slaughterhouse. We also hope to apply them to epidemiological surveys of, for example, soil and water in endemic areas.

  6. DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid

    NARCIS (Netherlands)

    A. Copani; J.J.M. Hoozemans; F. Caraci; M. Calafiore; E.S. van Haastert; R. Veerhuis; A.J.M. Rozemuller; E. Aronica; M.A. Sortino; F. Nicoletti

    2006-01-01

    Cultured neurons exposed to synthetic beta-amyloid (A beta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments

  7. A germline polymorphism of DNA polymerase beta induces genomic instability and cellular transformation.

    Directory of Open Access Journals (Sweden)

    Jennifer Yamtich

    Full Text Available Several germline single nucleotide polymorphisms (SNPs have been identified in the POLB gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β, encoded by the POLB gene, is the main gap-filling polymerase involved in base excision repair (BER, a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the POLB germline coding SNP (rs3136797 in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility.

  8. A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

    Science.gov (United States)

    Houtteman, S W; Elder, R T

    1993-03-01

    Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology. A spontaneous recessive mutation that restored normal colony morphology was identified. This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number. An ARS1 plasmid was also more stable in mutant cells than in wild-type cells. The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2). This gene encodes DNA polymerase delta, which is essential for DNA replication. These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.

  9. Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates.

    Science.gov (United States)

    Güixens-Gallardo, Pedro; Hocek, Michal; Perlíková, Pavla

    2016-01-15

    A simple and elegant method for inhibition of non-templated nucleotide addition by DNA polymerases and for following DNA 3'-heterogeneity in enzymatic DNA synthesis by primer extension (PEX) is described. When template bearing ortho-twisted intercalating nucleic acid (ortho-TINA) at the 5'-end is used, non-templated nucleotide addition is reduced in both the A- and B-family DNA polymerases (KOD XL, KOD (exo-), Bst 2.0, Therminator, Deep Vent (exo-) and Taq). Formation of a single oligonucleotide product was observed with ortho-TINA modified template and KOD XL, KOD (exo-), Bst 2.0, Deep Vent (exo-) and Taq DNA polymerases. This approach can be applied to the synthesis of both unmodified and base-modified oligonucleotides. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Human DNA polymerases catalyze lesion bypass across benzo[a]pyrene-derived DNA adduct clustered with an abasic site.

    Science.gov (United States)

    Starostenko, Lidia V; Rechkunova, Nadejda I; Lebedeva, Natalia A; Kolbanovskiy, Alexander; Geacintov, Nicholas E; Lavrik, Olga I

    2014-12-01

    The combined action of oxidative stress and genotoxic polycyclic aromatic hydrocarbons derivatives can lead to cluster-type DNA damage that includes both a modified nucleotide and a bulky lesion. As an example, we investigated the possibility of repair of an AP site located opposite a minor groove-positioned (+)-trans-BPDE-dG or a base-displaced intercalated (+)-cis-BPDE-dG adduct (BP lesion) by a BER system. Oligonucleotides with single uracil residue in the certain position were annealed with complementary oligonucleotides bearing either a cis- or trans-BP adduct. Digestion with uracil DNA glycosylase was utilized to generate an AP site which was then hydrolyzed by APE1, and the resulting gap was processed by X-family DNA polymerases β (Polβ) and λ (Polλ), or Y-family polymerase ι (Polι). By varying reaction conditions, namely, Mg2+/Mn2+ replacement/combination and ionic strength decrease, we found that under certain conditions both Polβ and Polι can catalyze lesion bypass across both cis- and trans-BP adducts in the presence of physiological dNTP concentrations. Polβ and Polι catalyze gap filling trans-lesion synthesis in an error prone manner. By contrast, Polλ selectively introduced the correct dCTP opposite the modified dG in the case of cis-BP-dG adduct only, and did not bypass the stereoisomeric trans-adduct under any of the conditions examined. The results suggest that Polλ is a specialized polymerase that can process these kinds of lesions.

  11. Novel Cytomegalovirus UL54 DNA Polymerase Gene Mutations Selected In Vitro That Confer Brincidofovir Resistance

    Science.gov (United States)

    Ercolani, Ronald J.; Lanier, E. Randall

    2016-01-01

    Eight in vitro selection experiments under brincidofovir pressure elicited the known cytomegalovirus DNA polymerase amino acid substitutions N408K and V812L and the novel exonuclease domain substitutions D413Y, E303D, and E303G, which conferred ganciclovir and cidofovir resistance with 6- to 11-fold resistance to brincidofovir or 17-fold when E303G was combined with V812L. The new exonuclease domain I resistance mutations selected under brincidofovir pressure add to the single instance previously reported and show the expected patterns of cross-resistance. PMID:27044553

  12. A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

    Science.gov (United States)

    Wang, Yingfei; An, Ran; Umanah, George K.; Park, Hyejin; Nambiar, Kalyani; Eacker, Stephen M.; Kim, BongWoo; Bao, Lei; Harraz, Maged M.; Chang, Calvin; Chen, Rong; Wang, Jennifer E.; Kam, Tae-In; Jeong, Jun Seop; Xie, Zhi; Neifert, Stewart; Qian, Jiang; Andrabi, Shaida A.; Blackshaw, Seth; Zhu, Heng; Song, Hongjun; Ming, Guo-li; Dawson, Valina L.; Dawson, Ted M.

    2016-01-01

    Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF's nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation. PMID:27846469

  13. PARP2 Is the Predominant Poly(ADP-Ribose Polymerase in Arabidopsis DNA Damage and Immune Responses.

    Directory of Open Access Journals (Sweden)

    Junqi Song

    2015-05-01

    Full Text Available Poly (ADP-ribose polymerases (PARPs catalyze the transfer of multiple poly(ADP-ribose units onto target proteins. Poly(ADP-ribosylation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390, rather than PARP1 (At2g31320, makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose glycohydrolase (PARG enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosylation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosylation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation.

  14. Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Kirouac, Kevin N.; Ling, Hong; (UWO)

    2009-06-30

    Human DNA polymerase iota (pol iota) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of pol complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson-Crick base pairing by pol. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template 'U-turn' is stabilized by pol and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping experiments indicate that the finger domain is responsible for pol's high error rates on pyrimidines and determines the incorporation specificity.

  15. Sliding Clamp–DNA Interactions Are Required for Viability and Contribute to DNA Polymerase Management in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Heltzel, J.; Scouten Ponticelli, S; Sanders, L; Duzen, J; Cody, V; Pace, J; Snell, E; Sutton, M

    2009-01-01

    Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli {beta} clamp interact physically with the DNA that it topologically encircles. We utilized mutant {beta} clamp proteins bearing G66E and G174A substitutions ({beta}159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 ({beta}148-152), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of {beta}148-152, which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both {beta}159 and {beta}148-152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of {beta}148-152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired {beta}148-152-DNA interactions. Once loaded, {beta}148-152 was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, {beta}148-152 was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, {beta}148-152 was unable to support viability of E. coli. Nevertheless, physiological levels of {beta}148-152 expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing {beta}159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA

  16. Crystallization and preliminary X-ray analysis of the Plasmodium falciparum apicoplast DNA polymerase

    Science.gov (United States)

    Milton, Morgan E.; Choe, Jun-yong; Honzatko, Richard B.; Nelson, Scott W.

    2015-01-01

    Infection by the parasite Plasmodium falciparum is the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment of Escherichia coli DNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals of P. falciparum apPOL are reported. Data complete to 3.5 Å resolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space group P6522 (unit-cell parameters a = b = 141.8, c = 149.7 Å, α = β = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress. PMID:25760711

  17. Nucleotide insertion initiated by van der Waals interaction during polymerase beta DNA replication

    CERN Document Server

    Arulsamy, Andrew Das

    2011-01-01

    Immortality will remain a fantasy for as long as aging is determined by the erroneous biochemical reactions during a particular DNA replication. The replication and base excision repair mechanism, associated to eukaryotic DNA polymerase-beta enzyme are central to maintaining a healthy cell. Here, we give a series of unambiguous theoretical analyses and prove that the exclusive biochemical reaction involved in a single nucleotide insertion into the DNA primer can be efficiently tracked using the renormalized van der Waals interaction of the stronger type, and the Hermansson blue-shifting hydrogen bond effect. We found that there are two biochemical steps involved to complete the insertion of a single dCTP into the 3' end of a DNA primer. First, the O3' (from a DNA primer) initiates the nucleophilic attack on P_alpha?(from an incoming dCTP), in response, O3_alpha (bonded to P_alpha) retaliates by interacting with H' (bonded to O3'). These interactions are shown to be strongly interdependent and require the form...

  18. Transcriptional analysis of the DNA polymerase gene of shrimp white spot syndrome virus.

    Science.gov (United States)

    Chen, Li-Li; Wang, Han-Ching; Huang, Chiu-Jung; Peng, Shao-En; Chen, Yen-Gu; Lin, Shin-Jen; Chen, Wei-Yu; Dai, Chang-Feng; Yu, Hon-Tsen; Wang, Chung-Hsiung; Lo, Chu-Fang; Kou, Guang-Hsiung

    2002-09-15

    The white spot syndrome virus DNA polymerase (DNA pol) gene (WSSV dnapol) has already been tentatively identified based on the presence of highly conserved motifs, but it shows low overall homology with other DNA pols and is also much larger (2351 amino acid residues vs 913-1244 aa). In the present study we perform a transcriptional analysis of the WSSV dnapol gene using the total RNA isolated from WSSV-infected shrimp at different times after infection. Northern blot analysis with a WSSV dnapol-specific riboprobe found a major transcript of 7.5 kb. 5'-RACE revealed that the major transcription start point is located 27 nucleotides downstream of the TATA box, at the nucleotide residue A within a CAGT motif, one of the initiator (Inr) motifs of arthropods. In a temporal expression analysis using differential RT-PCR, WSSV dnapol transcripts were detected at low levels at 2-4 h.p.i., increased at 6 h.p.i., and remained fairly constant thereafter. This is similar to the previously reported transcription patterns for genes encoding the key enzyme of nucleotide metabolism, ribonucleotide reductase. Phylogenetic analysis showed that the DNA pols from three different WSSV isolates form an extremely tight cluster. In addition, similar to an earlier phylogenetic analysis of WSSV protein kinase, the phylogenetic tree of viral DNA pols further supports the suggestion that WSSV is a distinct virus (likely at the family level) that does not belong to any of the virus families that are currently recognized.

  19. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

    Institute of Scientific and Technical Information of China (English)

    Scott D McCulloch; Thomas A Kunkel

    2008-01-01

    In their seminal publication describing the structure of the DNA double helix [1], Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.

  20. Detection of Toxoplasma gondii DNA by polymerase chain reaction in experimentally desiccated tissues

    Directory of Open Access Journals (Sweden)

    Márcia Andreia Barge Loução Terra

    2004-03-01

    Full Text Available Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations.Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG, or infected with tachyzoites (RH strain, the acute infection group (AIG. Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.

  1. Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

    Science.gov (United States)

    Shao, Zhiyong; Graf, Shannon; Chaga, Oleg Y; Lavrov, Dennis V

    2006-10-15

    The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

  2. Validation study of HPV DNA detection from stained FNA smears by polymerase chain reaction

    DEFF Research Database (Denmark)

    Channir, Hani Ibrahim; Grønhøj Larsen, Christian; Ahlborn, Lise Barlebo;

    2016-01-01

    BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) often presents with cystic cervical metastasis and a small primary tumor localized in the palatine tonsils or base of the tongue, which is diagnostically challenging. Testing for HPV DNA in fine......-needle aspiration (FNA) smears from metastases may facilitate a targeted diagnostic workup for identifying the primary tumor. This study was designed to assess the ability to detect HPV DNA in FNA smears with polymerase chain reaction (PCR). METHODS: May-Grünvald-Giemsa (MGG)-stained FNA smears from metastases...... and corresponding surgical specimens were collected from 71 patients with known HPV-positive OPSCC, 12 patients with oral squamous cell carcinoma (OSCC), 20 patients with branchial cleft cysts, and 20 patients with Warthin tumors. Thirty-eight patients with OPSCC and 7 patients with OSCC had FNA smears available...

  3. Replicative DNA polymerase δ but not ε proofreads errors in Cis and in Trans.

    Directory of Open Access Journals (Sweden)

    Carrie L Flood

    2015-03-01

    Full Text Available It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3'-terminal 8 oxoG, unlike a 3'-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.

  4. Poly(ADP-ribose polymerase (PARP-1 is not involved in DNA double-strand break recovery

    Directory of Open Access Journals (Sweden)

    Fernet Marie

    2003-07-01

    Full Text Available Abstract Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose polymerase (PARP-1 in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

  5. Allele-specific polymerase chain reaction for detection of a mutation in the relax circular DNA and the covalently closed circular DNA of hepatitis B virus.

    Science.gov (United States)

    Pan, Wan-Long; Hu, Jie-Li; Fang, Yan; Luo, Qiang; Xu, Ge; Xu, Lei; Jing, Zhou-Hong; Shan, Xue-Feng; Zhu, Yan-Ling; Huang, Ai-Long

    2013-12-01

    The relax circle DNA (rcDNA) sequence and the covalently closed circle DNA (cccDNA) sequence in hepatitis B virus (HBV) are crucial regions for HBV infections. To analyze mutations in rcDNA and cccDNA, DNA sequencing is often used, although it is time-consuming and expensive. Herein, we report a simple, economic, albeit accurate allele-specific polymerase chain reaction (AS-PCR) to detect mutations in these regions of HBV. This method can be extensively used to screen for mutations at specific positions of HBV genome.

  6. Competitive fitness during feast and famine: how SOS DNA polymerases influence physiology and evolution in Escherichia coli.

    Science.gov (United States)

    Corzett, Christopher H; Goodman, Myron F; Finkel, Steven E

    2013-06-01

    Escherichia coli DNA polymerases (Pol) II, IV, and V serve dual roles by facilitating efficient translesion DNA synthesis while simultaneously introducing genetic variation that can promote adaptive evolution. Here we show that these alternative polymerases are induced as cells transition from exponential to long-term stationary-phase growth in the absence of induction of the SOS regulon by external agents that damage DNA. By monitoring the relative fitness of isogenic mutant strains expressing only one alternative polymerase over time, spanning hours to weeks, we establish distinct growth phase-dependent hierarchies of polymerase mutant strain competitiveness. Pol II confers a significant physiological advantage by facilitating efficient replication and creating genetic diversity during periods of rapid growth. Pol IV and Pol V make the largest contributions to evolutionary fitness during long-term stationary phase. Consistent with their roles providing both a physiological and an adaptive advantage during stationary phase, the expression patterns of all three SOS polymerases change during the transition from log phase to long-term stationary phase. Compared to the alternative polymerases, Pol III transcription dominates during mid-exponential phase; however, its abundance decreases to <20% during long-term stationary phase. Pol IV transcription dominates as cells transition out of exponential phase into stationary phase and a burst of Pol V transcription is observed as cells transition from death phase to long-term stationary phase. These changes in alternative DNA polymerase transcription occur in the absence of SOS induction by exogenous agents and indicate that cell populations require appropriate expression of all three alternative DNA polymerases during exponential, stationary, and long-term stationary phases to attain optimal fitness and undergo adaptive evolution.

  7. A cDNA encoding RAP74, a general initiation factor for transcription by RNA polymerase II.

    Science.gov (United States)

    Finkelstein, A; Kostrub, C F; Li, J; Chavez, D P; Wang, B Q; Fang, S M; Greenblatt, J; Burton, Z F

    1992-01-30

    RAP30/74 (also known as TFIIF, beta gamma and FC is one of several general factors required for initiation by RNA polymerase II. The small RAP30 subunit of RAP30/74 binds directly to polymerase and appears structurally and functionally homologous to bacterial sigma factors in their RNA polymerase-binding region. RAP30/74 or recombinant RAP30 suppresses nonspecific binding of RNA polymerase II to DNA and is required for RNA polymerase II to assemble stably into a preinitiation complex containing promoter DNA and the general factors TFIID, TFIIA and TFIIB; both RAP30 and RAP74 are physical components of the preinitiation complex. A complementary DNA encoding human RAP30 has been isolated, and here we report the isolation of a cDNA encoding human RAP74. RAP30 and RAP74 produced in Escherichia coli can be used in place of natural human RAP30/74 to direct accurate transcription initiation by RNA polymerase II in vitro.

  8. Proofreading of DNA polymerase: a new kinetic model with higher-order terminal effects

    Science.gov (United States)

    Song, Yong-Shun; Shu, Yao-Gen; Zhou, Xin; Ou-Yang, Zhong-Can; Li, Ming

    2017-01-01

    The fidelity of DNA replication by DNA polymerase (DNAP) has long been an important issue in biology. While numerous experiments have revealed details of the molecular structure and working mechanism of DNAP which consists of both a polymerase site and an exonuclease (proofreading) site, there were quite a few theoretical studies on the fidelity issue. The first model which explicitly considered both sites was proposed in the 1970s and the basic idea was widely accepted by later models. However, all these models did not systematically investigate the dominant factor on DNAP fidelity, i.e. the higher-order terminal effects through which the polymerization pathway and the proofreading pathway coordinate to achieve high fidelity. In this paper, we propose a new and comprehensive kinetic model of DNAP based on some recent experimental observations, which includes previous models as special cases. We present a rigorous and unified treatment of the corresponding steady-state kinetic equations of any-order terminal effects, and derive analytical expressions for fidelity in terms of kinetic parameters under bio-relevant conditions. These expressions offer new insights on how the higher-order terminal effects contribute substantially to the fidelity in an order-by-order way, and also show that the polymerization-and-proofreading mechanism is dominated only by very few key parameters. We then apply these results to calculate the fidelity of some real DNAPs, which are in good agreements with previous intuitive estimates given by experimentalists.

  9. A parallel synthesis scheme for generating libraries of DNA polymerase substrates and inhibitors.

    Science.gov (United States)

    Strobel, Heike; Dugué, Laurence; Marlière, Philippe; Pochet, Sylvie

    2002-12-02

    We report a combinatorial approach aimed at producing in a single step a large family of nucleoside triphosphate derivatives that could be tested for their ability to be substrates for DNA polymerases. We propose as a unique triphosphate building block a nucleotide with a hydrazine function anchored to an imidazole ring. Condensation between the 5'-triphosphate derivative of 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH(2))TP) and any aldehyde or ketone, followed by reduction of the intermediate hydrazones dXmTP, resulted in the corresponding hydrazides (dXnTP). Following this scheme, a series of aldehydes having various aromatic parts yielded a number of adducts dY(NHR)TP. Vent (exo-) DNA polymerase is found to be able to catalyse the single incorporation of these bulky triphosphate derivatives. Subsequent extensions of the modified pairs with canonical triphosphates resulted mainly in abortive elongations at primer+2, except after the incorporation of dY(NHben)TP and, to a lesser extent, dY(NHphe)TP opposite C. These results illustrate the potential of this parallel synthetic scheme for generating new substrates or inhibitors of replication in a single step.

  10. DNA polymerase conformational dynamics and the role of fidelity-conferring residues: Insights from computational simulations

    Directory of Open Access Journals (Sweden)

    Massimiliano eMeli

    2016-05-01

    Full Text Available Herein we investigate the molecular bases of DNA polymerase I conformational dynamics that underlie the replication fidelity of the enzyme. Such fidelity is determined by conformational changes that promote the rejection of incorrect nucleotides before the chemical ligation step. We report a comprehensive atomic resolution study of wild type and mutant enzymes in different bound states and starting from different crystal structures, using extensive molecular dynamics (MD simulations that cover a total timespan of ~ 5 microseconds. The resulting trajectories are examined via a combination of novel methods of internal dynamics and energetics analysis, aimed to reveal the principal molecular determinants for the (destabilization of a certain conformational state. Our results show that the presence of fidelity-decreasing mutations or the binding of incorrect nucleotides in ternary complexes tend to favor transitions from closed towards open structures, passing through an ensemble of semi-closed intermediates. The latter ensemble includes the experimentally observed ajar conformation which, consistent with previous experimental observations, emerges as a molecular checkpoint for the selection of the correct nucleotide to incorporate. We discuss the implications of our results for the understanding of the relationships between the structure, dynamics and function of DNA polymerase I at the atomistic level.

  11. Conformational changes during nucleotide selection by Sulfolobus solfataricus DNA polymerase Dpo4.

    Science.gov (United States)

    Eoff, Robert L; Sanchez-Ponce, Raymundo; Guengerich, F Peter

    2009-07-31

    The mechanism of nucleotide selection by Y-family DNA polymerases has been the subject of intense study, but significant structural contacts and/or conformational changes that relate to polymerase fidelity have been difficult to identify. Here we report on the conformational dynamics of a model Y-family polymerase Dpo4 from Sulfolobus solfataricus. Hydrogen-deuterium exchange in tandem with mass spectrometry was used to monitor changes in Dpo4 structure as a function of time and the presence or absence of specific substrates and ligands. Analysis of the data revealed previously unrecognized structural changes that accompany steps in the catalytic cycle leading up to phosphoryl transfer. For example, the solvent accessibility of the alphaB-loop-alphaC region in the finger domain decreased in the presence of all four dNTP insertion events, but the rate of deuterium exchange, an indicator of conformational flexibility, only decreased during an accurate insertion event. Of particular note is a change in the region surrounding the H-helix of the thumb domain. Upon binding DNA and Mg2+, the H-helix showed a decrease in solvent accessibility and flexibility that was relaxed only upon addition of dCTP, which forms a Watson-Crick base pair with template dG and not during mispairing events. The current study expands upon a previous report from our group that used a fluorescent probe located near the thumb domain to measure the kinetic properties of Dpo4 conformational changes. We now present a model for nucleotide selection by Dpo4 that arises from a synthesis of both structural and kinetic data.

  12. A Label-Free, Sensitive, Real-Time, Semiquantitative Electrochemical Measurement Method for DNA Polymerase Amplification (ePCR).

    Science.gov (United States)

    Aydemir, Nihan; McArdle, Hazel; Patel, Selina; Whitford, Whitney; Evans, Clive W; Travas-Sejdic, Jadranka; Williams, David E

    2015-01-01

    Oligonucleotide hybridization to a complementary sequence that is covalently attached to an electrochemically active conducting polymer (ECP) coating the working electrode of an electrochemical cell causes an increase in reaction impedance for the ferro-ferricyanide redox couple. We demonstrate the use of this effect to measure, in real time, the progress of DNA polymerase chain reaction (PCR) amplification of a minor component of a DNA extract. The forward primer is attached to the ECP. The solution contains other PCR components and the redox couple. Each cycle of amplification gives an easily measurable impedance increase. Target concentration can be estimated by cycle count to reach a threshold impedance. As proof of principle, we demonstrate an electrochemical real-time quantitative PCR (e-PCR) measurement in the total DNA extracted from chicken blood of an 844 base pair region of the mitochondrial Cytochrome c oxidase gene, present at ∼1 ppm of total DNA. We show that the detection and semiquantitation of as few as 2 copies/μL of target can be achieved within less than 10 PCR cycles.

  13. The Miscoding Potential of 5-Hydroxycytosine Arises Due to Template Instability in the Replicative Polymerase Active Site

    Energy Technology Data Exchange (ETDEWEB)

    Zahn, Karl E.; Averill, April; Wallace, Susan S.; Doublié, Sylvie (Vermont)

    2012-07-18

    5-Hydroxycytosine (5-OHC) is a stable oxidation product of cytosine associated with an increased frequency of C {yields} T transition mutations. When this lesion escapes recognition by the base excision repair pathway and persists to serve as a templating base during DNA synthesis, replicative DNA polymerases often misincorporate dAMP at the primer terminus, which can lead to fixation of mutations and subsequent disease. To characterize the dynamics of DNA synthesis opposite 5-OHC, we initiated a comparison of unmodified dCMP to 5-OHC, 5-fluorocytosine (5-FC), and 5-methylcytosine (5-MEC) in which these bases act as templates in the active site of RB69 gp43, a high-fidelity DNA polymerase sharing homology with human replicative DNA polymerases. This study presents the first crystal structure of any DNA polymerase binding this physiologically important premutagenic DNA lesion, showing that while dGMP is stabilized by 5-OHC through normal Watson-Crick base pairing, incorporation of dAMP leads to unstacking and instability in the template. Furthermore, the electronegativity of the C5 substituent appears to be important in the miscoding potential of these cytosine-like templates. While dAMP is incorporated opposite 5-OHC {approx}5 times more efficiently than opposite unmodified dCMP, an elevated level of incorporation is also observed opposite 5-FC but not 5-MEC. Taken together, these data imply that the nonuniform templating by 5-OHC is due to weakened stacking capabilities, which allows dAMP incorporation to proceed in a manner similar to that observed opposite abasic sites.

  14. Quantitation of Genital Herpes Virus DNA by Polymerase Chain Reaction and ELISA

    Institute of Scientific and Technical Information of China (English)

    CHENG Peihua(程培华)

    2002-01-01

    Objective:To detect and quantitate genital herpes simplex virus (HSV) DNA in specimens from 100 patients clinically diagnosed with genital herpes.Methods: Polymerase Chain Reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used with a standard curve of DNA copies of HSV as quantitative contrast.Results: Ninety-three cases were confirmed HSV positive and 7 cases were found to be negative. There were 58 cases of HSV-2 (62.4%) and 35 cases of HSV-1 (37.6%) among the 93 positive cases. The number of DNA plasmids ranged from 115 to 1.1×105 per 250μL among the 93 positive samples (mean =7.1×104/250μL). The number of HSV DNA plasmids ranged from 136 to 1.1×105 copies per 250μL (mean =7.6×104) among those with HSV-2, and 115 to 9.4×104 per 250μL (mean =6.3×104) among those with HSV-1. Meanwhile 10μL of extracted and dissolved DNA randomly taken from 8 each of HSV-2 and HSV-1 samples were tested. The number of HSV-2 DNA plasmids ranged from 35 copies to 2.7×104 (Mean =1.8×104) and the number of HSV-1 DNA ranged from 29 to 2.5×104 (Mean = 1.6×104). In the 7 negative cases, the quantity of HSV plasmids was zero.Conclusion: The sensitivity of ELISA quantitation (93%) is equal to that of Southern blot. The sensitivity of PCR for diagnosis is 91%, and 88% for PCR typing.

  15. An aromatic residue switch in enhancer-dependent bacterial RNA polymerase controls transcription intermediate complex activity

    Science.gov (United States)

    Wiesler, Simone C.; Weinzierl, Robert O. J.; Buck, Martin

    2013-01-01

    The formation of the open promoter complex (RPo) in which the melted DNA containing the transcription start site is located at the RNA polymerase (RNAP) catalytic centre is an obligatory step in the transcription of DNA into RNA catalyzed by RNAP. In the RPo, an extensive network of interactions is established between DNA, RNAP and the σ-factor and the formation of functional RPo occurs via a series of transcriptional intermediates (collectively ‘RPi’). A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site. Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in σ54-regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo. Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation. PMID:23609536

  16. Comparison of DNA extraction methods for polymerase chain reaction amplification of guanaco (Lama guanicoe) fecal DNA samples.

    Science.gov (United States)

    Espinosa, M I; Bertin, A; Squeo, F A; Cortés, A; Gouin, N

    2015-01-23

    Feces-based population genetic studies have become increasingly popular. However, polymerase chain reaction (PCR) amplification rates from fecal material vary depending on the species, populations, loci, and extraction protocols. Here, we assessed the PCR amplification success of three microsatellite markers and a segment of the mitochondrial control region of DNA extracted from field-collected feces of guanaco (Lama guanicoe) using two protocols - Qiagen DNA Stool Kit and 2 cetyltrimethylammonium bromide/phenol:chloroform:isoamyl alcohol (2CTAB/PCI) method. Chelex resin treatment to remove inhibitors was also tested. Our results show that the mitochondrial locus was the most difficult to amplify. PCR success rates improved for all markers after Chelex treatment of extracted DNA, and 2CTAB/PCI method (95.83%) appeared to perform slightly better than stool kit (91.67%) for the nuclear markers. Amplification success was significantly influenced by the extraction method, Chelex treatment, and locus (P 0.89), but they decreased slightly after treatment for amplification of nuclear markers and markedly after treatment for amplification of the mitochondrial control region. Thus, we showed that Chelex treatment gives high PCR success, especially for nuclear markers, and adequate DNA extraction rates can be achieved from L. guanicoe feces even from non-fresh fecal material. Although not significant, 2CTAB/PCI method tended to provide higher successful amplification rates on a whole set of samples, suggesting that the method could be particularly useful when using small sample sizes.

  17. Helicase and Polymerase Move Together Close to the Fork Junction and Copy DNA in One-Nucleotide Steps

    Directory of Open Access Journals (Sweden)

    Manjula Pandey

    2014-03-01

    Full Text Available By simultaneously measuring DNA synthesis and dNTP hydrolysis, we show that T7 DNA polymerase and T7 gp4 helicase move in sync during leading-strand synthesis, taking one-nucleotide steps and hydrolyzing one dNTP per base-pair unwound/copied. The cooperative catalysis enables the helicase and polymerase to move at a uniformly fast rate without guanine:cytosine (GC dependency or idling with futile NTP hydrolysis. We show that the helicase and polymerase are located close to the replication fork junction. This architecture enables the polymerase to use its strand-displacement synthesis to increase the unwinding rate, whereas the helicase aids this process by translocating along single-stranded DNA and trapping the unwound bases. Thus, in contrast to the helicase-only unwinding model, our results suggest a model in which the helicase and polymerase are moving in one-nucleotide steps, DNA synthesis drives fork unwinding, and a role of the helicase is to trap the unwound bases and prevent DNA reannealing.

  18. Use of neuropathological tissue for molecular genetic studies: parameters affecting DNA extraction and polymerase chain reaction.

    Science.gov (United States)

    Kösel, S; Graeber, M B

    1994-01-01

    Nuclear and mitochondrial DNA were extracted from gray matter of human cerebral cortex which had either been formalin-fixed and embedded into paraffin or stored in formalin for up to 26 years. Extraction conditions were optimized for proteinase K digestion, i.e., enzyme concentration, digestion temperature and incubation time. Using the polymerase chain reaction (PCR), DNA was successfully amplified from archival material and sequenced employing a direct nonradioactive cycle sequencing protocol. In general, tissue embedded into paraffin following brief fixation in formalin gave good quantitative results, i.e., up to 1 microgram DNA/mg tissue were extracted. This yield was at least one order of magnitude higher than that obtained with tissue stored in formalin. However, paraffin-embedded neuropathological material was found to contain an as-yet-unidentified PCR inhibitor, and a deleterious effect of long-term fixation in unbuffered low-grade formalin was clearly detectable. Importantly, both paraffin-embedded tissue blocks and human brain that had been stored in formalin for many years yielded DNA sufficient for qualitative analysis. The implications of these findings for the use of neuropathological material in molecular genetic studies are discussed.

  19. RNA polymerase motors on DNA track: effects of traffic congestion on RNA synthesis

    CERN Document Server

    Tripathi, Tripti

    2007-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by a ssDNA. In some circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track. We refer to such collective movements of the RNAPs as RNAP traffic because of the similarities between the collective dynamics of the RNAPs on ssDNA track and that of vehicles in highway traffic. In this paper we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechano-chemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the ssDNA track. We also suggest novel experiments for testing o...

  20. Culture-Negative Endocarditis Diagnosed Using 16S DNA Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Stephen Duffett

    2012-01-01

    Full Text Available 16S DNA polymerase chain reaction (PCR is a molecular amplification technique that can be used to identify bacterial pathogens in culture-negative endocarditis. Bacterial DNA can be isolated from surgically excised valve tissue or from blood collected in EDTA vials. Use of this technique is particularly helpful in identifying the bacterial pathogen in cases of culture-negative endocarditis. A case involving a 48-year-old man who presented with severe aortic regurgitation and a four-month prodrome of low-grade fever is reported. Blood and valve tissue cultures following valve replacement were negative. A valve tissue sample was sent for investigation with 16S DNA PCR, which successfully identified Streptococcus salivarius and was interpreted as the true diagnosis. A review of the literature suggests that 16S DNA PCR from valve tissue is a more sensitive diagnostic test than culture. It is also extremely specific, based on a sequence match of at least 500 base pairs.

  1. Activation of Poly(ADP-Ribose) Polymerase by Myocardial Ischemia and Coronary Reperfusion in Human Circulating Leukocytes

    OpenAIRE

    Tóth-Zsámboki, Emese; Horváth, Eszter; Vargova, Katarina; Pankotai, Eszter; Murthy, Kanneganti; Zsengellér, Zsuzsanna; Bárány, Tamás; Pék, Tamás; Fekete, Katalin; Kiss, Róbert Gábor; Préda, István; Lacza, Zsombor; Gerö, Domokos; SzabÓ, Csaba

    2006-01-01

    Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 2...

  2. The main early and late promoters of Bacillus subtilis phage phi 29 form unstable open complexes with sigma A-RNA polymerase that are stabilized by DNA supercoiling.

    Science.gov (United States)

    Rojo, F; Nuez, B; Mencía, M; Salas, M

    1993-02-25

    Most Escherichia coli promoters studied so far form stable open complexes with sigma 70-RNA polymerase which have relatively long half-lives and, therefore, are resistant to a competitor challenge. A few exceptions are nevertheless known. The analysis of a number of promoters in Bacillus subtilis has suggested that the instability of open complexes formed by the vegetative sigma A-RNA polymerase may be a more general phenomenon than in Escherichia coli. We show that the main early and late promoters from the Bacillus subtilis phage phi 29 form unstable open complexes that are stabilized either by the formation of the first phosphodiester bond between the initiating nucleoside triphosphates or by DNA supercoiling. The functional characteristics of these two strong promoters suggest that they are not optimized for a tight and stable RNA polymerase binding. Their high activity is probably the consequence of the efficiency of further steps leading to the formation of an elongation complex.

  3. Mutating Asn-666 to Glu in the O-helix region of the taq DNA polymerase gene.

    Science.gov (United States)

    Sadeghi, H Mir Mohammad; Rajaei, R; Moazen, F; Rabbani, M; Jafarian-Dehkordi, A

    2010-01-01

    Taq DNA polymerase is widely used in laboratories and for this reason many investigators have focused their attention on understanding the role of various regions and amino acids in this enzyme. O-helix is a part of taq polymerase suggested to play an important role in the enzyme fidelity. The influence of Asn666 in this helix on the enzyme function has never been investigated, and therefore by using nested PCR, a portion of taq DNA polymerase gene containing Asn666Glu mutation was amplified. This DNA was digested with Eco RI restriction enzyme to confirm the presence of Asn666Glu mutation. After digesting this product and the wild type taq-pET-15b plasmid with NheI and BamHI restriction enzymes, they were ligated and used for the transformation of E. coli DH5α competent cells. The obtained colonies were screened for the presence of the mutated taq polymerase gene using EcoRI, NdeI and BamHI restriction enzymes. In conclusion, with the use of the obtained recombinant plasmid it is possible to study the role of this amino acid on taq DNA polymerase function.

  4. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV.

    Directory of Open Access Journals (Sweden)

    Julie A Law

    2011-07-01

    Full Text Available DNA methylation is an evolutionarily conserved epigenetic modification that is critical for gene silencing and the maintenance of genome integrity. In Arabidopsis thaliana, the de novo DNA methyltransferase, domains rearranged methyltransferase 2 (DRM2, is targeted to specific genomic loci by 24 nt small interfering RNAs (siRNAs through a pathway termed RNA-directed DNA methylation (RdDM. Biogenesis of the targeting siRNAs is thought to be initiated by the activity of the plant-specific RNA polymerase IV (Pol-IV. However, the mechanism through which Pol-IV is targeted to specific genomic loci and whether factors other than the core Pol-IV machinery are required for Pol-IV activity remain unknown. Through the affinity purification of nuclear RNA polymerase D1 (NRPD1, the largest subunit of the Pol-IV polymerase, we found that several previously identified RdDM components co-purify with Pol-IV, namely RNA-dependent RNA polymerase 2 (RDR2, CLASSY1 (CLSY1, and RNA-directed DNA methylation 4 (RDM4, suggesting that the upstream siRNA generating portion of the RdDM pathway may be more physically coupled than previously envisioned. A homeodomain protein, SAWADEE homeodomain homolog 1 (SHH1, was also found to co-purify with NRPD1; and we demonstrate that SHH1 is required for de novo and maintenance DNA methylation, as well as for the accumulation of siRNAs at specific loci, confirming it is a bonafide component of the RdDM pathway.

  5. Effect of SOS-induced Pol II, Pol IV, and Pol V DNA polymerases on UV-induced mutagenesis and MFD repair in Escherichia coli cells.

    Science.gov (United States)

    Wrzesiński, Michał; Nowosielska, Anetta; Nieminuszczy, Jadwiga; Grzesiuk, Elzbieta

    2005-01-01

    Irradiation of organisms with UV light produces genotoxic and mutagenic lesions in DNA. Replication through these lesions (translesion DNA synthesis, TSL) in Escherichia coli requires polymerase V (Pol V) and polymerase III (Pol III) holoenzyme. However, some evidence indicates that in the absence of Pol V, and with Pol III inactivated in its proofreading activity by the mutD5 mutation, efficient TSL takes place. The aim of this work was to estimate the involvement of SOS-inducible DNA polymerases, Pol II, Pol IV and Pol V, in UV mutagenesis and in mutation frequency decline (MFD), a mechanism of repair of UV-induced damage to DNA under conditions of arrested protein synthesis. Using the argE3-->Arg(+) reversion to prototrophy system in E. coli AB1157, we found that the umuDC-encoded Pol V is the only SOS-inducible polymerase required for UV mutagenesis, since in its absence the level of Arg(+) revertants is extremely low and independent of Pol II and/or Pol IV. The low level of UV-induced Arg(+) revertants observed in the AB1157mutD5DumuDC strain indicates that under conditions of disturbed proofreading activity of Pol III and lack of Pol V, UV-induced lesions are bypassed without inducing mutations. The presented results also indicate that Pol V may provide substrates for MFD repair; moreover, we suggest that only those DNA lesions which result from umuDC-directed UV mutagenesis are subject to MFD repair.

  6. A Major Role of DNA Polymerase δ in Replication of Both the Leading and Lagging DNA Strands.

    Science.gov (United States)

    Johnson, Robert E; Klassen, Roland; Prakash, Louise; Prakash, Satya

    2015-07-16

    Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M-generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. A major role of DNA polymerase δ in replication of both the leading and lagging DNA strands

    Science.gov (United States)

    Prakash, Louise; Prakash, Satya

    2015-01-01

    SUMMARY Genetic studies with S. cerevisiae Polδ (pol3-L612M) and Polε (pol2-M644G) mutant alleles, each of which display a higher rate for the generation of a specific mismatch, have led to the conclusion that Polε is the primary leading strand replicase and that Polδ is restricted to replicating the lagging strand template. Contrary to this widely accepted view, here we show that Polδ plays a major role in the replication of both DNA strands, and that the paucity of pol3-L612M generated errors on the leading strand results from their more proficient removal. Thus, the apparent lack of Polδ contribution to leading strand replication is due to differential mismatch removal rather than differential mismatch generation. Altogether, our genetic studies with Pol3 and Pol2 mutator alleles support the conclusion that Polδ, and not Polε, is the major DNA polymerase for carrying out both leading and lagging DNA synthesis. PMID:26145172

  8. Nested polymerase chain reaction (PCR) targeting 16S rDNA for bacterial identification in empyema.

    Science.gov (United States)

    Prasad, Rajniti; Kumari, Chhaya; Das, B K; Nath, Gopal

    2014-05-01

    Empyema in children causes significant morbidity and mortality. However, identification of organisms is a major concern. To detect bacterial pathogens in pus specimens of children with empyema by 16S rDNA nested polymerase chain reaction (PCR) and correlate it with culture and sensitivity. Sixty-six children admitted to the paediatric ward with a diagnosis of empyema were enrolled prospectively. Aspirated pus was subjected to cytochemical examination, culture and sensitivity, and nested PCR targeting 16S rDNA using a universal eubacterial primer. Mean (SD) age was 5·8 (1·8) years (range 1-13). Analysis of aspirated pus demonstrated total leucocyte count >1000×10(6)/L, elevated protein (≧20 g/L) and decreased glucose (≤2·2 mmol/L) in 80·3%, 98·5% and 100%, respectively. Gram-positive cocci were detected in 29 (43·9%) and Gram-negative bacilli in two patients. Nested PCR for the presence of bacterial pathogens was positive in 50·0%, compared with 36·3% for culture. 16S rDNA PCR improves rates of detection of bacteria in pleural fluid, and can detect bacterial species in a single assay as well as identifying unusual and unexpected causal agents.

  9. Insights into the error bypass of 1-Nitropyrene DNA adduct by DNA polymerase ι: A QM/MM study

    Science.gov (United States)

    Li, Yanwei; Bao, Lei; Zhang, Ruiming; Tang, Xiaowen; Zhang, Qingzhu; Wang, Wenxing

    2017-10-01

    The error bypass mechanism of DNA polymerase ι toward N-(deoxyguanosin-8-yl)-1-aminopyrene adduction was studied by using quantum mechanics/molecular mechanics method. The most favorable mechanism highlights three elementary steps: proton transfer from dC to dATP, phosphoryl transfer, and deprotonation of dAMP. The phosphoryl transfer step was found to be rate-determining. The calculated average barrier (23.8 kcal mol-1) is in accordance with the experimental value (21.5 kcal mol-1). Electrostatic influence analysis indicates that residues Asp126 and Lys207 significantly suppress the error bypass while Glu127 facilitates the process. These results highlight the origins of the mutagenicity of nitrated polycyclic aromatic hydrocarbons in molecular detail.

  10. A Micro Polymerase Chain Reaction Module for Integrated and Portable DNA Analysis Systems

    Directory of Open Access Journals (Sweden)

    Elisa Morganti

    2011-01-01

    Full Text Available This work deals with the design, fabrication, and thermal characterization of a disposable miniaturized Polymerase Chain Reaction (PCR module that will be integrated in a portable and fast DNA analysis system. It is composed of two independent parts: a silicon substrate with embedded heater and thermometers and a PDMS (PolyDiMethylSiloxane chamber reactor as disposable element; the contact between the two parts is assured by a mechanical clamping obtained using a Plastic Leaded Chip Carrier (PLCC. This PLCC is also useful, avoid the PCR mix evaporation during the thermal cycles. Finite Element Analysis was used to evaluate the thermal requirements of the device. The thermal behaviour of the device was characterized revealing that the temperature can be controlled with a precision of ±0.5°C. Different concentrations of carbon nanopowder were mixed to the PDMS curing agent in order to increase the PDMS thermal conductivity and so the temperature control accuracy.

  11. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    Science.gov (United States)

    Langston, Lance D; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E; Finkelstein, Jeff; Yao, Nina Y; Indiani, Chiara; O'Donnell, Mike E

    2014-10-28

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.

  12. 重组Taq DNA聚合酶的表达和纯化鉴定%Expression, Purification and Identification of Recombinant Taq DNA Polymerase

    Institute of Scientific and Technical Information of China (English)

    刘钦松; 刘孟刚; 张丛丛; 张程; 许彤

    2012-01-01

    [目的] 表达、纯化Taq DNA聚合酶,并检测其扩增性能.[方法] 活化含Taq DNA聚合酶基因的菌株JM109,IPTG诱导表达.目的蛋白利用AKTA蛋白纯化系统,依次过Affi - Gel Blue Sepharose,Heparin Sepharnse Fast Flow,Q - Sepharose Fast Flow,经SDS - PAGE分析,以国外进口Taq酶为标准,采用对比法初略测定酶活性,验证产品质量.[结果] 制备的Taq DNA聚合酶具有扩增效率高、纯度高、特异性强、无核酸酶污染、活性稳定等优点.[结论] 成功表达纯化Taq DNA聚合酶,其扩增性能达到甚至超过国外同类产品.%In order to express and purify Taq DNA polymerase, and detect its amplification performance, E. coli strain JM109 containing the gene of Taq DNA polymerase was activated, and the gene was expressed under the inducement of IPTG. The expressed protein was purified by AKTA protein purification system through passing Affi - Gel Blue Sepharose, Heparin Sepharose Fast Flow and Q - Sepharose Fast Flow in turn- Taking Taq enzyme imported from abroad as the standard, the enzyme activity was roughly determined through antitheses and SDS - PAGE analysis, and the product quality was verified finally. The results revealed that the prepared Taq DNA polymerase had the following good characteristics: high amplification efficiency, high purity, strong specificity, no nuclease pollution and stable activity. In conclusion, the recombinant Taq DNA polymerase was successfully expressed and purified, and its amplification performance could reach or exceed that of the similar products imported from abroad.

  13. A broadly applicable method to characterize large DNA viruses and adenoviruses based on the DNA polymerase gene

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    Montgomery Roy D

    2006-04-01

    Full Text Available Abstract Background Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. Results We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus from cell culture and lymphocystis disease virus (iridovirus and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1. The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. Conclusion The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified

  14. Synthesis of deoxynucleoside triphosphates that include proline, urea, or sulfonamide groups and their polymerase incorporation into DNA.

    Science.gov (United States)

    Hollenstein, Marcel

    2012-10-15

    To expand the chemical array available for DNA sequences in the context of in vitro selection, I present herein the synthesis of five nucleoside triphosphate analogues containing side chains capable of organocatalysis. The synthesis involved the coupling of L-proline-containing residues (dU(tP)TP and dU(cP)TP), a dipeptide (dU(FP)TP), a urea derivative (dU(Bpu)TP), and a sulfamide residue (dU(Bs)TP) to a suitably protected common intermediate, followed by triphosphorylation. These modified dNTPs were shown to be excellent substrates for the Vent (exo(-)) and Pwo DNA polymerases, as well as the Klenow fragment of E. coli DNA polymerase I, although they were only acceptable substrates for the 9°N(m) polymerase. All of the modified dNTPs, with the exception of dU(Bpu)TP, were readily incorporated into DNA by the polymerase chain reaction (PCR). Modified oligonucleotides efficiently served as templates for PCR for the regeneration of unmodified DNA. Thermal denaturation experiments showed that these modifications are tolerated in the major groove. Overall, these heavily modified dNTPs are excellent candidates for SELEX.

  15. Comparative molecular dynamics studies of heterozygous open reading frames of DNA polymerase eta (η) in pathogenic yeast Candida albicans

    Science.gov (United States)

    Satpati, Suresh; Manohar, Kodavati; Acharya, Narottam; Dixit, Anshuman

    2017-01-01

    Genomic instability in Candida albicans is believed to play a crucial role in fungal pathogenesis. DNA polymerases contribute significantly to stability of any genome. Although Candida Genome database predicts presence of S. cerevisiae DNA polymerase orthologs; functional and structural characterizations of Candida DNA polymerases are still unexplored. DNA polymerase eta (Polη) is unique as it promotes efficient bypass of cyclobutane pyrimidine dimers. Interestingly, C. albicans is heterozygous in carrying two Polη genes and the nucleotide substitutions were found only in the ORFs. As allelic differences often result in functional differences of the encoded proteins, comparative analyses of structural models and molecular dynamic simulations were performed to characterize these orthologs of DNA Polη. Overall structures of both the ORFs remain conserved except subtle differences in the palm and PAD domains. The complementation analysis showed that both the ORFs equally suppressed UV sensitivity of yeast rad30 deletion strain. Our study has predicted two novel molecular interactions, a highly conserved molecular tetrad of salt bridges and a series of π-π interactions spanning from thumb to PAD. This study suggests these ORFs as the homologues of yeast Polη, and due to its heterogeneity in C. albicans they may play a significant role in pathogenicity.

  16. Replication bypass of the trans-4-Hydroxynonenal-derived (6S,8R,11S)-1,N(2)-deoxyguanosine DNA adduct by the sulfolobus solfataricus DNA polymerase IV.

    Science.gov (United States)

    Banerjee, Surajit; Christov, Plamen P; Kozekova, Albena; Rizzo, Carmelo J; Egli, Martin; Stone, Michael P

    2012-02-20

    trans-4-Hydroxynonenal (HNE) is the major peroxidation product of ω-6 polyunsaturated fatty acids in vivo. Michael addition of the N(2)-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N(2)-dGuo (1,N(2)-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N(2)-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N(2)-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua → Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N(2)-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N(2)-dGuo adduct, the (6S,8R,11S)-1,N(2)-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either d

  17. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

    Science.gov (United States)

    Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

    2016-01-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  18. RNA Polymerase II Regulates Topoisomerase 1 Activity to Favor Efficient Transcription.

    Science.gov (United States)

    Baranello, Laura; Wojtowicz, Damian; Cui, Kairong; Devaiah, Ballachanda N; Chung, Hye-Jung; Chan-Salis, Ka Yim; Guha, Rajarshi; Wilson, Kelli; Zhang, Xiaohu; Zhang, Hongliang; Piotrowski, Jason; Thomas, Craig J; Singer, Dinah S; Pugh, B Franklin; Pommier, Yves; Przytycka, Teresa M; Kouzine, Fedor; Lewis, Brian A; Zhao, Keji; Levens, David

    2016-04-01

    We report a mechanism through which the transcription machinery directly controls topoisomerase 1 (TOP1) activity to adjust DNA topology throughout the transcription cycle. By comparing TOP1 occupancy using chromatin immunoprecipitation sequencing (ChIP-seq) versus TOP1 activity using topoisomerase 1 sequencing (TOP1-seq), a method reported here to map catalytically engaged TOP1, TOP1 bound at promoters was discovered to become fully active only after pause-release. This transition coupled the phosphorylation of the carboxyl-terminal-domain (CTD) of RNA polymerase II (RNAPII) with stimulation of TOP1 above its basal rate, enhancing its processivity. TOP1 stimulation is strongly dependent on the kinase activity of BRD4, a protein that phosphorylates Ser2-CTD and regulates RNAPII pause-release. Thus the coordinated action of BRD4 and TOP1 overcame the torsional stress opposing transcription as RNAPII commenced elongation but preserved negative supercoiling that assists promoter melting at start sites. This nexus between transcription and DNA topology promises to elicit new strategies to intercept pathological gene expression.

  19. A Role for DNA Polymerase μ in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo▿ †

    Science.gov (United States)

    Gozalbo-López, Beatriz; Andrade, Paula; Terrados, Gloria; de Andrés, Belén; Serrano, Natalia; Cortegano, Isabel; Palacios, Beatriz; Bernad, Antonio; Blanco, Luis; Marcos, Miguel A. R.; Gaspar, María Luisa

    2009-01-01

    The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJH joints and compare them with those present in later life. The embryo DJH joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase μ (Polμ), which was widely expressed in the early ontogeny, as shown by analysis of Polμ−/− embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polμ also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polμ participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development. PMID:19103746

  20. Identification of histidine residue in active center of Escherichia coli RNA-polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Grachev, M.A.; Mustaev, A.A.; Kolocheva, T.I.

    1985-03-01

    Affinity labeling with beta-imidazole GDP, DNA phage T7 promotor, and alpha-/sup 32/P-UTP were used to mark the histidine residue in the active site of E. coli RNA-polymerase. Mild acid treatment led to complete demodification, while kinetic demodification with dodecylsulfate corresponded to that obtained with model modified histidine, and differed sharply from kinetic hydrolysis of a phosphamide bond. The histidine residue was identified by degrading the labeled peptide with cyanogen bromide and measuring the length of the radioactive peptides obtained by SDS disc-electrophoresis. This indicated that the labeled histidine was between methionines 1107 and 1119. The only histidine in this location is number 1116. This method for partial protein sequencing has general applicability. 10 references, 3 figures.

  1. Variations of Human DNA Polymerase Genes as Biomarkers of Prostate Cancer Progression

    Science.gov (United States)

    2011-07-01

    analysis was done by single - strand conformation polymorphism , a technique that can miss muta- tions [Pearce et al., 2008]. We recently sequenced the complete...7) mice exhibit increased single - stranded DNA breaks, chromosomal aberrations, and mutagenicity compared to normal animals [Cabelof et al., 2003...a single -nucleotide gapped DNA template (the natural pol b substrate), and that this interaction is essential for pol b conformational activation

  2. Genotyping the hepatitis B virus with a fragment of the HBV DNA polymerase gene in Shenyang, China

    Directory of Open Access Journals (Sweden)

    Juan Feng

    2011-06-01

    Full Text Available Abstract The hepatitis B virus (HBV has been classified into eight genotypes (A-H based on intergenotypic divergence of at least 8% in the complete nucleotide sequence or more than 4% in the S gene. To facilitate the investigation of the relationship between the efficacy of drug treatment and the mutation with specific genotype of HBV, we have established a new genotyping strategy based on a fragment of the HBV DNA polymerase gene. Pairwise sequence and phylogenetic analyses were performed using CLUSTAL V (DNASTAR on the eight (A-H standard full-length nucleotide sequences of HBV DNA from GenBank (NCBI and the corresponding semi-nested PCR products from the HBV DNA polymerase gene. The differences in the semi-nested PCR fragments of the polymerase genes among genotypes A through F were greater than 4%, which is consistent with the intergenotypic divergence of at least 4% in HBV DNA S gene sequences. Genotyping using the semi-nested PCR products of the DNA polymerase genes revealed that only genotypes B, C, and D were present in the 50 cases, from Shenyang, China, with a distribution of 11 cases (22%, 25 cases (50%, and 14 cases (28% respectively. These results demonstrate that our new genotyping method utilizing a fragment of the HBV DNA polymerase gene is valid and can be employed as a general genotyping strategy in areas with prevalent HBV genotypes A through F. In Shenyang, China, genotypes C, B, and D were identified with this new genotyping method, and genotype C was demonstrated to be the dominant genotype.

  3. A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

    Directory of Open Access Journals (Sweden)

    Veloso IF

    2000-01-01

    Full Text Available Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870 followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2 leptospira, using the silver stain procedure.

  4. A power-efficient thermocycler based on induction heating for DNA amplification by polymerase chain reaction

    Science.gov (United States)

    Pal, Debjani; Venkataraman, V.; Mohan, K. Naga; Chandra, H. Sharat; Natarajan, Vasant

    2004-09-01

    We have built a thermocycler based on the principles of induction heating for polymerase chain reaction (PCR) of target sequences in DNA samples of interest. The cycler has an average heating rate of ˜0.8 °C/s and a cooling rate of ˜0.5 °C/s, and typically takes ˜4 h to complete a 40-cycle PCR protocol. It is power-efficient (˜6 W per reaction tube), micro-processor controlled, and can be adapted for battery operation. Using this instrument, we have successfully amplified a 350 bp segment from a plasmid and SRY, the human sex determining gene, which occurs as a single-copy sequence in genomic DNA of human males. The PCR products from this thermocycler are comparable to those obtained by the use of commercially available machines. Its easy front-end operation, low-power design, portability and low cost makes it suitable for diagnostic field applications of PCR.

  5. Modulation of Pleurodeles waltl DNA polymerase mu expression by extreme conditions encountered during spaceflight.

    Directory of Open Access Journals (Sweden)

    Véronique Schenten

    Full Text Available DNA polymerase µ is involved in DNA repair, V(DJ recombination and likely somatic hypermutation of immunoglobulin genes. Our previous studies demonstrated that spaceflight conditions affect immunoglobulin gene expression and somatic hypermutation frequency. Consequently, we questioned whether Polμ expression could also be affected. To address this question, we characterized Polμ of the Iberian ribbed newt Pleurodeles waltl and exposed embryos of that species to spaceflight conditions or to environmental modifications corresponding to those encountered in the International Space Station. We noted a robust expression of Polμ mRNA during early ontogenesis and in the testis, suggesting that Polμ is involved in genomic stability. Full-length Polμ transcripts are 8-9 times more abundant in P. waltl than in humans and mice, thereby providing an explanation for the somatic hypermutation predilection of G and C bases in amphibians. Polμ transcription decreases after 10 days of development in space and radiation seem primarily involved in this down-regulation. However, space radiation, alone or in combination with a perturbation of the circadian rhythm, did not affect Polμ protein levels and did not induce protein oxidation, showing the limited impact of radiation encountered during a 10-day stay in the International Space Station.

  6. Detection of Mycobacterium tuberculosis DNA in clinical samples by using a simple lysis method and polymerase chain reaction.

    Science.gov (United States)

    Folgueira, L; Delgado, R; Palenque, E; Noriega, A R

    1993-01-01

    We have evaluated the polymerase chain reaction for detection of Mycobacterium tuberculosis in clinical samples from patients with tuberculous infection. Two simple methods for mycobacterial DNA release have been compared: sonication and lysis with nonionic detergents and proteinase K. The more effective method was the enzymatic technique. By using this protocol with 75 specimens we detected M. tuberculosis DNA in all of the samples, whereas only 48 and 71 samples were positive by acid-fast staining and culture, respectively. Images PMID:8463383

  7. Activation and modulation of cardiac poly-adenosine diphosphate ribose polymerase activity in a rat model of brain death.

    Science.gov (United States)

    Brain, John G; Rostron, Anthony J; Dark, John H; Kirby, John A

    2008-05-15

    DNA damage during transplantation can activate poly-adenosine diphosphate ribose polymerase (PARP) resulting in the generation of polymers of adenosine diphosphate-ribose (PAR). Excessive linkage of PAR to nuclear proteins can induce cell death, thereby limiting the function of transplanted organs. This study uses a rat model of brain death to determine the profile of PARP activation and whether mechanisms that lead to cell death can be ameliorated by appropriate donor resuscitation. The expression of PAR-linked nuclear proteins within cardiac myocytes was greatly increased after the induction of donor brain death. Importantly, infusion of noradrenaline or vasopressin to normalize the chronic hypotension produced by brain death reduced the expression of PAR to a level below baseline. These data suggest that chronic hypotension after donor brain death has the potential to limit cardiac function through the activation of PARP; however, this early cause of graft damage can be mitigated by appropriate donor resuscitation.

  8. Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA Polymerase-η.

    Science.gov (United States)

    Genna, Vito; Gaspari, Roberto; Dal Peraro, Matteo; De Vivo, Marco

    2016-04-07

    Trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), are essential for cell survival. Pol-η bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-η function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-η catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-η for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases.

  9. Comparison of specific binding sites for Escherichia coli RNA polymerase with naturally occurring hairpin regions in single-stranded DNA of coliphage M13. [Aspergillus oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Niyogi, S.K.; Mitra, S.

    1978-08-25

    Escherichia coli RNA polymerase binds specifically to the single-stranded circular DNA of coliphage M13 in the presence of a saturating concentration of the bacterial DNA binding protein presumably as an essential step in the synthesis of the RNA primer required for synthesizing the complementary DNA strand in parental replicative-form DNA. The RNA polymerase-protected DNA regions were isolated after extensive digestion with pancreatic DNase, S1 endonuclease of Aspergillus oryzae, and exonuclease I of E. coli. The physicochemical properties of the RNA polymerase-protected segments (called PI and PII) were compared with those of the naturally occurring hairpin regions.

  10. Extraction of DNA from exfoliative cytology specimens and its suitability for analysis by the polymerase chain reaction.

    Science.gov (United States)

    Jackson, D P; Payne, J; Bell, S; Lewis, F A; Taylor, G R; Peel, K R; Sutton, J; Quirke, P

    1990-01-01

    The extraction of DNA from archival exfoliative cytology samples would allow the molecular biological analysis of this readily available material using the polymerase chain reaction (PCR). We have quantitatively and qualitatively studied the extraction of DNA from a variety of cytological preparations. For both fresh and archival cervical smears, overnight incubation with proteinase K produces high yields of high molecular weight DNA, but simply boiling the samples produces DNA suitable for PCR amplification of a single copy gene. Increasing the proteinase K incubation to several days allows the extraction of DNA from fixed and stained archival cytology slides from a variety of sites. The extracted DNA was again suitable for PCR analysis. Fresh and archival cytological material can be utilized for molecular biological study of disease processes using PCR. Archival cytological material is probably the best source of DNA and RNA after stored frozen tissue.

  11. The DNA/RNA-dependent RNA polymerase QDE-1 generates aberrant RNA and dsRNA for RNAi in a process requiring replication protein A and a DNA helicase.

    Directory of Open Access Journals (Sweden)

    Heng-Chi Lee

    Full Text Available The production of aberrant RNA (aRNA is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs to generate double-stranded RNA (dsRNA is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP. Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA, a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways.

  12. Nucleotide sequence of the DNA polymerase gene of herpes simplex virus type 2 and comparison with the type 1 counterpart.

    Science.gov (United States)

    Tsurumi, T; Maeno, K; Nishiyama, Y

    1987-01-01

    The complete nucleotide sequence of the DNA polymerase gene of herpes simplex virus (HSV) type 2 strain 186 has been determined. The gene included a 3720-bp major open reading frame capable of encoding 1240 amino acids. The predicted primary translation product had an Mr of 137,354, which was slightly larger than its HSV-1 counterpart. A comparison of the predicted functional amino acid sequences of the HSV-1 and HSV-2 DNA polymerases revealed 95.5% overall amino acid homology, the value of which was the highest among those of the other known polypeptides encoded by HSV-1 and HSV-2. The functional amino acid changes were spread in the N-terminal one-third of the protein, whereas the C-terminal two-third was almost identical between the two types except a particular hydrophilic region. A highly conserved sequence of 6 aa, YGDTDS, which has been observed in DNA polymerases of HSV-1, Epstein-Barr virus, adenovirus, and vaccinia virus, was also present at positions 889 to 894 in the C-terminal region of HSV-2 DNA polymerase.

  13. Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase.

    Science.gov (United States)

    Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek; Dewhurst, Stephen

    2012-10-01

    Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.

  14. Problem-Solving Test: Real-Time Polymerase Chain Reaction

    Science.gov (United States)

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: polymerase chain reaction, DNA amplification, electrophoresis, breast cancer, "HER2" gene, genomic DNA, "in vitro" DNA synthesis, template, primer, Taq polymerase, 5[prime][right arrow]3[prime] elongation activity, 5[prime][right arrow]3[prime] exonuclease activity, deoxyribonucleoside…

  15. Biochemical analysis of DNA polymerase η fidelity in the presence of replication protein A.

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    Samuel C Suarez

    Full Text Available DNA polymerase η (pol η synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS, is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA, when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD. We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.

  16. Structure and mechanism of error-free replication past the major benzo[a]pyrene adduct by human DNA polymerase κ.

    Science.gov (United States)

    Jha, Vikash; Bian, Chuanbing; Xing, Guangxin; Ling, Hong

    2016-06-02

    Benzo[a]pyrene (BP) is a well-known and frequently encountered carcinogen which generates a bulky DNA adduct (+)-trans-10S-BP-N(2)-dG (BP-dG) in cells. DNA polymerase kappa (polκ) is the only known Y-family polymerase that bypasses BP-dG accurately and thus protects cells from genotoxic BP. Here, we report the structures of human polκ in complex with DNA containing either a normal guanine (G) base or a BP-dG adduct at the active site and a correct deoxycytidine. The structures and supporting biochemical data reveal a unique mechanism for accurate replication by translesion synthesis past the major bulky adduct. The active site of polκ opens at the minor groove side of the DNA substrate to accommodate the bulky BP-dG that is attached there. More importantly, polκ stabilizes the lesion DNA substrate in the same active conformation as for regular B-form DNA substrates and the bulky BPDE ring in a 5' end pointing conformation. The BP-dG adducted DNA substrate maintains a Watson-Crick (BP-dG:dC) base pair within the active site, governing correct nucleotide insertion opposite the bulky adduct. In addition, polκ's unique N-clasp domain supports the open conformation of the enzyme and the extended conformation of the single-stranded template to allow bypass of the bulky lesion. This work illustrates the first molecular mechanism for how a bulky major adduct is replicated accurately without strand misalignment and mis-insertion.

  17. Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

    Science.gov (United States)

    Lee, Ai-Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

    2008-12-15

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

  18. Characterization of soluble RNA-dependent RNA polymerase from dengue virus serotype 2: The polyhistidine tag compromises the polymerase activity.

    Science.gov (United States)

    Kamkaew, Maliwan; Chimnaronk, Sarin

    2015-08-01

    The viral RNA polymerase is an attractive target for inhibition in the treatment of viral infections. In the case of dengue virus (DENV), a member of the genus Flavivirus, the RNA-dependent RNA polymerase (RdRp) activity resides in the C-terminal two-thirds of non-structural protein (NS) 5 responsible for the de novo synthesis of the viral RNA genome. Among four distinct, but closely related dengue serotypes, serotype 2 (DENV-2) produces more severe diseases than other serotypes. It has been reported that bacterial production of the recombinant DENV-2 RdRp was difficult due to its low expression and solubility levels. To facilitate functional and structural analyses, we here demonstrate complete protocols for overexpression and purification of soluble DENV-2 RdRp, increasing protein yields by a remarkable 10 times compared to earlier reports. Three different forms of DENV-2 RdRp as either N- or C-terminally His-tagged fusions, or without tag, were purified to homogeneity. We show here that the presence of both the N- and C-terminal His-tag had a deleterious effect on polymerase activity and, in contrast to earlier studies, our non-tagged RdRp did not require manganese ions to activate RNA polymerization. We also determined an apparent Kd value of 53nM for binding to the 5'-UTR RNA by surface plasmon resonance (SPR). Our work provide a more suitable material for basic research of viral RdRp and for drug development.

  19. DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Alden C Klarer

    Full Text Available Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta, is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE, the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.

  20. Cell proliferation in Allium cepa L. meristems under 8-hydroxyquinoline, a chelating agent that affects DNA and RNA polymerases.

    Science.gov (United States)

    Ferrero, M L; De la Torre, C

    1986-02-01

    8-Hydroxyquinoline (HQ) chelates Mg2+ and Mn2+ and, secondarily, affects the activities of DNA and RNA polymerases. The in vivo effect of HQ has been estimated in Allium cepa L. meristems growing under new growth kinetics in the presence of this agent. HQ (at both 5 X 10(-5) M and 10(-4) M) depressed incorporation of [3H]uridine much more effectively than that of [3H]-thymidine. Cycle kinetics in meristems behaved as if they were independent of the rates of synthesis or accumulation of RNA since, under HQ, cycle time was only moderately modified and the new cycle kinetics achieved could be explained by the new rates of [3H]thymidine incorporation. Lengthened S periods were partially compensated for by shortened G2 phases, suggesting that, in these cells, both the growth cycle and its coupling with the DNA-division cycle were not disturbed by a decreased amount of RNA. Finally, the nucleolar cycle during mitosis, but not the interphase nucleolus, was modified under the new rates of RNA synthesis.

  1. Characterization of family IV UDG from Aeropyrum pernix and its application in hot-start PCR by family B DNA polymerase.

    Directory of Open Access Journals (Sweden)

    Xi-Peng Liu

    Full Text Available Recombinant uracil-DNA glycosylase (UDG from Aeropyrum pernix (A. pernix was expressed in E. coli. The biochemical characteristics of A. pernix UDG (ApeUDG were studied using oligonucleotides carrying a deoxyuracil (dU base. The optimal temperature range and pH value for dU removal by ApeUDG were 55-65°C and pH 9.0, respectively. The removal of dU was inhibited by the divalent ions of Zn, Cu, Co, Ni, and Mn, as well as a high concentration of NaCl. The opposite base in the complementary strand affected the dU removal by ApeUDG as follows: U/C≈U/G>U/T≈U/AP≈U/->U/U≈U/I>U/A. The phosphorothioate around dU strongly inhibited dU removal by ApeUDG. Based on the above biochemical characteristics and the conservation of amino acid residues, ApeUDG was determined to belong to the IV UDG family. ApeUDG increased the yield of PCR by Pfu DNA polymerase via the removal of dU in amplified DNA. Using the dU-carrying oligonucleotide as an inhibitor and ApeUDG as an activator of Pfu DNA polymerase, the yield of undesired DNA fragments, such as primer-dimer, was significantly decreased, and the yield of the PCR target fragment was increased. This strategy, which aims to amplify the target gene with high specificity and yield, can be applied to all family B DNA polymerases.

  2. The Y-Family DNA Polymerase Dpo4 Uses a Template Slippage Mechanism To Create Single-Base Deletions

    Energy Technology Data Exchange (ETDEWEB)

    Y Wu; R Wilson; J Pata

    2011-12-31

    The Y-family polymerases help cells tolerate DNA damage by performing translesion synthesis, yet they also can be highly error prone. One distinctive feature of the DinB class of Y-family polymerases is that they make single-base deletion errors at high frequencies in repetitive sequences, especially those that contain two or more identical pyrimidines with a 5? flanking guanosine. Intriguingly, different deletion mechanisms have been proposed, even for two archaeal DinB polymerases that share 54% sequence identity and originate from two strains of Sulfolobus. To reconcile these apparent differences, we have characterized Dpo4 from Sulfolobus solfataricus using the same biochemical and crystallographic approaches that we have used previously to characterize Dbh from Sulfolobus acidocaldarius. In contrast to previous suggestions that Dpo4 uses a deoxynucleoside triphosphate (dNTP)-stabilized misalignment mechanism when creating single-base deletions, we find that Dpo4 predominantly uses a template slippage deletion mechanism when replicating repetitive DNA sequences, as was previously shown for Dbh. Dpo4 stabilizes the skipped template base in an extrahelical conformation between the polymerase and the little-finger domains of the enzyme. This contrasts with Dbh, in which the extrahelical base is stabilized against the surface of the little-finger domain alone. Thus, despite sharing a common deletion mechanism, these closely related polymerases use different contacts with the substrate to accomplish the same result.

  3. NanoPCR observation: different levels of DNA replication fidelity in nanoparticle-enhanced polymerase chain reactions

    Science.gov (United States)

    Shen, Cenchao; Yang, Wenjuan; Ji, Qiaoli; Maki, Hisaji; Dong, Anjie; Zhang, Zhizhou

    2009-11-01

    Nanoparticle-assisted PCR (polymerase chain reaction) technology is getting more and more attention recently. It is believed that some of the DNA recombinant technologies will be upgraded by nanotechnology in the near future, among which DNA replication is one of the core manipulation techniques. So whether or not the DNA replication fidelity is compromised in nanoparticle-assisted PCR is a question. In this study, a total of 16 different metallic and non-metallic nanoparticles (NPs) were tested for their effects on DNA replication fidelity in vitro and in vivo. Sixteen types of nanomaterials were distinctly different in enhancing the PCR efficiency, and their relative capacity to retain DNA replication fidelity was largely different from each other based on rpsL gene mutation assay. Generally speaking, metallic nanoparticles induced larger error rates in DNA replication fidelity than non-metallic nanoparticles, and non-metallic nanomaterials such as carbon nanopowder or nanotubes were still safe as PCR enhancers because they did not compromise the DNA replication fidelity in the Taq DNA polymerase-based PCR system.

  4. INCURVATA2 Encodes the Catalytic Subunit of DNA Polymerase α and Interacts with Genes Involved in Chromatin-Mediated Cellular Memory in Arabidopsis thaliana

    Science.gov (United States)

    Barrero, José María; González-Bayón, Rebeca; del Pozo, Juan Carlos; Ponce, María Rosa; Micol, José Luis

    2007-01-01

    Cell type–specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase α of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity. PMID:17873092

  5. INCURVATA2 encodes the catalytic subunit of DNA Polymerase alpha and interacts with genes involved in chromatin-mediated cellular memory in Arabidopsis thaliana.

    Science.gov (United States)

    Barrero, José María; González-Bayón, Rebeca; del Pozo, Juan Carlos; Ponce, María Rosa; Micol, José Luis

    2007-09-01

    Cell type-specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase alpha of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity.

  6. The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation*

    Science.gov (United States)

    Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F.

    2015-01-01

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. PMID:25814667

  7. The roles of family B and D DNA polymerases in Thermococcus species 9°N Okazaki fragment maturation.

    Science.gov (United States)

    Greenough, Lucia; Kelman, Zvi; Gardner, Andrew F

    2015-05-15

    During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Cost-effective optimization of real-time PCR based detection of Campylobacter and Salmonella with inhibitor tolerant DNA polymerases

    DEFF Research Database (Denmark)

    Fachmann, Mette Sofie Rousing; Josefsen, Mathilde Hasseldam; Hoorfar, Jeffrey

    2015-01-01

    PCR master mix performed best for both meat and fecal samples (LODs of 102 and 104 CFU ml-1 in the purest and crudest DNA extractions, respectively) compared with Tth (LOD=102 -103 and 105 -106 CFU ml-1 ). AmpliTaqGold and HotMasterTaq both performed well (LOD=102 -104 CFU ml-1 ) with meat samples and poorly......AIMS: The aim of this study was to cost-effectively improve detection of foodborne pathogens in PCR inhibitory samples through the use of alternative DNA polymerases. METHODS AND RESULTS: Commercially available polymerases (n=16) and PCR master mixes (n=4) were screened on DNA purified from...... bacterial cells in two validated real-time PCR assays for Campylobacter and Salmonella. The five best performing (based on: limit of detection (LOD), maximum fluorescence, shape of amplification curves, and amplification efficiency) were subsequently applied to meat and fecal samples. The VeriQuest q...

  9. Mutations of mitochondrial DNA polymerase gammaA are a frequent cause of autosomal dominant or recessive progressive external ophthalmoplegia.

    Science.gov (United States)

    Lamantea, Eleonora; Tiranti, Valeria; Bordoni, Andreina; Toscano, Antonio; Bono, Francesco; Servidei, Serena; Papadimitriou, Alex; Spelbrink, Hans; Silvestri, Laura; Casari, Giorgio; Comi, Giacomo P; Zeviani, Massimo

    2002-08-01

    One form of familial progressive external ophthalmoplegia with multiple mitochondrial DNA deletions recently has been associated with mutations in POLG1, the gene encoding pol gammaA, the catalytic subunit of mitochondrial DNA polymerase. We screened the POLG1 gene in several PEO families and identified five different heterozygous missense mutations of POLG1 in 10 autosomal dominant families. Recessive mutations were found in three families. Our data show that mutations of POLG1 are the most frequent cause of familial progressive external ophthalmoplegia associated with accumulation of multiple mitochondrial DNA deletions, accounting for approximately 45% of our family cohort.

  10. Two modes of interaction of the single-stranded DNA-binding protein of bacteriophage T7 with the DNA polymerase-thioredoxin complex

    KAUST Repository

    Ghosh, Sharmistha

    2010-04-06

    The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD.gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. DNA Polymerases η and ζ Combine to Bypass O(2)-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens.

    Science.gov (United States)

    Gowda, A S Prakasha; Spratt, Thomas E

    2016-03-21

    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are important human carcinogens in tobacco products. They are metabolized to produce a variety 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts including O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT), the most abundant POB adduct in NNK- and NNN-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past O(2)-POB-dT and O(2)-Me-dT by purified human DNA polymerases η, κ, ι, and yeast polymerase ζ in vitro. Under conditions of polymerase in excess, polymerase η was most effective at the insertion of dNTPs opposite O(2)-alkyl-dTs. The time courses were biphasic suggesting the formation of inactive DNA-polymerase complexes. The kpol parameter was reduced approximately 100-fold in the presence of the adduct for pol η, κ, and ι. Pol η was the most reactive polymerase for the adducts due to a higher burst amplitude. For all three polymerases, the nucleotide preference was dATP > dTTP ≫ dGTP and dCTP. Yeast pol ζ was most effective in bypassing the adducts; the kcat/Km values were reduced only 3-fold in the presence of the adducts. The identity of the nucleotide opposite the O(2)-alkyl-dT did not significantly affect the ability of pol ζ to bypass the adducts. The data support a model in which pol η inserts ATP or dTTP opposite O(2)-POB-dT, and then, pol ζ extends past the adduct.

  12. Domain Structures and Inter-Domain Interactions Defining the Holoenzyme Architecture of Archaeal D-Family DNA Polymerase

    OpenAIRE

    Hideshi Yokoyama; Kazuhiko Yamasaki; Ikuo Matsui; Eriko Matsui

    2013-01-01

    Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large sub...

  13. Uniform Free-Energy Profiles of the P-O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ.

    Science.gov (United States)

    Klvaňa, Martin; Bren, Urban; Florián, Jan

    2016-12-29

    Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3'-OH group of the primer DNA strand, and the subsequent formation and cleavage of P-O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol(-1) for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P-O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pKa of the 3'-OH group as a predictor of the catalytic rate of X-family DNA polymerases.

  14. Different applications of polymerases with and without proofreading activity in single-nucleotide polymorphism analysis.

    Science.gov (United States)

    Zhang, Jia; Li, Kai; Liao, Duanfang; Pardinas, Jose R; Chen, Linling; Zhang, Xu

    2003-08-01

    With the completion of the human genome project, single-nucleotide polymorphisms (SNPs) have become the focus of intense study in biomedical research. Polymerase-mediated primer extension has been employed in a variety of SNP assays. However, these SNP assays using polymerase without proofreading function are compromised by their low reliability. Using a newly developed short amplicon harboring restriction enzyme site, EcoR-I, we were able to compare the single-base discrimination abilities of polymerases with and without proofreading function in primer extension in a broad range of annealing temperatures. Thermodynamic analysis demonstrated a striking single-nucleotide discrimination ability of polymerases with proofreading function. Using unmodified 3'-end allele-specific primers, only template-dependent products were generated by polymerase with proofreading activity. This powerful single-base discrimination ability of exo(+) polymerases was further evaluated in primer extension using three types of 3' terminally modified allele-specific primers. As compared with the poor fidelity in primer extension of polymerases lacking 3' exonuclease activity, this study provides convincing evidence that the use of proofreading polymerases in combination with 3'-end modified allele-specific primers can be a powerful new strategy for the development of SNP assays.

  15. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

    Science.gov (United States)

    Langston, Lance D.; Zhang, Dan; Yurieva, Olga; Georgescu, Roxana E.; Finkelstein, Jeff; Yao, Nina Y.; Indiani, Chiara; O’Donnell, Mike E.

    2014-01-01

    DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol α/primase initiates primers on both strands that are extended by Pol ε on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG–Pol ε complex and showed that it is a functional polymerase–helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes. PMID:25313033

  16. Structures of an apo and a binary complex of an evolved archeal B family DNA polymerase capable of synthesising highly cy-dye labelled DNA.

    Directory of Open Access Journals (Sweden)

    Samantha A Wynne

    Full Text Available Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10 of Pyrococcus furiosus (Pfu polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP in PCR and synthesise highly fluorescent "CyDNA" densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers.

  17. Analysis ulcerative colitis for presence Epstein-Barr virus DNA sequences by polymerase chain reaction technique

    Directory of Open Access Journals (Sweden)

    Sahar Mehrabani khasraghi

    2016-02-01

    Full Text Available Introduction: Ulcerative colitis (UC is one type of inflammatory bowel disease (IBD. The purpose of this study is to explore the prevalence of Epstein–Barr virus (EBV in UC patients in comparison with healthy subjects using the polymerase chain reaction (PCR method. Methods: In this case-control study, five biopsies of patients with UC and 30 healthy people as controls were selected. Sampling was performed by endoscopic biopsy operation. After DNA extraction, PCR was used to determine EBV genome by specific primers. Statistical analysis was performed using the chi-square test. Results: The results of PCR indicated that EBV genome was detected in 60.0% of samples in the case group, and 36.7% of samples in the control group were positive for EBV. Thus, no significant association was observed between the prevalence of EBV and incidence of UC in comparison with the control group (P = 0.36. Conclusion: The findings presented herein demonstrate no direct molecular evidence to support an association of EBV with UC. These results, do not exclude the possibility oncogenic role of EBV to infect the different colon cell.

  18. Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase δ

    Directory of Open Access Journals (Sweden)

    Yuh Shiwa

    2012-01-01

    Full Text Available A novel mutagenesis technique using error-prone DNA polymerase δ (polδ, the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of the polδMKII mutator, which expresses the proofreading-deficient and low-fidelity polδ, in Saccharomyces cerevisiae haploid strain with that of the commonly used chemical mutagen ethyl methanesulfonate (EMS. This mutator strain possesses exogenous mutant polδ supplied from a plasmid, tthereby leaving the genomic one intact. We measured the mutation rate achieved by each mutagen and performed high-throughput next generation sequencing to analyze the genome-wide mutation spectra produced by the 2 mutagenesis methods. The mutation frequency of the mutator was approximately 7 times higher than that of EMS. Our analysis confirmed the strong G/C to A/T transition bias of EMS, whereas we found that the mutator mainly produces transversions, giving rise to more diverse amino acid substitution patterns. Our present study demonstrated that the polδMKII mutator is a useful and efficient method for rapid strain improvement based on in vivo mutagenesis.

  19. Cattle fetal sex determination by polymerase chain reaction using DNA isolated from maternal plasma.

    Science.gov (United States)

    da Cruz, A S; Silva, D C; Costa, E O A; De M-Jr, P; da Silva, C C; Silva, D M; da Cruz, A D

    2012-03-01

    The objective of this study was to evaluate the use of polymerase chain reaction analysis (PCR) of fetal cells/DNA in the maternal plasma of pregnant cows to determine the sex of the fetus. Plasma was harvested from 35 cows of mixed genotype at different stages of pregnancy ranging from 5 to 35 weeks. A male calf and a heifer calf provided the control samples. Fetal sex was determined by amplification of Y-specific sequences. For the 35 cows, the fetal sex predicted by this technique was in accordance with the sex of the calf at birth in 88.6% of cases. The agreement between predicted and observed fetal sex was less for cows with a gestational length of 35-48 days (63.6%). Regression analysis showed that there was a strong relationship between the probability of correctly predicting fetal sex and the stage of gestation. It was estimated that the test performed at 43.8 days post fertilization would have 95% accuracy, increasing to 99% accuracy for testing at 48.4 days and 99.9% accuracy for tests at 55.0 days or later. It was concluded that PCR analysis of fetal cells in maternal plasma can be used to predict successfully the sex of the fetus in cattle.

  20. Interaction of 2'-deoxyguanosine triphosphate analogue inhibitors of HIV reverse transcriptase with human mitochondrial DNA polymerase gamma.

    Science.gov (United States)

    Ray, Adrian S; Feng, Joy Y; Murakami, Eisuke; Chu, Chung K; Schinazi, Raymond F; Anderson, Karen S

    2007-01-01

    Mitochondrial toxicity is a limiting factor in the use of some nucleoside reverse transcriptase inhibitors of HIV. To further understand the impact of structural features on the incorporation and exonuclease removal of nucleoside monophosphate (MP) analogues by human mitochondrial DNA polymerase (pol gamma), transient kinetic studies were done with analogues of 2'-deoxyguanosine triphosphate. The kinetic parameters for the incorporation and removal of carbovir (CBV)-MP, dioxolane guanosine (DXG)-MP and 2',3'-dideoxy-2',3'-didehydroguanosine (d4G)-MP were studied with pol gamma holoenzyme. The importance of the ribose oxygen in incorporation by pol gamma was illustrated by an approximate 3,000-fold decrease in the incorporation efficiency of an analogue lacking the ribose oxygen (CBV-TP) relative to those containing a ribose oxygen (DXG-TP and d4G-TP). As a result, a comparison with previous data for the incorporation by HIV reverse transcriptase showed CBV-TP to be approximately 800-8,000-fold more selective for its antiviral target over pol gamma relative to the other guanosine analogues. However, DXG-TP and d4G-TP were found to be much more selective than previously reported values for mitochondrial toxic nucleoside analogues. Structural modelling based on sequence homology with other polymerase A family members suggests that an interaction between the ribose oxygen and arginine 853 in pol gamma may play a critical role in causing this differential incorporation. Exonuclease removal of a chain-terminating CBV-MP was also found to be more efficient by pol gamma. These results help to further elucidate the structure activity relationships for pol gamma and should aid in the design of more selective antiviral agents.

  1. A DNA-Based Encryption Method Based on Two Biological Axioms of DNA Chip and Polymerase Chain Reaction (PCR) Amplification Techniques.

    Science.gov (United States)

    Zhang, Yunpeng; Wang, Zhiwen; Wang, Zhenzhen; Liu, Xin; Yuan, Xiaojing

    2017-09-27

    Researchers have gained a deeper understanding of DNA-based encryption and its effectiveness in enhancing information security in recent years. However, there are many theoretical and technical issues about DNA-based encryption that need to be addressed before it can be effectively used in the field of security. Currently, the most popular DNA-based encryption schemes are based on traditional cryptography and the integration of existing DNA technology. These schemes are not completely based on DNA computing and biotechnology. Herein, as inspired by nature, encryption based on DNA has been developed, which is, in turn, based on two fundamental biological axioms about DNA sequencing: 1) DNA sequencing is difficult under the conditions of not knowing the correct sequencing primers and probes, and 2) without knowing the correct probe, it is difficult to decipher precisely and sequence the information of unknown and mixed DNA/peptide nucleic acid (PNA) probes, which only differ in nucleotide sequence, arranged on DNA chips (microarrays). In essence, when creating DNA-based encryption by means of biological technologies, such as DNA chips and polymerase chain reaction (PCR) amplification, the encryption method discussed herein cannot be decrypted, unless the DNA/PNA probe or PCR amplification is known. The biological analysis, mathematical analysis, and simulation results demonstrate the feasibility of the method, which provides much stronger security and reliability than that of traditional encryption methods. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Catalytic effects of mutations of distant protein residues in human DNA polymerase β: theory and experiment.

    Science.gov (United States)

    Klvaňa, Martin; Murphy, Drew L; Jeřábek, Petr; Goodman, Myron F; Warshel, Arieh; Sweasy, Joann B; Florián, Jan

    2012-11-06

    We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase β (Pol β). Since the mutated residues in the point mutants, I174S, I260Q, M282L, H285D, E288K, and K289M, were not located in the Pol β catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition-state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate. On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Ω = (k(pol)/K(D))(mut)/(k(pol)/K(D))(WT) using free-energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences. We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol β using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Ω values. The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Ω values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Ω values. These scaled FEP and FEP/LIE methods were also used to predict log Ω for R283A and R283L Pol

  3. Association Between Single Nucleotide Polymorphisms in DNA Polymerase Kappa Gene and Breast Cancer Risk in Chinese Han Population

    Science.gov (United States)

    Dai, Zhi-Jun; Liu, Xing-Han; Ma, Yun-Feng; Kang, Hua-Feng; Jin, Tian-Bo; Dai, Zhi-Ming; Guan, Hai-Tao; Wang, Meng; Liu, Kang; Dai, Cong; Yang, Xue-Wen; Wang, Xi-Jing

    2016-01-01

    Abstract DNA polymerases are responsible for ensuring stability of the genome and avoiding genotoxicity caused by a variety of factors during DNA replication. Consequently, these proteins have been associated with an increased cancer risk. DNA polymerase kappa (POLK) is a specialized DNA polymerase involved in translesion DNA synthesis (TLS) that allows DNA synthesis over the damaged DNA. Recently, some studies investigated relationships between POLK polymorphisms and cancer risk, but the role of POLK genetic variants in breast cancer (BC) remains to be defined. In this study, we aimed to evaluate the effects of POLK polymorphisms on BC risk. We used the Sequenom MassARRAY method to genotype 3 single nucleotide polymorphisms (SNPs) in POLK (rs3213801, rs10077427, and rs5744533), in order to determine the genotypes of 560 BC patients and 583 controls. The association of genotypes and BC was assessed by computing the odds ratio (OR) and 95% confidence intervals (95% CIs) from logistic regression analyses. We found a statistically significant difference between patient and control groups in the POLK rs10077427 genotypic groups, excluding the recessive model. A positive correlation was also found between positive progesterone receptor (PR) status, higher Ki67 index, and rs10077427 polymorphism. For rs5744533 polymorphism, the codominant, dominant, and allele models frequencies were significantly higher in BC patients compared to healthy controls. Furthermore, our results indicated that rs5744533 SNP has a protective role in the postmenopausal women. However, we failed to find any associations between rs3213801 polymorphism and susceptibility to BC. Our results indicate that POLK polymorphisms may influence the risk of developing BC, and, because of this, may serve as a prognostic biomarker among Chinese women. PMID:26765445

  4. Structural coupling between RNA polymerase composition and DNA supercoiling in coordinating transcription: a global role for the omega subunit?

    Science.gov (United States)

    Geertz, Marcel; Travers, Andrew; Mehandziska, Sanja; Sobetzko, Patrick; Chandra-Janga, Sarath; Shimamoto, Nobuo; Muskhelishvili, Georgi

    2011-01-01

    In growing bacterial cells, the global reorganization of transcription is associated with alterations of RNA polymerase composition and the superhelical density of the DNA. However, the existence of any regulatory device coordinating these changes remains elusive. Here we show that in an exponentially growing Escherichia coli rpoZ mutant lacking the polymerase ω subunit, the impact of the Eσ(38) holoenzyme on transcription is enhanced in parallel with overall DNA relaxation. Conversely, overproduction of σ(70) in an rpoZ mutant increases both overall DNA supercoiling and the transcription of genes utilizing high negative superhelicity. We further show that transcription driven by the Eσ(38) and Eσ(70) holoenzymes from cognate promoters induces distinct superhelical densities of plasmid DNA in vivo. We thus demonstrate a tight coupling between polymerase holoenzyme composition and the supercoiling regimen of genomic transcription. Accordingly, we identify functional clusters of genes with distinct σ factor and supercoiling preferences arranging alternative transcription programs sustaining bacterial exponential growth. We propose that structural coupling between DNA topology and holoenzyme composition provides a basic regulatory device for coordinating genome-wide transcription during bacterial growth and adaptation. IMPORTANCE Understanding the mechanisms of coordinated gene expression is pivotal for developing knowledge-based approaches to manipulating bacterial physiology, which is a problem of central importance for applications of biotechnology and medicine. This study explores the relationships between variations in the composition of the transcription machinery and chromosomal DNA topology and suggests a tight interdependence of these two variables as the major coordinating principle of gene regulation. The proposed structural coupling between the transcription machinery and DNA topology has evolutionary implications and suggests a new methodology for

  5. Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE).

    Science.gov (United States)

    Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Watanabe, Kousuke; Emoto, Noriko; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2011-07-01

    Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.

  6. Docking of anti-HIV-1 oxoquinoline-acylhydrazone derivatives as potential HSV-1 DNA polymerase inhibitors

    Science.gov (United States)

    Yoneda, Julliane Diniz; Albuquerque, Magaly Girão; Leal, Kátia Zaccur; Santos, Fernanda da Costa; Batalha, Pedro Netto; Brozeguini, Leonardo; Seidl, Peter R.; de Alencastro, Ricardo Bicca; Cunha, Anna Cláudia; de Souza, Maria Cecília B. V.; Ferreira, Vitor F.; Giongo, Viveca A.; Cirne-Santos, Cláudio; Paixão, Izabel C. P.

    2014-09-01

    Although there are many antiviral drugs available for the treatment of herpes simplex virus (HSV) infections, still the synthesis of new anti-HSV candidates is an important strategy to be pursued, due to the emergency of resistant HSV strains mainly in human immunodeficiency virus (HIV) co-infected patients. Some 1,4-dihydro-4-oxoquinolines, such as PNU-183792 (1), show a broad spectrum antiviral activity against human herpes viruses, inhibiting the viral DNA polymerase (POL) without affecting the human POLs. Thus, on an ongoing antiviral research project, our group has synthesized ribonucleosides containing the 1,4-dihydro-4-oxoquinoline (quinolone) heterocyclic moiety, such as the 6-Cl derivative (2), which is a dual antiviral agent (HSV-1 and HIV-1). Molecular dynamics simulations of the complexes of 1 and 2 with the HSV-1 POL suggest that structural modifications of 2 should increase its experimental anti-HSV-1 activity, since its ribosyl and carboxyl groups are highly hydrophilic to interact with a hydrophobic pocket of this enzyme. Therefore, in this work, comparative molecular docking simulations of 1 and three new synthesized oxoquinoline-acylhydrazone HIV-1 inhibitors (3-5), which do not contain those hydrophilic groups, were carried out, in order to access these modifications in the proposition of new potential anti-HSV-1 agents, but maintaining the anti-HIV-1 activity. Among the docked compounds, the oxoquinoline-acylhydrazone 3 is the best candidate for an anti-HSV-1 agent, and, in addition, it showed anti-HIV-1 activity (EC50 = 3.4 ± 0.3 μM). Compounds 2 and 3 were used as templates in the design of four new oxoquinoline-acylhydrazones (6-9) as potential anti-HSV-1 agents to increase the antiviral activity of 2. Among the docked compounds, oxoquinoline-acylhydrazone 7 was selected as the best candidate for further development of dual anti-HIV/HSV activity.

  7. Biological response to ionizing radiation in mouse embryo fibroblasts with a targeted disruption of the DNA polymerase beta gene.

    Science.gov (United States)

    Miura, M; Watanabe, H; Okochi, K; Sasaki, T; Shibuya, H

    2000-06-01

    Base excision repair (BER) is carried out by two distinct pathways in mammalian cells, one dependent on DNA polymerase beta (Polb) and the other on proliferating cell nuclear antigen (Pcna). We studied whether the Polb-dependent pathway plays an important role in BER in vivo after exposure to ionizing radiation. For this purpose, we used mouse embryo fibroblasts derived from wild-type and Polb gene knockout littermates. Both cell lines had essentially the same clonogenic cell survival and low levels of apoptosis as determined by a colony formation assay and by a change in mitochondrial membrane potential, respectively. No significant cleavage of protein kinase C delta (Pkcd) in vivo, which is a substrate for caspase 3, was detected, and intact Pkcd was retained in both cell lines for at least 72 h after irradiation. Similar significant increases in caspase 3-like activities as measured by Asp-Glu-Val-Asp (DEVD) cleaving activity in vitro were observed in both cell lines after irradiation. Radiation induced cell cycle arrest in the form of a G(2)-phase block, and G(2)/M-phase fractions reached a peak approximately 10 h after irradiation and decreased thereafter with a similar time course in both cell lines. Similar levels of chromatin-bound Pcna were observed immediately after irradiation in non-S-phase cells of both cell lines and disappeared by 4 h after irradiation. We conclude that the deficiency in Polb does not have a significant influence on the radiation responses of these cells. Together with evidence accumulated in vitro, these results strongly support the idea that the Pcna-dependent pathway predominantly acts in BER of radiation-induced DNA damage in vivo.

  8. Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase δ revealed in individual cells by cytometry.

    Science.gov (United States)

    Darzynkiewicz, Zbigniew; Zhao, Hong; Zhang, Sufang; Lee, Marietta Y W T; Lee, Ernest Y C; Zhang, Zhongtao

    2015-05-20

    During our recent studies on mechanism of the regulation of human DNA polymerase δ in preparation for DNA replication or repair, multiparameter imaging cytometry as exemplified by laser scanning cytometry (LSC) has been used to assess changes in expression of the following nuclear proteins associated with initiation of DNA replication: cyclin A, PCNA, Ki-67, p21(WAF1), DNA replication factor Cdt1 and the smallest subunit of DNA polymerase δ, p12. In the present review, rather than focusing on Pol δ, we emphasize the application of LSC in these studies and outline possibilities offered by the concurrent differential analysis of DNA replication in conjunction with expression of the nuclear proteins. A more extensive analysis of the data on a correlation between rates of EdU incorporation, likely reporting DNA replication, and expression of these proteins, is presently provided. New data, specifically on the expression of cyclin D1 and cyclin E with respect to EdU incorporation as well as on a relationship between expression of cyclin A vs. p21(WAF1) and Ki-67 vs. Cdt1, are also reported. Of particular interest is the observation that this approach makes it possible to assess the temporal sequence of degradation of cyclin D1, p21(WAF1), Cdt1 and p12, each with respect to initiation of DNA replication and with respect to each other. Also the sequence or reappearance of these proteins in G2 after termination of DNA replication is assessed. The reviewed data provide a more comprehensive presentation of potential markers, whose presence or absence marks the DNA replicating cells. Discussed is also usefulness of these markers as indicators of proliferative activity in cancer tissues that may bear information on tumor progression and have a prognostic value.

  9. Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

    Institute of Scientific and Technical Information of China (English)

    XU Chun-hai; LI Zhao-shen; DAI Jun-ying; ZHU Hai-yang; YU Jian-wu; L(U) Shu-Ian

    2011-01-01

    peripheral blood mononuclear cells; marrow mononuclear cells Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).Methods Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.Results The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0x102 copies/ml to 3.9x108 copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.Conclusion The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

  10. HeLa Cells Containing a Truncated Form of DNA Polymerase Beta are More Sensitized to Alkylating Agents than to Agents Inducing Oxidative Stress.

    Science.gov (United States)

    Khanra, Kalyani; Chakraborty, Anindita; Bhattacharyya, Nandan

    2015-01-01

    The present study was aimed at determining the effects of alkylating and oxidative stress inducing agents on a newly identified variant of DNA polymerase beta (polβ Δ208-304) specific for ovarian cancer. Pol β Δ208-304 has a deletion of exons 11-13 which lie in the catalytic part of enzyme. We compared the effect of these chemicals on HeLa cells and HeLa cells stably transfected with this variant cloned into in pcDNAI/neo vector by MTT, colony forming and apoptosis assays. Polβ Δ208-304 cells exhibited greater sensitivity to an alkylating agent and less sensitivity towards H2O2 and UV when compared with HeLa cells alone. It has been shown that cell death in Pol β Δ208-304 transfected HeLa cells is mediated by the caspase 9 cascade. Exon 11 has nucleotidyl selection activity, while exons 12 and 13 have dNTP selection activity. Hence deletion of this part may affect polymerizing activity although single strand binding and double strand binding activity may remain same. The lack of this part may adversely affect catalytic activity of DNA polymerase beta so that the variant may act as a dominant negative mutant. This would represent clinical significance if translated into a clinical setting because resistance to radiation or chemotherapy during the relapse of the disease could be potentially overcome by this approach.

  11. Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

    OpenAIRE

    Meier, A.; Persing, D. H.; Finken, M; Böttger, E C

    1993-01-01

    Analysis based on comparisons of 16S rRNA sequences provides a rapid and reliable approach to identifying human pathogens. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, bacterial 16S ribosomal DNA sequences of virtually any member of the eubacterial kingdom can be amplified by polymerase chain reaction and subsequently analyzed by sequence determination. Indeed, automated systems for broad-range amplification, sequencing, and data analysis are...

  12. DNA polymerase ɛ, acetylases and remodellers cooperate to form a specialized chromatin structure at a tRNA insulator

    OpenAIRE

    Dhillon, Namrita; Raab, Jesse; Guzzo, Julie; Szyjka, Shawn J.; Gangadharan, Sunil; Aparicio, Oscar M.; Andrews, Brenda; Kamakaka, Rohinton T.

    2009-01-01

    Insulators bind transcription factors and use chromatin remodellers and modifiers to mediate insulation. In this report, we identified proteins required for the efficient formation and maintenance of a specialized chromatin structure at the yeast tRNA insulator. The histone acetylases, SAS-I and NuA4, functioned in insulation, independently of tRNA and did not participate in the formation of the hypersensitive site at the tRNA. In contrast, DNA polymerase ɛ, functioned with the chromatin remo...

  13. Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity in HER2 overexpressing breast cancer.

    Science.gov (United States)

    García-Parra, Jetzabel; Dalmases, Alba; Morancho, Beatriz; Arpí, Oriol; Menendez, Silvia; Sabbaghi, MohammadA; Zazo, Sandra; Chamizo, Cristina; Madoz, Juan; Eroles, Pilar; Servitja, Sonia; Tusquets, Ignasi; Yelamos, Jose; Lluch, Ana; Arribas, Joaquin; Rojo, Federico; Rovira, Ana; Albanell, Joan

    2014-10-01

    Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Cytonuclear conflict in interpopulation hybrids: the role of RNA polymerase in mtDNA transcription and replication.

    Science.gov (United States)

    Ellison, C K; Burton, R S

    2010-03-01

    Organismal fitness requires functional integration of nuclear and mitochondrial genomes. Structural and regulatory elements coevolve within lineages and several studies have found that interpopulation hybridization disrupts mitonuclear interactions. Because mitochondrial RNA polymerase (mtRPOL) plays key roles in both mitochondrial DNA (mtDNA) replication and transcription, the interaction between mtRPOL and coevolved regulatory sites in the mtDNA may be central to mitonuclear integration. Here, we generate interpopulation hybrids between divergent populations of the copepod Tigriopus californicus to obtain lines having different combinations of mtRPOL and mtDNA. Lines were scored for mtDNA copy number and ATP6 (mtDNA) gene expression. We find that there is a genotype-dependent negative association between mitochondrial transcriptional response and mtDNA copy number. We argue that an observed increase in mtDNA copy number and reduced mtDNA transcription in hybrids reflects the regulatory role of mtRPOL; depending on the mitonuclear genotype, hybridization may disrupt the normal balance between transcription and replication of the mitochondrial genome.

  15. Viruses Infecting a Freshwater Filamentous Cyanobacterium (Nostoc sp.) Encode a Functional CRISPR Array and a Proteobacterial DNA Polymerase B.

    Science.gov (United States)

    Chénard, Caroline; Wirth, Jennifer F; Suttle, Curtis A

    2016-06-14

    Here we present the first genomic characterization of viruses infecting Nostoc, a genus of ecologically important cyanobacteria that are widespread in freshwater. Cyanophages A-1 and N-1 were isolated in the 1970s and infect Nostoc sp. strain PCC 7210 but remained genomically uncharacterized. Their 68,304- and 64,960-bp genomes are strikingly different from those of other sequenced cyanophages. Many putative genes that code for proteins with known functions are similar to those found in filamentous cyanobacteria, showing a long evolutionary history in their host. Cyanophage N-1 encodes a CRISPR array that is transcribed during infection and is similar to the DR5 family of CRISPRs commonly found in cyanobacteria. The presence of a host-related CRISPR array in a cyanophage suggests that the phage can transfer the CRISPR among related cyanobacteria and thereby provide resistance to infection with competing phages. Both viruses also encode a distinct DNA polymerase B that is closely related to those found in plasmids of Cyanothece sp. strain PCC 7424, Nostoc sp. strain PCC 7120, and Anabaena variabilis ATCC 29413. These polymerases form a distinct evolutionary group that is more closely related to DNA polymerases of proteobacteria than to those of other viruses. This suggests that the polymerase was acquired from a proteobacterium by an ancestral virus and transferred to the cyanobacterial plasmid. Many other open reading frames are similar to a prophage-like element in the genome of Nostoc sp. strain PCC 7524. The Nostoc cyanophages reveal a history of gene transfers between filamentous cyanobacteria and their viruses that have helped to forge the evolutionary trajectory of this previously unrecognized group of phages. Filamentous cyanobacteria belonging to the genus Nostoc are widespread and ecologically important in freshwater, yet little is known about the genomic content of their viruses. Here we report the first genomic analysis of cyanophages infecting

  16. A simplified arthropod genomic-DNA extraction protocol for polymerase chain reaction (PCR)-based specimen identification through barcoding.

    Science.gov (United States)

    Margam, Venu M; Gachomo, Emma W; Shukle, John H; Ariyo, Oluwole O; Seufferheld, Manfredo J; Kotchoni, Simeon O

    2010-10-01

    Genomic DNA extraction protocols generally require the use of expensive and hazardous reagents necessary for decontamination of phenolic compounds from the extracts. In addition, they are lengthy, hindering large-scale sample extractions necessary for high-throughput analyses. Here we describe a simple, time and cost-efficient method for genomic DNA extraction from insects. The extracted DNA was successfully used in a Polymerase Chain Reaction (PCR), making it suitable for automation for large-scale genetic analysis and barcoding studies. The protocol employs a single purification step to remove polysaccharides and other contaminating compounds using a non-hazardous reagent buffer. In addition, we conducted a bioinformatics database analysis as proof of concept for the efficiency of the DNA extraction protocol by using universal barcoding primers specific for cytochrome c oxidase I gene to identify different arthropod specimens through Barcode of Life Database (B