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Sample records for specific dna polymerased

  1. Synthetic Nucleotides as Probes of DNA Polymerase Specificity

    PubMed Central

    Walsh, Jason M.; Beuning, Penny J.

    2012-01-01

    The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example, Escherichia coli DNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides. PMID:22720133

  2. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  3. Measurement of the sequence specificity of covalent DNA modification by antineoplastic agents using Taq DNA polymerase.

    PubMed Central

    Ponti, M; Forrow, S M; Souhami, R L; D'Incalci, M; Hartley, J A

    1991-01-01

    A polymerase stop assay has been developed to determine the DNA nucleotide sequence specificity of covalent modification by antineoplastic agents using the thermostable DNA polymerase from Thermus aquaticus and synthetic labelled primers. The products of linear amplification are run on sequencing gels to reveal the sites of covalent drug binding. The method has been studied in detail for a number of agents including nitrogen mustards, platinum analogues and mitomycin C, and the sequence specificities obtained accord with those obtained by other procedures. The assay is advantageous in that it is not limited to a single type of DNA lesion (as in the piperidine cleavage assay for guanine-N7 alkylation), does not require a strand breakage step, and is more sensitive than other primer extension procedures which have only one cycle of polymerization. In particular the method has considerable potential for examining the sequence selectivity of damage and repair in single copy gene sequences in genomic DNA from cells. Images PMID:2057351

  4. The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

    PubMed Central

    Lange, Sabine S.; Tomida, Junya; Boulware, Karen S.; Bhetawal, Sarita; Wood, Richard D.

    2016-01-01

    DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents. PMID:26727495

  5. Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I.

    PubMed Central

    O'Connor, D; Stöhrer, G

    1985-01-01

    Oligodeoxyribonucleotides with site-specific modifications have been used as substrates for Escherichia coli DNA polymerase I holoenzyme and Klenow fragment. Modifications included the bulky guanine-8-aminofluorene adduct and a guanine oxidation product resembling the product of photosensitized DNA oxidation. By a combination of primers and "nick-mers", conditions of single-strand-directed DNA synthesis and nick-translation could be created. Our results show that the polymerase can bypass both types of lesions. Bypass occurs on a single-stranded template but is facilitated on a nicked, double-stranded template. Only purines, with guanine more favored than adenine, are incorporated across both lesions. Hesitation during bypass could not be detected. The results indicate that site-specifically modified oligonucleotides can be sensitive probes for the action of polymerases on damaged templates. They also suggest a function for polymerase I, in its nick-translation capacity, during DNA repair and mutagenesis. Images PMID:3887400

  6. Specific antigenic relationships between the RNA-dependent DNA polymerases of avian reticuloendotheliosis viruses and mammalian type C retroviruses.

    PubMed Central

    Bauer, G; Temin, H M

    1980-01-01

    Immunoglobulin G directed against the DNA polymerase of Rauscher murine leukemia virus (R-MuLV) could bind to 125I-labeled DNA polymerase of spleen necrosis virus (SNV), a member of the reticuloendotheliosis virus (REV) species. Competition radioimmunoassays showed the specificity of this cross-reaction. The antigenic determinants common to SNV and R-MuLV DNA polymerases were shared completely by the DNA polymerases of Gross MuLV, Moloney MuLV, RD 114 virus, REV-T, and duck infectious anemia virus. Baboon endogenous virus and chicken syncytial virus competed partially for antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. DNA polymerases of avian leukosis viruses, pheasant viruses, and mammalian type B and D retroviruses and particles with RNA-dependent DNA polymerase activity from the allantoic fluid of normal chicken eggs and from the medium of a goose cell culture did not compete for the antibodies directed against the common antigenic determinants of SNV and R-MuLV DNA polymerases. We also present data about a factor in normal mammalian immunoglobulin G that specifically inhibits the DNA polymerases of REV and mammalian type C retrovirus DNA polymerases. PMID:6154804

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

    PubMed Central

    Schiebel, W; Pélissier, T; Riedel, L; Thalmeir, S; Schiebel, R; Kempe, D; Lottspeich, F; Sänger, H L; Wassenegger, M

    1998-01-01

    A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed. PMID:9836747

  9. Sequence-Specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases.

    PubMed

    Welter, Moritz; Verga, Daniela; Marx, Andreas

    2016-08-16

    DNA polymerases select the right nucleotide for the growing polynucleotide chain based on the shape and geometry of the nascent nucleotide pairs and thereby ensure high DNA replication selectivity. High-fidelity DNA polymerases are believed to possess tight active sites that allow little deviation from the canonical structures. However, DNA polymerases are known to use nucleotides with small modifications as substrates, which is key for numerous core biotechnology applications. We show that even high-fidelity DNA polymerases are capable of efficiently using nucleotide chimera modified with a large protein like horseradish peroxidase as substrates for template-dependent DNA synthesis, despite this "cargo" being more than 100-fold larger than the natural substrates. We exploited this capability for the development of systems that enable naked-eye detection of DNA and RNA at single nucleotide resolution. PMID:27392211

  10. Specific Nucleotide Binding and Rebinding to Individual DNA Polymerase Complexes Captured on a Nanopore

    PubMed Central

    Hurt, Nicholas; Wang, Hongyun; Akeson, Mark; Lieberman, Kate R.

    2009-01-01

    Nanoscale pores are a tool for single molecule analysis of DNA or RNA processing enzymes. Monitoring catalytic activity in real time using this technique requires that these enzymes retain function while held atop a nanopore in an applied electric field. Using an α-hemolysin nanopore, we measured the dwell time for complexes of DNA with the Klenow fragment of Escherichia coli DNA polymerase I (KF) as a function of the concentration of deoxynucleoside triphosphate (dNTP) substrate. We analyzed these dwell time measurements in the framework of a two-state model for captured complexes (DNA-KF binary and DNA-KF-dNTP ternary states). Average nanopore dwell time increased without saturating as a function of correct dNTP concentration across four orders of magnitude. This arises from two factors that are proportional to dNTP concentration: 1) The fraction of complexes that are in the ternary state when initially captured predominantly affects dwell time at low dNTP concentrations; 2) The rate of binding and rebinding of dNTP to captured complexes affects dwell time at higher dNTP concentrations. Thus there are two regimes that display a linear relationship between average dwell time and dNTP concentration. The transition from one linear regime to the other occurs near the equilibrium dissociation constant (Kd) for dNTP binding to KF-DNA complexes in solution. We conclude from the combination of titration experiments and modeling that DNA-KF complexes captured atop the nanopore retain iterative, sequence-specific dNTP binding, as required for catalysis and fidelity in DNA synthesis. PMID:19275265

  11. Replicative DNA polymerases.

    PubMed

    Johansson, Erik; Dixon, Nicholas

    2013-06-01

    In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3'-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein-protein and protein-DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact. PMID:23732474

  12. Anti-tumor effects of dehydroaltenusin, a specific inhibitor of mammalian DNA polymerase {alpha}

    SciTech Connect

    Maeda, Naoki; Kokai, Yasuo; Ohtani, Seiji; Sahara, Hiroeki; Kuriyama, Isoko; Kamisuki, Shinji; Takahashi, Shunya; Sakaguchi, Kengo; Sugawara, Fumio; Yoshida, Hiromi; Sato, Noriyuki; Mizushina, Yoshiyuki . E-mail: mizushin@nutr.kobegakuin.ac.jp

    2007-01-12

    In the screening of selective inhibitors of eukaryotic DNA polymerases (pols), dehydroaltenusin was found to be an inhibitor of pol {alpha} from a fungus (Alternaria tennuis). We succeeded in chemically synthesizing dehydroaltenusin, and the compound inhibited only mammalian pol {alpha} with IC{sub 50} value of 0.5 {mu}M, and did not influence the activities of other replicative pols such as pols {delta} and {epsilon}, but also showed no effect on pol {alpha} activity from another vertebrate, fish, or from a plant species. Dehydroaltenusin also had no influence on the other pols and DNA metabolic enzymes tested. The compound also inhibited the proliferation of human cancer cells with LD{sub 50} values of 38.0-44.4 {mu}M. In an in vivo anti-tumor assay on nude mice bearing solid tumors of HeLa cells, dehydroaltenusin was shown to be a promising suppressor of solid tumors. Histopathological examination revealed that increased tumor necrosis and decreased mitotic index were apparently detected by the compound in vivo. Therefore, dehydroaltenusin could be of interest as not only a mammalian pol {alpha}-specific inhibitor, but also as a candidate drug for anti-cancer treatment.

  13. Elongation factor SII-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein.

    PubMed Central

    Reines, D; Mote, J

    1993-01-01

    In eukaryotes the genetic material is contained within a coiled, protein-coated structure known as chromatin. RNA polymerases must recognize specific nucleoprotein assemblies and maintain contact with the underlying DNA duplex for many thousands of base pairs. Template-bound lac operon repressor from Escherichia coli arrests RNA polymerase II in vitro and in vivo [Kuhn, A., Bartsch, I. & Grummt, I. (1990) Nature (London) 344, 559-562; Deuschele, U., Hipskind, R. A. & Bujard, H. (1990) Science 248, 480-483]. We show that in a reconstituted transcription system, elongation factor SII enables RNA polymerase II to proceed through this blockage at high efficiency. lac repressor-arrested elongation complexes display an SII-activated transcript cleavage reaction, an activity associated with transcriptional read-through of a previously characterized region of bent DNA. This demonstrates factor-dependent transcription by RNA polymerase II through a sequence-specific DNA-binding protein. Nascent transcript cleavage may be a general mechanism by which RNA polymerase II can bypass many transcriptional impediments. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8446609

  14. Specific binding of the adenovirus terminal protein precursor-DNA polymerase complex to the origin of DNA replication.

    PubMed Central

    Rijnders, A W; van Bergen, B G; van der Vliet, P C; Sussenbach, J S

    1983-01-01

    Initiation of adenovirus DNA replication is dependent on a complex of the precursor of the terminal protein and the adenovirus-coded DNA polymerase (pTP-pol complex). This complex catalyzes the formation of a covalent linkage between dCMP and pTP in the presence of a functional origin of DNA replication residing in the terminal nucleotide sequence of adenovirus DNA. We have purified the pTP-pol complex of adenovirus type 5 and studied its binding to double-stranded DNA. Using DNA-cellulose chromatography it could be shown that the pTP-pol complex has a higher affinity for adenovirus DNA than for calf thymus or pBR322 DNA. From the differential binding of the pTP-pol complex to plasmids containing adenovirus terminal sequences with different deletions, it has been concluded that a sequence of 14 nucleotide pairs at positions 9-22 plays a crucial role in the binding of pTP-pol to adenovirus DNA. This region is conserved in the DNA's of all human adenovirus serotypes and is obviously an important structural element of the adenovirus origin of DNA replication. Comparative binding studies with adenovirus DNA polymerase and pTP-pol indicated that pTP is responsible for the binding. The nature of the binding of pTP-pol to the conserved sequence will be discussed. Images PMID:6672772

  15. Mouse models of DNA polymerases.

    PubMed

    Menezes, Miriam R; Sweasy, Joann B

    2012-12-01

    In 1956, Arthur Kornberg discovered the mechanism of the biological synthesis of DNA and was awarded the Nobel Prize in Physiology or Medicine in 1959 for this contribution, which included the isolation and characterization of Escherichia coli DNA polymerase I. Now there are 15 known DNA polymerases in mammalian cells that belong to four different families. These DNA polymerases function in many different cellular processes including DNA replication, DNA repair, and damage tolerance. Several biochemical and cell biological studies have provoked a further investigation of DNA polymerase function using mouse models in which polymerase genes have been altered using gene-targeting techniques. The phenotypes of mice harboring mutant alleles reveal the prominent role of DNA polymerases in embryogenesis, prevention of premature aging, and cancer suppression. PMID:23001998

  16. Purified canola lutein selectively inhibits specific isoforms of mammalian DNA polymerases and reduces inflammatory response.

    PubMed

    Horie, Sho; Okuda, Chiaki; Yamashita, Takatoshi; Watanabe, Kenichi; Kuramochi, Kouji; Hosokawa, Masashi; Takeuchi, Toshifumi; Kakuda, Makiko; Miyashita, Kazuo; Sugawara, Fumio; Yoshida, Hiromi; Mizushina, Yoshiyuki

    2010-08-01

    In the screening of DNA polymerase (pol) inhibitor, we isolated lutein, a carotenoid, from the crude (unrefined) pressed oil of canola (low erucic acid rapeseed, Brassica napus L.). Commercially prepared carotenoids such as lutein (1), zeaxanthin (2), beta-cryptoxanthin (3), astaxanthin (4), canthaxanthin (5), beta-carotene (6), lycopene (7), capsanthin (8), fucoxanthin (9) and fucoxanthinol (10), were investigated for the inhibitory activities of pols. Compounds 1, 2 and 8 exhibited strong inhibition of the activities of mammalian pols beta and lambda, which are DNA repair- and/or recombination-related pols. On the other hand, all carotenoids tested had no influence on the activity of a mammalian pol alpha, which is a DNA replicative pol. Lutein (1) was the strongest pol inhibitor of mammalian pols beta and lambda in the prepared ten carotenoids tested, but did not influence of the activities of mammalian pols alpha, gamma, delta and epsilon. The tendency for pols beta and lambda inhibition by these carotenoids showed a positive correlation with the suppression of TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation. These results suggest that cold pressed unrefined canola/rapeseed oil, or other oils with high levels of lutein and other carotenoids, may be useful for their anti-inflammatory properties. PMID:20669052

  17. A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation.

    PubMed

    Arakawa, Toshiya; Ohta, Tohru; Abiko, Yoshihiro; Okayama, Miki; Mizoguchi, Itaru; Takuma, Taishin

    2011-09-15

    DNA methylation is an important epigenetic modification that leads to a wide variety of biological functions, including transcription, growth and development, and diseases associated with altered gene expression such as cancers. However, tools to insert site-specific methylation into DNA for analyzing epigenetic functions are limited. Here we describe a novel polymerase chain reaction (PCR)-based approach to provide site-specific DNA methylation at any site, including CpG or CpNpG islands. This method is simple and versatile, and it consists of four steps to construct the DNA methylation vector: (I) design and synthesis of methylated primers, (II) PCR amplification, (III) isolation of single-stranded DNA, and (IV) annealing and ligation of isolated single-stranded DNAs. First we produced and validated a linear green fluorescence protein (GFP) vector by this method. Next we applied this method to introduce methyl groups into the promoter of the cyclooxygenase-2 (COX-2) gene and found that site-specific DNA methylation at the CRE element significantly altered COX-2 gene expression. These results demonstrate that this PCR-based approach is useful for the analysis of biological functions that depend on DNA methylation. PMID:21669180

  18. Mechanism of polyoxometalate-mediated inactivation of DNA polymerases: an analysis with HIV-1 reverse transcriptase indicates specificity for the DNA-binding cleft.

    PubMed Central

    Sarafianos, S G; Kortz, U; Pope, M T; Modak, M J

    1996-01-01

    The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12O36]16- (polyoxometalate I) and [(O3PCH2PO3)4W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 microM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme-polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules. PMID:8912703

  19. Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides.

    PubMed Central

    Ren, Yan-Guo; Martínez, Javier; Kirsebom, Leif A; Virtanen, Anders

    2002-01-01

    Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed. PMID:12458793

  20. Antimutator Variants of DNA Polymerases

    PubMed Central

    Herr, Alan J.; Williams, Lindsey N.; Preston, Bradley D.

    2011-01-01

    Evolution balances DNA replication speed and accuracy to optimize replicative fitness and genetic stability. There is no selective pressure to improve DNA replication fidelity beyond the background mutation rate from other sources, such as DNA damage. However, DNA polymerases remain amenable to amino-acid substitutions that lower intrinsic error rates. Here, we review these ‘antimutagenic’ changes in DNA polymerases and discuss what they reveal about mechanisms of replication fidelity. Pioneering studies with bacteriophage T4 DNA polymerase (T4 Pol) established the paradigm that antimutator amino-acid substitutions reduce replication errors by increasing proofreading efficiency at the expense of polymerase processivity. The discoveries of antimutator substitutions in proofreading-deficient ‘mutator’ derivatives of bacterial Pols I and III and yeast Pol δ suggest there must be additional antimutagenic mechanisms. Remarkably, many of the affected amino-acid positions from Pol I, Pol III, and Pol δ are similar to the original T4 Pol substitutions. The locations of antimutator substitutions within DNA polymerase structures suggest that they may increase nucleotide selectivity and/or promote dissociation of primer termini from polymerases poised for misincorporation, leading to expulsion of incorrect nucleotides. If misincorporation occurs, enhanced primer dissociation from polymerase domains may improve proofreading in cis by an intrinsic exonuclease or in trans by alternate cellular proofreading activities. Together, these studies reveal that natural selection can readily restore replication error rates to sustainable levels following an adaptive mutator phenotype. PMID:21977975

  1. DNA Polymerase β Ribonucleotide Discrimination

    PubMed Central

    Cavanaugh, Nisha A.; Beard, William A.; Wilson, Samuel H.

    2010-01-01

    DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxy-counterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase β to utilize nucleotides with modified sugars. DNA polymerase β readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3′-endo sugar pucker facilitates nucleotide insertion, whereas a 2′-endo conformation inhibits insertion. PMID:20519499

  2. Aphidicolin resistance in herpes simplex virus type 1 appears to alter substrate specificity in the DNA polymerase

    SciTech Connect

    Hall, J.D.; Woodward, S.

    1989-06-01

    The authors describe novel mutants of herpes simplex virus which are resistant to aphidicolin. Their mutant phenotypes suggest that they encode DNA polymerases with altered substrate recognition. This conclusion is based on their abnormal sensitivity to polymerase inhibitors and to the abnormal mutation rates exhibited by two of the mutants.

  3. Amplification and analysis of specific DNA and RNA sequences of bovine leukemia virus from infected cows by polymerase chain reaction.

    PubMed Central

    Sherman, M P; Ehrlich, G D; Ferrer, J F; Sninsky, J J; Zandomeni, R; Dock, N L; Poiesz, B

    1992-01-01

    Bovine leukemia virus (BLV) is the etiologic agent of leukemia in cattle and is believed to cause decreases in milk productivity, fertility, and life span in infected cows. BLV is a type C retrovirus in the Oncovirinae subfamily. It is most closely related to human T-cell lymphoma/leukemia virus type I (HTLV-I) and type II (HTLV-II). Since the polymerase chain reaction (PCR) provides rapid and efficient amplification of DNA sequences, primers were designed to amplify regions of the polymerase (pol) and pX genes specific for BLV targets. These sets of primers consistently amplified as few as 10 copies of BLV DNA contained in a plasmid in the background of 1 microgram of either human or bovine chromosomal DNA. In addition, no amplification products were detected from cell lines infected with HTLV-I, HTLV-II, or human immunodeficiency virus type 1 or 2 by the BLV PCR systems. Samples of peripheral blood mononuclear cells from 18 cows, previously determined to be serologically positive or negative, were correctly identified in a blind study as containing proviral DNA by use of the BLV primers and probes. Cloning and sequencing of amplified products revealed finite sequence variations among a previously cloned BLV isolate, the wild-type virus, and the published genome. Reverse transcriptase-directed PCR with the primers for both BLV pol and BLV pX was performed on plasma from a BLV-infected cow and detected in vivo BLV RNA expression. In summary, we have developed a specific and sensitive assay using PCR for the detection and identification of BLV infections; this assay can now be applied to clinical and basic research questions in veterinary medicine. Images PMID:1370847

  4. Detection of Nesopora caninum-specific DNA from cerebrospinal fluid by polymerase chain reaction in a dog with confirmed neosporosis.

    PubMed

    Ishigaki, Kyohei; Noya, Masahiko; Kagawa, Yumiko; Ike, Kazunori; Orima, Hiromitsu; Imai, Soichi

    2012-08-01

    A one-month male Greyhound dog presented with a swinging gait of the hindlimbs, and later developed muscular atrophy of the femoral region and hyperextension of hindlimbs. The dog had positive serum IFAT titers to Neospora caninum, but a negative titer in the cerebrospinal fluid (CSF). N. caninum-specific DNA was amplified from the CSF using a semi-nested polymerase chain reaction assay. Clusters of protozoa in biopsied muscle fibers were subsequently confirmed as N. caninum tachyzoites by immunohistochemical examination. Early recognition and treatment are necessary for effective recovery of clinical canine neosporosis, but antemortem diagnosis is difficult. We suggest that the detection of parasite deoxyribonucleic acid in the CSF is a useful antemortem diagnostic method in facilitating treatment of this disease. PMID:22446406

  5. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

    A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

  6. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  7. Archaeal DNA polymerases in biotechnology.

    PubMed

    Zhang, Likui; Kang, Manyu; Xu, Jiajun; Huang, Yanchao

    2015-08-01

    DNA polymerase (pol) is a ubiquitous enzyme that synthesizes DNA strands in all living cells. In vitro, DNA pol is used for DNA manipulation, including cloning, PCR, site-directed mutagenesis, sequencing, and several other applications. Family B archaeal DNA pols have been widely used for molecular biological methods. Biochemical and structural studies reveal that each archaeal DNA pol has different characteristics with respect to fidelity, processivity and thermostability. Due to their high fidelity and strong thermostability, family B archaeal DNA pols have the extensive application on high-fidelity PCR, DNA sequencing, and site-directed mutagenesis while family Y archaeal DNA pols have the potential for error-prone PCR and random mutagenesis because of their low fidelity and strong thermostability. This information combined with mutational analysis has been used to construct novel DNA pols with altered properties that enhance their use as biotechnological reagents. In this review, we focus on the development and use of family B archaeal DNA pols. PMID:26150245

  8. The evolutionary conservation of DNA polymerase. alpha

    SciTech Connect

    Miller, M.A.; Korn, D.; Wang, T.S.F. )

    1988-08-25

    The evolutionary conservation of DNA polymerase {alpha} was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase {alpha} monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase {alpha} catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.

  9. Segregation of relaxed replicated dimers when DNA ligase and DNA polymerase I are limited during oriC-specific DNA replication.

    PubMed Central

    Munson, B R; Maier, P G; Greene, R S

    1989-01-01

    An in vitro Escherichia coli oriC-specific DNA replication system was used to investigate the DNA replication pathways of oriC plasmids. When this system was perturbed by the DNA ligase inhibitor nicotinamide mononucleotide (NMN), alterations occurred in the initiation of DNA synthesis and processing of intermediates and DNA products. Addition of high concentrations of NMN soon after initiation resulted in the accumulation of open circular dimers (OC-OC). These dimers were decatenated to open circular monomers (form II or OC), which were then processed to closed circular supercoiled monomers (form I or CC) products. After a delay, limited ligation of the interlinked dimers (OC-OC to CC-OC and CC-CC) also occurred. Similar results were obtained with replication protein extracts from polA mutants. The presence of NMN before any initiation events took place prolonged the existence of nicked template DNA and promoted, without a lag period, limited incorporation into form II molecules. This DNA synthesis was nonspecific with respect to oriC, as judged by DnaA protein dependence, and presumably occurred at nicks in the template DNA. These results are consistent with oriC-specific initiation requiring closed supercoiled molecules dependent on DNA ligase activity. The results also show that decatenation of dimers occurs readily on nicked dimer and represents an efficient pathway for processing replication intermediates in vitro. Images PMID:2544556

  10. Direct and site-specific quantification of RNA 2'-O-methylation by PCR with an engineered DNA polymerase.

    PubMed

    Aschenbrenner, Joos; Marx, Andreas

    2016-05-01

    Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  11. Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

    PubMed Central

    Aschenbrenner, Joos; Marx, Andreas

    2016-01-01

    Methylation of the 2′-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2′-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2′-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2′-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  12. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

    PubMed

    McInerney, Peter; Adams, Paul; Hadi, Masood Z

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. PMID:25197572

  13. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    DOE PAGESBeta

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Errormore » rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu , Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.« less

  14. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    PubMed Central

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. PMID:25197572

  15. Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases.

    PubMed

    Loh, Ern; Loeb, Lawrence A

    2005-12-01

    DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity. PMID:16230053

  16. Human DNA polymerase α in binary complex with a DNA:DNA template-primer

    PubMed Central

    Coloma, Javier; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2016-01-01

    The Polα/primase complex assembles the short RNA-DNA fragments for priming of lagging and leading strand DNA replication in eukaryotes. As such, the Polα polymerase subunit encounters two types of substrates during primer synthesis: an RNA:DNA helix and a DNA:DNA helix. The engagement of the polymerase subunit with the DNA:DNA helix has been suggested as the of basis for primer termination in eukaryotes. However, there is no structural information on how the Polα polymerase subunit actually engages with a DNA:DNA helix during primer synthesis. We present here the first crystal structure of human Polα polymerase subunit in complex with a DNA:DNA helix. Unexpectedly, we find that portion of the DNA:DNA helix in contact with the polymerase is not in a B-form but in a hybrid A-B form. Almost all of the contacts observed previously with an RNA primer are preserved with a DNA primer – with the same set of polymerase residues tracking the sugar-phosphate backbone of the DNA or RNA primer. Thus, rather than loss of specific contacts, the free energy cost of distorting DNA from B- to hybrid A-B form may augur the termination of primer synthesis in eukaryotes. PMID:27032819

  17. Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.

    PubMed Central

    Seal, S E; Jackson, L A; Daniels, M J

    1992-01-01

    A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers. Images PMID:1482193

  18. A fast one-step reverse transcription and polymerase chain reaction (RT-PCR) amplification procedure providing highly specific complementary DNA from plant virus RNA.

    PubMed

    Sambade, A; Martín, S; Olmos, A; García, M L; Cambra, M; Grau, O; Guerri, J; Moreno, P

    2000-06-01

    Reverse transcription and polymerase chain reaction (RT-PCR) are being used increasingly for detection and typing RNA viruses. For this purpose, metal block thermal cyclers (MBTC) are considered to provide higher DNA yield, whereas air thermal cyclers (ATC) allow PCR amplification in a much shorter time. A fast ATC protocol (0 s denaturation, 0 s annealing, and 4-8 s elongation) was developed to amplify genomic segments from two RNA viruses, which allowed increasing the number of cycles without a parallel increase of non-specific DNA fragments. Under these conditions, 80-90 cycles with the ATC provided a DNA yield close to that of a standard 40-cycles MBTC protocol in about half the time. The DNA synthesised by the new procedure was highly specific and could be cloned readily. PMID:10856749

  19. Specific contacts of the −35 region of the galP1 promoter by RNA polymerase inhibit GalR-mediated DNA looping repression

    PubMed Central

    Csiszovszki, Zsolt; Lewis, Dale E. A.; Le, Phuoc; Sneppen, Kim; Semsey, Szabolcs

    2012-01-01

    The P1 promoter of the galactose operon in Escherichia coli is one of the best studied examples of ‘extended −10’ promoters. Recognition of the P1 promoter does not require specific contacts between RNA polymerase and its poor −35 element. To investigate whether specific recognition of the −35 element would affect the regulation of P1 by GalR, we mutagenized the −35 element of P1, isolated variants of the −35 element and studied the regulation of the mutant promoters by in vitro transcription assays and by mathematical modeling. The results show that the GalR-mediated DNA loop is less efficient in repressing P1 transcription when RNA polymerase binds to the −10 and −35 elements concomitantly. Our results suggest that promoters that lack specific −35 element recognition allow decoupling of local chromosome structure from transcription initiation. PMID:22941635

  20. Specific contacts of the -35 region of the galP1 promoter by RNA polymerase inhibit GalR-mediated DNA looping repression.

    PubMed

    Csiszovszki, Zsolt; Lewis, Dale E A; Le, Phuoc; Sneppen, Kim; Semsey, Szabolcs

    2012-11-01

    The P1 promoter of the galactose operon in Escherichia coli is one of the best studied examples of 'extended -10' promoters. Recognition of the P1 promoter does not require specific contacts between RNA polymerase and its poor -35 element. To investigate whether specific recognition of the -35 element would affect the regulation of P1 by GalR, we mutagenized the -35 element of P1, isolated variants of the -35 element and studied the regulation of the mutant promoters by in vitro transcription assays and by mathematical modeling. The results show that the GalR-mediated DNA loop is less efficient in repressing P1 transcription when RNA polymerase binds to the -10 and -35 elements concomitantly. Our results suggest that promoters that lack specific -35 element recognition allow decoupling of local chromosome structure from transcription initiation. PMID:22941635

  1. Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strand-specific nucleotide excision repair of RNA polymerase I transcribed genes.

    PubMed Central

    Verhage, R A; Van de Putte, P; Brouwer, J

    1996-01-01

    Removal of UV-induced pyrimidine dimers from the individual strands of the rDNA locus in Saccharomyces cerevisiae was studied. Yeast rDNA, that is transcribed by RNA polymerase I(RNA pol I), is repaired efficiently, slightly strand-specific and independently of RAD26, which has been implicated in transcription-coupled repair of the RNA pol II transcribed RPB2 gene. No repair of rDNA is observed in rad1,2,3 and 14 mutants, demonstrating that dimer removal from this highly repetitive DNA is accomplished by nucleotide excision repair (NER). In rad7 and rad16 mutants, which are specifically deficient in repair of non-transcribed DNA, there is a clear preferential repair of the transcribed strand of rDNA, indicating that strand-specific and therefore probably transcription-coupled repair of RNA pol I transcribed genes does exist in yeast. Unexpectedly, the transcribed but not the non-transcribed strand of rDNA can be repaired in rad4 mutants, which seem otherwise completely NER-deficient. PMID:8604332

  2. NusG Is a Sequence-specific RNA Polymerase Pause Factor That Binds to the Non-template DNA within the Paused Transcription Bubble.

    PubMed

    Yakhnin, Alexander V; Murakami, Katsuhiko S; Babitzke, Paul

    2016-03-01

    NusG, referred to as Spt5 in archaeal and eukaryotic organisms, is the only transcription factor conserved in all three domains of life. This general transcription elongation factor binds to RNA polymerase (RNAP) soon after transcription initiation and dissociation of the RNA polymerase σ factor. Escherichia coli NusG increases transcription processivity by suppressing RNAP pausing, whereas Bacillus subtilis NusG dramatically stimulates pausing at two sites in the untranslated leader of the trpEDCFBA operon. These two regulatory pause sites participate in transcription attenuation and translational control mechanisms, respectively. Here we report that B. subtilis NusG makes sequence-specific contacts with a T-rich sequence in the non-template DNA (ntDNA) strand within the paused transcription bubble. NusG protects T residues of the recognition sequence from permanganate oxidation, and these T residues increase the affinity of NusG to the elongation complex. Binding of NusG to RNAP does not require interaction with RNA. These results indicate that bound NusG prevents forward movement of RNA polymerase by simultaneously contacting RNAP and the ntDNA strand. Mutational studies indicate that amino acid residues of two short regions within the NusG N-terminal domain are primarily responsible for recognition of the trp operon pause signals. Structural modeling indicates that these two regions are adjacent to each another in the protein. We propose that recognition of specific sequences in the ntDNA and stimulation of RNAP pausing is a conserved function of NusG-like transcription factors. PMID:26742846

  3. Mechanism for priming DNA synthesis by yeast DNA Polymerase α

    PubMed Central

    Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

    2013-01-01

    The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

  4. Effect of surface charge of PDDA-protected gold nanoparticles on the specificity and efficiency of DNA polymerase chain reaction.

    PubMed

    Yuan, Longfei; He, Yujian

    2013-01-21

    The polymerase chain reaction (PCR) has become an indispensable technique in molecular biology, however, it suffers from low efficiency and specificity problems. Developing suitable additives to effectively avoid nonspecific PCR reactions and explore the mechanism for PCR enhancing is a significant challenge. In this paper, we report three different modified gold nanoparticles (AuNPs) with different surface charge polarities and poly (diallyl dimethylammonium) chloride (PDDA) for use as novel PCR enhancers to improve the efficiency and specificity. These AuNPs included the positively charged PDDA protected AuNPs (PDDA-AuNPs), the neutral PDDA-AuNPs modified with excess chloride ion (PDDA.C-AuNPs), and the negatively charged sodium citrate (Na(3)Ct) protected AuNPs (Na(3)Ct-AuNPs). Our data clearly suggests that the positively charged PDDA-AuNPs with an optimum concentration as low as 1.54 pM could significantly enhance the specificity and efficiency of PCR, however, the optimum concentration of the negatively charged Na(3)Ct-AuNPs (2.02 nM) was more than 3 orders of magnitude higher than that of positively charged PDDA-AuNPs. The PCR specificity and efficiency are also improved by the neutral PDDA.C-AuNPs with an optimum concentration, much more than that of the PDDA-AuNPs. This suggests that there should be an electrostatic interaction between the positively charged PDDA-AuNPs and the negatively charged PCR components, and the surface charge polarities of PDDA-AuNPs may play an important role in improving the PCR specificity and efficiency. PMID:23170311

  5. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  6. Sphingosine, a modulator of human translesion DNA polymerase activity.

    PubMed

    Kamath-Loeb, Ashwini S; Balakrishna, Sharath; Whittington, Dale; Shen, Jiang-Cheng; Emond, Mary J; Okabe, Takayoshi; Masutani, Chikahide; Hanaoka, Fumio; Nishimura, Susumu; Loeb, Lawrence A

    2014-08-01

    Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress. PMID:24928506

  7. Directed evolution of DNA polymerase, RNA polymerase and reverse transcriptase activity in a single polypeptide.

    PubMed

    Ong, Jennifer L; Loakes, David; Jaroslawski, Szymon; Too, Kathleen; Holliger, Philipp

    2006-08-18

    DNA polymerases enable key technologies in modern biology but for many applications, native polymerases are limited by their stringent substrate recognition. Here we describe short-patch compartmentalized self-replication (spCSR), a novel strategy to expand the substrate spectrum of polymerases in a targeted way. spCSR is based on the previously described CSR, but unlike CSR only a short region (a "patch") of the gene under investigation is diversified and replicated. This allows the selection of polymerases under conditions where catalytic activity and processivity are compromised to the extent that full self-replication is inefficient. We targeted two specific motifs involved in substrate recognition in the active site of DNA polymerase I from Thermus aquaticus (Taq) and selected for incorporation of both ribonucleotide- (NTP) and deoxyribonucleotide-triphosphates (dNTPs) using spCSR. This allowed the isolation of multiple variants of Taq with apparent dual substrate specificity. They were able to synthesize RNA, while still retaining essentially wild-type (wt) DNA polymerase activity as judged by PCR. One such mutant (AA40: E602V, A608V, I614M, E615G) was able to incorporate both NTPs and dNTPs with the same catalytic efficiency as the wt enzyme incorporates dNTPs. AA40 allowed the generation of mixed RNA-DNA amplification products in PCR demonstrating DNA polymerase, RNA polymerase as well as reverse transcriptase activity within the same polypeptide. Furthermore, AA40 displayed an expanded substrate spectrum towards other 2'-substituted nucleotides and was able to synthesize nucleic acid polymers in which each base bore a different 2'-substituent. Our results suggest that spCSR will be a powerful strategy for the generation of polymerases with altered substrate specificity for applications in nano- and biotechnology and in the enzymatic synthesis of antisense and RNAi probes. PMID:16859707

  8. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. PMID:18834537

  9. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases

    PubMed Central

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. This article was reviewed by Eugene Koonin and Mark Ragan. PMID:18834537

  10. Effect of. gamma. -irradiated DNA on the activity of DNA polymerase. [/sup 60/Co

    SciTech Connect

    Leadon, S.A.; Ward, J.F.

    1981-06-01

    A cell-free assay was developed to measure the effect of ..gamma..-irradiated DNA template on the ability of DNA polymerase to copy unirradiated template. Doses as low as 1 krad were able to decrease (approx. 15%) the activity of both bacterial and mammalian DNA polymerases in the assay. The percentage of polymerase activity decreased as the dose received by the template increased. The reduction in DNA polymerase activity was shown to be due to an inhibition of the enzyme by the irradiated DNA. Irradiated poly(dA-dT) was more effective in reducing polymerase activity than calf thymus DNA. Thus the polymerase-inhibition site(s) appears to be associated with base damage, specifically adenine or thymine. Using a free-radical scavenger, OH radicals were found to be involved in producing the damage sites. The interaction between irradiated DNA and DNA polymerase was found to be specific for the enzyme and not for other proteins present in the assay. The inhibition of DNA polymerase occurred prior to or during the initiation of DNA synthesis rather than after initiation of synthesis, i.e., during elongation.

  11. T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

    PubMed Central

    Smale, S T; Tjian, R

    1986-01-01

    We have combined in vitro DNA replication reactions and immunological techniques to analyze biochemical interactions between simian virus (SV40) large T antigen and components of the cellular replication apparatus. First, in vitro SV40 DNA replication was characterized with specific origin mutants. Next, monoclonal antibodies were used to demonstrate that a specific domain of T antigen formed a complex with cellular DNA polymerase alpha. Several antibodies were identified that coprecipitated T antigen and DNA polymerase alpha, while others were found to selectively prevent this interaction and concomitantly inhibit DNA replication. DNA polymerase alpha also bound efficiently to a T-antigen affinity column, confirming the immunoprecipitation results and providing a useful method for purification of the complete protein complex. Taken together, these results suggest that the T-antigen-polymerase association may be a key step in the initiation of SV40 DNA replication. Images PMID:3025630

  12. Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase

    PubMed Central

    Xia, Gang; Chen, Liangjing; Sera, Takashi; Fa, Ming; Schultz, Peter G.; Romesberg, Floyd E.

    2002-01-01

    The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of Thermus aquaticus DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates. PMID:12011423

  13. Lability of DNA polymerase alpha correlated with decreased DNA synthesis and increased age in human cells

    SciTech Connect

    Busbee, D.; Sylvia, V.; Stec, J.; Cernosek, Z.; Norman, J.

    1987-12-01

    DNA excision repair and mitogen-initiated blastogenesis in human cells declined in efficiency as an apparent function of decreased DNA polymerase alpha specific activity with increased age of the cell donor. DNA polymerase alpha isolated from fetal cells contained a single, high-specific-activity enzyme form that could not be further activated and that was stable with regard to enzyme activity and affinity for DNA template-primer. DNA polymerase alpha isolated from adult-derived cells contained both low-specific-activity and high-specific-activity forms. The low-activity enzyme form, which showed low affinity of binding to DNA template-primer, was activated by treatment with phosphatidylinositol, /sup 32/P-ATP, and phosphatidylinositol kinase, resulting in a /sup 32/P-labeled enzyme that exhibited high affinity of binding to DNA template-primer. The activated enzyme was unstable, exhibiting a loss of /sup 32/P-label correlated with the loss of both specific activity and high affinity of binding to DNA template-primer. The data suggest that DNA polymerase alpha isolated from adult-derived human cells has low-activity and high-activity forms. Decreased specific activity of DNA polymerase alpha correlated with increased age of the donor appears to be a function of loss of an enzyme activator molecule resulting in diminished ability of the enzyme to bind DNA template-primer.

  14. DNA polymerase mu, a candidate hypermutase?

    PubMed Central

    Ruiz, J F; Domínguez, O; Laín de Lera, T; Garcia-Díaz, M; Bernad, A; Blanco, L

    2001-01-01

    A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining. PMID:11205337

  15. Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication

    PubMed Central

    Lapenta, Fabio; Montón Silva, Alejandro; Brandimarti, Renato; Lanzi, Massimiliano; Gratani, Fabio Lino; Vellosillo Gonzalez, Perceval; Perticarari, Sofia; Hochkoeppler, Alejandro

    2016-01-01

    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. PMID:27050298

  16. Fluorescence resonance energy transfer analysis of escherichia coli RNA polymerase and polymerase-DNA complexes.

    PubMed

    Heyduk, T; Niedziela-Majka, A

    Fluorescence resonance energy transfer (FRET) is a technique allowing measurements of atomic-scale distances in diluted solutions of macromolecules under native conditions. This feature makes FRET a powerful tool to study complicated biological assemblies. In this report we review the applications of FRET to studies of transcription initiation by Escherichia coli RNA polymerase. The versatility of FRET for studies of a large macromolecular assembly such as RNA polymerase is illustrated by examples of using FRET to address several different aspects of transcription initiation by polymerase. FRET has been used to determine the architecture of polymerase, its complex with single-stranded DNA, and the conformation of promoter fragment bound to polymerase. FRET has been also used as a binding assay to determine the thermodynamics of promoter DNA fragment binding to the polymerase. Functional conformational changes in the specificity subunit of polymerase responsible for the modulation of the promoter binding activity of the enzyme and the mechanistic aspects of the transition from the initiation to the elongation complex were also investigated. PMID:11987181

  17. Human DNA polymerase. alpha. : Predicted functional domains and relationships with viral DNA polymerases

    SciTech Connect

    Wang, T.S.F.; Wong, S.W.; Korn, D. )

    1989-01-01

    The primary sequence of human DNA polymerase {alpha} deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage {phi}19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DBA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.

  18. Physical Interactions between Mcm10, DNA, and DNA Polymerase [alpha

    SciTech Connect

    Warren, Eric M.; Huang, Hao; Fanning, Ellen; Chazin, Walter J.; Eichman, Brandt F.

    2009-10-21

    Mcm10 is an essential eukaryotic protein required for the initiation and elongation phases of chromosomal replication. Specifically, Mcm10 is required for the association of several replication proteins, including DNA polymerase {alpha} (pol {alpha}), with chromatin. We showed previously that the internal (ID) and C-terminal (CTD) domains of Mcm10 physically interact with both single-stranded (ss) DNA and the catalytic p180 subunit of pol {alpha}. However, the mechanism by which Mcm10 interacts with pol {alpha} on and off DNA is unclear. As a first step toward understanding the structural details for these critical intermolecular interactions, x-ray crystallography and NMR spectroscopy were used to map the binary interfaces between Mcm10-ID, ssDNA, and p180. The crystal structure of an Mcm10-ID {center_dot} ssDNA complex confirmed and extended our previous evidence that ssDNA binds within the oligonucleotide/oligosaccharide binding-fold cleft of Mcm10-ID. We show using NMR chemical shift perturbation and fluorescence spectroscopy that p180 also binds to the OB-fold and that ssDNA and p180 compete for binding to this motif. In addition, we map a minimal Mcm10 binding site on p180 to a small region within the p180 N-terminal domain (residues 286-310). These findings, together with data for DNA and p180 binding to an Mcm10 construct that contains both the ID and CTD, provide the first mechanistic insight into how Mcm10 might use a handoff mechanism to load and stabilize pol {alpha} within the replication fork.

  19. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens.

    PubMed

    Kersting, Sebastian; Rausch, Valentina; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2014-01-01

    We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in <20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics. FigureThe combination of multiplex isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides. PMID:25253912

  20. DNA polymerases drive DNA sequencing-by-synthesis technologies: both past and present

    PubMed Central

    Chen, Cheng-Yao

    2014-01-01

    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. Escherichia 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 ϕ29 DNA polymerase copies long stretches of DNA and possesses a unique capability to efficiently incorporate terminal phosphate-labeled nucleoside polyphosphates. Furthermore, ϕ29 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. PMID:25009536

  1. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • 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. PMID:25202519

  2. Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

    PubMed Central

    Diez, Cristina; Lorenz, Virginia; Ortiz, Silvia; Gonzalez, Verónica; Racca, Andrea; Bontempi, Iván; Manattini, Silvia; Solari, Aldo; Marcipar, Iván

    2010-01-01

    We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe. PMID:20064998

  3. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    NASA Astrophysics Data System (ADS)

    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.

  4. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1995-01-01

    Two Genes for DNA polymerase delta were identified from the wild type Chinese hamster ovary cells. These genes were cloned via RT-PCR from mRNA prepared the Chinese hamster ovary cells using primers specific to conserved sequences of the DNA polymerase {delta} gene. The first gene encodes a PCNA dependent DNA polymerase {delta} gene whereas the second gene encodes a PCNA independent DNA polymerase {delta} gene. Methods were developed to clone these genes in expression vector and host systems. The role of the two genes in DNA replication and repair was determined.

  5. Antimutator Mutations in the α Subunit of Escherichia Coli DNA Polymerase III: Identification of the Responsible Mutations and Alignment with Other DNA Polymerases

    PubMed Central

    Fijalkowska, I. J.; Schaaper, R. M.

    1993-01-01

    The dnaE gene of Escherichia coli encodes the DNA polymerase (α subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions. PMID:8375647

  6. Interactions between the Werner syndrome helicase and DNA polymerase delta specifically facilitate copying of tetraplex and hairpin structures of the d(CGG)n trinucleotide repeat sequence.

    PubMed

    Kamath-Loeb, A S; Loeb, L A; Johansson, E; Burgers, P M; Fry, M

    2001-05-11

    Werner syndrome (WS) is an inherited disorder characterized by premature aging and genomic instability. The protein encoded by the WS gene, WRN, possesses intrinsic 3' --> 5' DNA helicase and 3' --> 5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report here that hairpin and G'2 bimolecular tetraplex structures of the fragile X expanded sequence, d(CGG)(n), effectively impede synthesis by three eukaryotic replicative DNA polymerases (pol): pol alpha, pol delta, and pol epsilon. The constraints imposed on pol delta-catalyzed synthesis are relieved, however, by WRN; WRN facilitates pol delta to traverse these template secondary structures to synthesize full-length DNA products. The alleviatory effect of WRN is limited to pol delta; neither pol alpha nor pol epsilon can traverse template d(CGG)(n) hairpin and tetraplex structures in the presence of WRN. Alleviation of pausing by pol delta is observed with Escherichia coli RecQ but not with UvrD helicase, suggesting a concerted action of RecQ helicases and pol delta. Our findings suggest a possible role of WRN in rescuing pol delta-mediated replication at forks stalled by unusual DNA secondary structures. PMID:11279038

  7. Posttranslational Regulation of Human DNA Polymerase ι*

    PubMed Central

    McIntyre, Justyna; McLenigan, Mary P.; Frank, Ekaterina G.; Dai, Xiaoxia; Yang, Wei; Wang, Yinsheng; Woodgate, Roger

    2015-01-01

    Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys11- and Lys48-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys11 and Lys48 rather than oxidative damage per se. PMID:26370087

  8. Hepatitis B virus: DNA polymerase activity of deletion mutants.

    PubMed

    Kim, Y; Hong, Y B; Jung, G

    1999-02-01

    The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain. PMID:10205676

  9. Overexpression of DNA polymerase beta: a genomic instability enhancer process.

    PubMed

    Canitrot, Y; Frechet, M; Servant, L; Cazaux, C; Hoffmann, J S

    1999-06-01

    DNA polymerase beta (Pol beta) is the most inaccurate of the six DNA polymerases found in mammalian cells. In a normal situation, it is expressed at a constant low level and its role is believed to be restricted to repair synthesis in the base excision repair pathway participating to the genome stability. However, excess of Pol beta, found in some human tumors, could confer an increase in spontaneous mutagenesis and result in a highly mutagenic tolerance phenotype toward bifunctional DNA cross-linking anticancer drugs. Here, we present a hypothesis on the mechanisms used by Pol beta to be a genetic instability enhancer through its overexpression. We hypothesize that an excess of Pol beta perturbs the well-defined specific functions of DNA polymerases developed by the cell and propose Pol beta-mediated gap fillings during DNA transactions like repair, replication, or recombination pathways as key processes to introduce illegitimate deoxyribonucleotides or mutagenic base analogs like those produced by intracellular oxidative processes. These mechanisms may predominate during cellular nonproliferative phases in the absence of DNA replication. PMID:10336894

  10. Guanine-rich sequences inhibit proofreading DNA polymerases

    PubMed Central

    Zhu, Xiao-Jing; Sun, Shuhui; Xie, Binghua; Hu, Xuemei; Zhang, Zunyi; Qiu, Mengsheng; Dai, Zhong-Min

    2016-01-01

    DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading DNA polymerases, they are more susceptible to be failed in PCR than non-proofreading DNA polymerases. Here we showed that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequences such as GGGGG and GGGGHGG can cause PCR failure using proofreading DNA polymerases but not Taq DNA polymerase. The inhibitory effect of these G-rich sequences is caused by G-quadruplex and is dose dependent. G-rich inhibitory sequence-containing primers can be used in PCR at a lower concentration to amplify its target DNA fragment. PMID:27349576

  11. Mitochondrial Disorders of DNA Polymerase γ Dysfunction

    PubMed Central

    Zhang, Linsheng; Chan, Sherine S. L.; Wolff, Daynna J.

    2011-01-01

    Context Primary mitochondrial dysfunction is one of the most common causes of inherited disorders predominantly involving the neuromuscular system. Advances in the molecular study of mitochondrial DNA have changed our vision and our approach to primary mitochondrial disorders. Many of the mitochondrial disorders are caused by mutations in nuclear genes and are inherited in an autosomal recessive pattern. Among the autosomal inherited mitochondrial disorders, those related to DNA polymerase γ dysfunction are the most common and the best studied. Understanding the molecular mechanisms and being familiar with the recent advances in laboratory diagnosis of this group of mitochondrial disorders are essential for pathologists to interpret abnormal histopathology and laboratory results and to suggest further studies for a definitive diagnosis. Objectives To help pathologists better understand the common clinical syndromes originating from mutations in DNA polymerase γ and its associated proteins and use the stepwise approach of clinical, laboratory, and pathologic diagnosis of these syndromes. Data Sources Review of pertinent published literature and relevant Internet databases. Conclusions Mitochondrial disorders are now better recognized with the development of molecular tests for clinical diagnosis. A cooperative effort among primary physicians, diagnostic pathologists, geneticists, and molecular biologists with expertise in mitochondrial disorders is required to reach a definitive diagnosis. PMID:21732785

  12. The primary structure of Plasmodium falciparum DNA polymerase delta is similar to drug sensitive delta-like viral DNA polymerases.

    PubMed

    Fox, B A; Bzik, D J

    1991-12-01

    We report the isolation and sequencing of genomic DNA clones that encode the 1094-amino acid catalytic subunit of DNA polymerase delta from the human malaria parasite Plasmodium falciparum. Protein sequence comparison to other DNA polymerases revealed the presence of six highly conserved regions found in alpha-like DNA polymerases from different prokaryotic, viral, and eukaryotic sources. Five additional regions of amino acid sequence similarity that are only conserved in delta and delta-like DNA polymerases, so far, were present in P. falciparum DNA polymerase delta. P. falciparum DNA polymerase delta was highly similar to both Saccharomyces cerevisiae DNA polymerase delta (DNA polymerase III; CDC2) and Epstein-Barr virus DNA polymerase at the amino acid sequence, and the predicted protein secondary structure levels. The gene that encodes DNA polymerase delta resides as a single copy on chromosome 10, and is expressed as a 4.5-kb mRNA during the trophozoite and schizont stages when parasite chromosomal DNA synthesis is active. PMID:1775172

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

    PubMed

    Mentegari, Elisa; Kissova, Miroslava; Bavagnoli, Laura; Maga, Giovanni; Crespan, Emmanuele

    2016-01-01

    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. PMID:27589807

  14. The Closing Mechanism of DNA Polymerase I at Atomic Resolution.

    PubMed

    Miller, Bill R; Beese, Lorena S; Parish, Carol A; Wu, Eugene Y

    2015-09-01

    DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase I bound to DNA in the open conformation with a dNTP present in the active site. We use this structure to computationally simulate the open to closed transition of DNA polymerase in the presence of a Watson-Crick base pair. Our microsecond simulations allowed us to characterize the key steps involved in active site assembly, and propose the sequence of events involved in the prechemistry steps of DNA polymerase catalysis. They also reveal new features of the polymerase mechanism, such as a conserved histidine as a potential proton acceptor from the primer 3'-hydroxyl. PMID:26211612

  15. A Sequence-Specific Interaction between the Saccharomyces cerevisiae rRNA Gene Repeats and a Locus Encoding an RNA Polymerase I Subunit Affects Ribosomal DNA Stability

    PubMed Central

    Cahyani, Inswasti; Cridge, Andrew G.; Engelke, David R.; Ganley, Austen R. D.

    2014-01-01

    The spatial organization of eukaryotic genomes is linked to their functions. However, how individual features of the global spatial structure contribute to nuclear function remains largely unknown. We previously identified a high-frequency interchromosomal interaction within the Saccharomyces cerevisiae genome that occurs between the intergenic spacer of the ribosomal DNA (rDNA) repeats and the intergenic sequence between the locus encoding the second largest RNA polymerase I subunit and a lysine tRNA gene [i.e., RPA135-tK(CUU)P]. Here, we used quantitative chromosome conformation capture in combination with replacement mapping to identify a 75-bp sequence within the RPA135-tK(CUU)P intergenic region that is involved in the interaction. We demonstrate that the RPA135-IGS1 interaction is dependent on the rDNA copy number and the Msn2 protein. Surprisingly, we found that the interaction does not govern RPA135 transcription. Instead, replacement of a 605-bp region within the RPA135-tK(CUU)P intergenic region results in a reduction in the RPA135-IGS1 interaction level and fluctuations in rDNA copy number. We conclude that the chromosomal interaction that occurs between the RPA135-tK(CUU)P and rDNA IGS1 loci stabilizes rDNA repeat number and contributes to the maintenance of nucleolar stability. Our results provide evidence that the DNA loci involved in chromosomal interactions are composite elements, sections of which function in stabilizing the interaction or mediating a functional outcome. PMID:25421713

  16. DNA Bending and Wrapping around RNA Polymerase: a “Revolutionary” Model Describing Transcriptional Mechanisms

    PubMed Central

    Coulombe, Benoit; Burton, Zachary F.

    1999-01-01

    A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription. PMID:10357858

  17. Mechanism of Suppression of Chromosomal Instability by DNA Polymerase POLQ

    PubMed Central

    Yousefzadeh, Matthew J.; Wyatt, David W.; Takata, Kei-ichi; Mu, Yunxiang; Hensley, Sean C.; Tomida, Junya; Bylund, Göran O.; Doublié, Sylvie; Johansson, Erik; Ramsden, Dale A.; McBride, Kevin M.; Wood, Richard D.

    2014-01-01

    Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3′ single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone. PMID:25275444

  18. Taq DNA Polymerase Mutants and 2'-Modified Sugar Recognition.

    PubMed

    Schultz, Hayley J; Gochi, Andrea M; Chia, Hannah E; Ogonowsky, Alexie L; Chiang, Sharon; Filipovic, Nedim; Weiden, Aurora G; Hadley, Emma E; Gabriel, Sara E; Leconte, Aaron M

    2015-09-29

    Chemical modifications to DNA, such as 2' modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2'-modified DNA nucleotides. While these mutant enzymes recognize these modified nucleotides, they are not capable of synthesizing full length modified DNA; thus, further engineering is required for these enzymes. Here, we describe comparative biochemical studies that identify useful, but previously uncharacterized, properties of these enzymes; one enzyme, SFM19, is able to recognize a range of 2'-modified nucleotides much wider than that previously examined, including fluoro, azido, and amino modifications. To understand the molecular origins of these differences, we also identify specific amino acids and combinations of amino acids that contribute most to the previously evolved unnatural activity. Our data suggest that a negatively charged amino acid at 614 and mutation of the steric gate residue, E615, to glycine make up the optimal combination for modified oligonucleotide synthesis. These studies yield an improved understanding of the mutational origins of 2'-modified substrate recognition as well as identify SFM19 as the best candidate for further engineering, whether via rational design or directed evolution. PMID:26334839

  19. A DNA polymerase activity is associated with Cauliflower Mosaic Virus.

    PubMed Central

    Menissier, J; Laquel, P; Lebeurier, G; Hirth, L

    1984-01-01

    A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa. Images PMID:6514573

  20. Incorporation of reporter-labeled nucleotides by DNA polymerases.

    PubMed

    Anderson, Jon P; Angerer, Bernhard; Loeb, Lawrence A

    2005-02-01

    The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA. PMID:15727132

  1. Structure–Function Studies of DNA Polymerase λ

    PubMed Central

    2015-01-01

    DNA polymerase λ (pol λ) functions in DNA repair with its main roles considered to be filling short gaps during repair of double-strand breaks by nonhomologous end joining and during base excision repair. As indicated by structural and biochemical studies over the past 10 years, pol λ shares many common properties with other family X siblings (pol β, pol μ, and terminal deoxynucleotidyl transferase) but also has unique structural features that determine its specific functions. In this review, we consider how structural studies over the past decade furthered our understanding of the behavior and biological roles of pol λ. PMID:24716527

  2. A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II[OPEN

    PubMed Central

    Qu, Jie; Ji, Shaoyi; Wallace, Andrew J.; Wu, Jian; Li, Yi; Gopalan, Venkat; Ding, Biao

    2016-01-01

    Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP. PSTVd replication in the nucleoplasm generates (−)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (−)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes. PMID:27113774

  3. A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II.

    PubMed

    Wang, Ying; Qu, Jie; Ji, Shaoyi; Wallace, Andrew J; Wu, Jian; Li, Yi; Gopalan, Venkat; Ding, Biao

    2016-05-01

    Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP PSTVd replication in the nucleoplasm generates (-)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (-)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes. PMID:27113774

  4. Dynamic Localization of Trypanosoma brucei Mitochondrial DNA Polymerase ID

    PubMed Central

    Concepción-Acevedo, Jeniffer; Luo, Juemin

    2012-01-01

    Trypanosomes contain a unique form of mitochondrial DNA called kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Several proteins are essential for network replication, and most of these localize to the antipodal sites or the kinetoflagellar zone. Essential components for kDNA synthesis include three mitochondrial DNA polymerases TbPOLIB, TbPOLIC, and TbPOLID). In contrast to other kDNA replication proteins, TbPOLID was previously reported to localize throughout the mitochondrial matrix. This spatial distribution suggests that TbPOLID requires redistribution to engage in kDNA replication. Here, we characterize the subcellular distribution of TbPOLID with respect to the Trypanosoma brucei cell cycle using immunofluorescence microscopy. Our analyses demonstrate that in addition to the previously reported matrix localization, TbPOLID was detected as discrete foci near the kDNA. TbPOLID foci colocalized with replicating minicircles at antipodal sites in a specific subset of the cells during stages II and III of kDNA replication. Additionally, the TbPOLID foci were stable following the inhibition of protein synthesis, detergent extraction, and DNase treatment. Taken together, these data demonstrate that TbPOLID has a dynamic localization that allows it to be spatially and temporally available to perform its role in kDNA replication. PMID:22286095

  5. Structure and mechanism of human DNA polymerase [eta

    SciTech Connect

    Biertümpfel, Christian; Zhao, Ye; Kondo, Yuji; Ramón-Maiques, Santiago; Gregory, Mark; Lee, Jae Young; Masutani, Chikahide; Lehmann, Alan R.; Hanaoka, Fumio; Yang, Wei

    2010-11-03

    The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase {eta} (Pol{eta}), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol{eta} at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol{eta} acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol{eta} orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol{eta} missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol{eta} in replicating through D loop and DNA fragile sites.

  6. Conformational selection and induced fit for RNA polymerase and RNA/DNA hybrid backtracked recognition

    PubMed Central

    Wu, Jian; Ye, Wei; Yang, Jingxu; Chen, Hai-Feng

    2015-01-01

    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. PMID:26594643

  7. Terminal Deoxynucleotidyl Transferase: The Story of a Misguided DNA Polymerase

    PubMed Central

    Motea, Edward A.; Berdis, Anthony J.

    2009-01-01

    Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis. PMID:19596089

  8. An Overview of Y-Family DNA Polymerases and a Case Study of Human DNA Polymerase η

    PubMed Central

    2015-01-01

    Y-Family DNA polymerases specialize in translesion synthesis, bypassing damaged bases that would otherwise block the normal progression of replication forks. Y-Family polymerases have unique structural features that allow them to bind damaged DNA and use a modified template base to direct nucleotide incorporation. Each Y-Family polymerase is unique and has different preferences for lesions to bypass and for dNTPs to incorporate. Y-Family polymerases are also characterized by a low catalytic efficiency, a low processivity, and a low fidelity on normal DNA. Recruitment of these specialized polymerases to replication forks is therefore regulated. The catalytic center of the Y-Family polymerases is highly conserved and homologous to that of high-fidelity and high-processivity DNA replicases. In this review, structural differences between Y-Family and A- and B-Family polymerases are compared and correlated with their functional differences. A time-resolved X-ray crystallographic study of the DNA synthesis reaction catalyzed by the Y-Family DNA polymerase human polymerase η revealed transient elements that led to the nucleotidyl-transfer reaction. PMID:24716551

  9. Iridium Complexes as a Roadblock for DNA Polymerase during Amplification.

    PubMed

    Chandra, Falguni; Kumar, Prashant; Tripathi, Suman Kumar; Patra, Srikanta; Koner, Apurba L

    2016-07-01

    Iridium-based metal complexes containing polypyridyl-pyrazine ligands show properties of DNA intercalation. They serve as roadblocks to DNA polymerase activity, thereby inhibiting the polymerization process. Upon the addition of increasing concentrations of these iridium complexes, a rapid polymerase chain reaction (PCR)-based assay reveals the selective inhibition of the DNA polymerization process. This label-free approach to study the inhibition of fundamental cellular processes via physical roadblock can offer an alternative route toward cancer therapy. PMID:27240728

  10. Phosphoesterase domains associated with DNA polymerases of diverse origins.

    PubMed Central

    Aravind, L; Koonin, E V

    1998-01-01

    Computer analysis of DNA polymerase protein sequences revealed previously unidentified conserved domains that belong to two distinct superfamilies of phosphoesterases. The alpha subunits of bacterial DNA polymerase III and two distinct family X DNA polymerases are shown to contain an N-terminal domain that defines a novel enzymatic superfamily, designated PHP, after polymerase and histidinol phosphatase. The predicted catalytic site of the PHP superfamily consists of four motifs containing conserved histidine residues that are likely to be involved in metal-dependent catalysis of phosphoester bond hydrolysis. The PHP domain is highly conserved in all bacterial polymerase III alpha subunits, but in proteobacteria and mycoplasmas, the conserved motifs are distorted, suggesting a loss of the enzymatic activity. Another conserved domain, found in the small subunits of archaeal DNA polymerase II and eukaryotic DNA polymerases alpha and delta, is shown to belong to the superfamily of calcineurin-like phospho-esterases, which unites a variety of phosphatases and nucleases. The conserved motifs required for phospho-esterase activity are intact in the archaeal DNA polymerase subunits, but are disrupted in their eukaryotic orthologs. A hypothesis is proposed that bacterial and archaeal replicative DNA polymerases possess intrinsic phosphatase activity that hydrolyzes the pyrophosphate released during nucleotide polymerization. As proposed previously, pyrophosphate hydrolysis may be necessary to drive the polymerization reaction forward. The phosphoesterase domains with disrupted catalytic motifs may assume an allosteric, regulatory function and/or bind other subunits of DNA polymerase holoenzymes. In these cases, the pyrophosphate may be hydrolyzed by a stand-alone phosphatase, and candidates for such a role were identified among bacterial PHP superfamily members. PMID:9685491

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

    SciTech Connect

    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.

  12. Identification of a New Motif in Family B DNA Polymerases by Mutational Analyses of the Bacteriophage T4 DNA Polymerase

    PubMed Central

    Li, Vincent; Hogg, Matthew; Reha-Krantz, Linda J.

    2011-01-01

    Structure-based protein sequence alignments of family B DNA polymerases revealed a conserved motif that is formed from interacting residues between loops from the N-terminal and palm domains and between the N-terminal loop and a conserved proline residue. The importance of the motif for function of the bacteriophage T4 DNA polymerase was revealed by suppressor analysis. T4 DNA polymerases that form weak replicating complexes cannot replicate DNA when the dGTP pool is reduced. The conditional lethality provides the means to identify amino acid substitutions that restore replication activity under low dGTP conditions by either correcting the defect produced by the first amino acid substitution or by generally increasing the stability of polymerase complexes; the second type are global suppressors that can effectively counter the reduced stability caused by a variety of amino acid substitutions. Some amino acid substitutions that increase the stability of polymerase complexes produce a new phenotype - sensitivity to the antiviral drug phosphonoacetic acid. Amino acid substitutions that confer decreased ability to replicate DNA under low dGTP conditions or drug sensitivity were identified in the new motif, which suggests that the motif functions in regulating the stability of polymerase complexes. Additional suppressor analyses revealed an apparent network of interactions that link the new motif to the fingers domain and to two patches of conserved residues that bind DNA. The collection of mutant T4 DNA polymerases provides a foundation for future biochemical studies to determine how DNA polymerases remain stably associated with DNA while waiting for the next available dNTP, how DNA polymerases translocate, and the biochemical basis for sensitivity to antiviral drugs. PMID:20493878

  13. Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA

    SciTech Connect

    Xing, G.; Kirouac, K.; Shin, Y.J.; Bell, S.D.; Ling, H.

    2009-09-16

    DNA polymerases are co-ordinated by sliding clamps (PCNA/{beta}-clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full-length Y-family polymerase, Dpo4, in complex with heterodimeric PCNA1-PCNA2 at 2.05 {angstrom} resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo- or DNA-bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C-terminal peptide of Dpo4 becomes structured with a 3{sub 10} helix and dominates the specific binding. The Y-family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation-dependent protein-protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding-coupled multiple conformations of a Y-family polymerase facilitate its recruitment and co-ordination on the sliding clamp. The conformational flexibility would turn the error-prone Y-family polymerase off when more efficient high-fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.

  14. DNA replication defect in Salmonella typhimurium mutants lacking the editing (epsilon) subunit of DNA polymerase III.

    PubMed Central

    Lifsics, M R; Lancy, E D; Maurer, R

    1992-01-01

    In Salmonella typhimurium, dnaQ null mutants (encoding the epsilon editing subunit of DNA polymerase III [Pol III]) exhibit a severe growth defect when the genetic background is otherwise wild type. Suppression of the growth defect requires both a mutation affecting the alpha (polymerase) subunit of DNA polymerase III and adequate levels of DNA polymerase I. In the present paper, we report on studies that clarify the nature of the physiological defect imposed by the loss of epsilon and the mechanism of its suppression. Unsuppressed dnaQ mutants exhibited chronic SOS induction, indicating exposure of single-stranded DNA in vivo, most likely as gaps in double-stranded DNA. Suppression of the growth defect was associated with suppression of SOS induction. Thus, Pol I and the mutant Pol III combined to reduce the formation of single-stranded DNA or accelerate its maturation to double-stranded DNA. Studies with mutants in major DNA repair pathways supported the view that the defect in DNA metabolism in dnaQ mutants was at the level of DNA replication rather than of repair. The requirement for Pol I was satisfied by alleles of the gene for Pol I encoding polymerase activity or by rat DNA polymerase beta (which exhibits polymerase activity only). Consequently, normal growth is restored to dnaQ mutants when sufficient polymerase activity is provided and this compensatory polymerase activity can function independently of Pol III. The high level of Pol I polymerase activity may be required to satisfy the increased demand for residual DNA synthesis at regions of single-stranded DNA generated by epsilon-minus pol III. The emphasis on adequate polymerase activity in dnaQ mutants is also observed in the purified alpha subunit containing the suppressor mutation, which exhibits a modestly elevated intrinsic polymerase activity relative to that of wild-type alpha. Images PMID:1400246

  15. DNA Polymerase δ Is Preferentially Recruited during Homologous Recombination To Promote Heteroduplex DNA Extension▿

    PubMed Central

    Maloisel, Laurent; Fabre, Francis; Gangloff, Serge

    2008-01-01

    DNA polymerases play a central role during homologous recombination (HR), but the identity of the enzyme(s) implicated remains elusive. The pol3-ct allele of the gene encoding the catalytic subunit of DNA polymerase δ (Polδ) has highlighted a role for this polymerase in meiotic HR. We now address the ubiquitous role of Polδ during HR in somatic cells. We find that pol3-ct affects gene conversion tract length during mitotic recombination whether the event is initiated by single-strand gaps following UV irradiation or by site-specific double-strand breaks. We show that the pol3-ct effects on gene conversion are completely independent of mismatch repair, indicating that shorter gene conversion tracts in pol3-ct correspond to shorter extensions of primed DNA synthesis. Interestingly, we find that shorter repair tracts do not favor synthesis-dependent strand annealing at the expense of double-strand-break repair. Finally, we show that the DNA polymerases that have been previously suspected to mediate HR repair synthesis (Polɛ and Polη) do not affect gene conversion during induced HR, including in the pol3-ct background. Our results argue strongly for the preferential recruitment of Polδ during HR. PMID:18086882

  16. Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants

    PubMed Central

    Morley, Stewart A.; Nielsen, Brent L.

    2016-01-01

    Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles. PMID:26870072

  17. Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA).

    PubMed Central

    Focher, F; Gassmann, M; Hafkemeyer, P; Ferrari, E; Spadari, S; Hübscher, U

    1989-01-01

    DNA polymerase delta from calf thymus was purified under conditions that minimized proteolysis to a specific activity of 27,000 units/mg. The four step isolation procedure included phosphocellulose, hydroxyapatite, heparin-Sepharose and FPLC-MonoS. This enzyme consists of four polypeptides with Mr of 140, 125, 48 and 40 kilodaltons. Velocity gradient sedimentation in glycerol removed the 48 kDa polypeptide while the other three sedimented with the DNA polymerase activity. The biochemical properties of the three subunit enzyme and the copurification of 3'----5' exonuclease activity were typical for a bona fide DNA polymerase delta. Tryptic peptide analysis showed that the 140 kDa polypeptide was different from the catalytic 180 kDa polypeptide of calf thymus DNA polymerase alpha. Both high Mr polypeptides (140 and 125 kDa) were catalytically active as analysed in an activity gel. Four templates were used by DNA polymerase delta with different preferences, namely poly(dA)/oligo(dT)12-18 much much greater than activated DNA greater than poly(dA-dT) greater than primed single-stranded M13DNA. Calf thymus proliferating cell nuclear antigen (PCNA) could not stimulated this DNA polymerase delta in any step of the isolation procedure. If tested on poly(dA)/oligo(dT)12-18 (base ratio 10:1), PCNA had no stimulatory effect on DNA polymerase delta when tested with low enzyme DNA ratio nor did it change the kinetic behaviour of the enzyme. DNA polymerase delta itself did not contain PCNA. The enzyme had an intrinsic processivity of several thousand bases, when tested either on the homopolymer poly(dA)/oligo(dT)12-18 (base ratio 64:1) or on primed single-stranded M13DNA. Contrary to DNA polymerase alpha, no pausing sites were seen with DNA polymerase delta. Under optimal in vitro replication conditions the enzyme could convert primed single-stranded circular M13 DNA of 7,200 bases to its double-stranded form in less than 10 min. This supports that a PCNA independent DNA

  18. The eureka enzyme: the discovery of DNA polymerase.

    PubMed

    Friedberg, Errol C

    2006-02-01

    The identification and partial purification by Arthur Kornberg and his colleagues in 1956 of an enzyme - DNA polymerase I of Escherichia coli - that catalysed the stable incorporation of deoxyribonucleotides into DNA in vitro came as a surprise. At the time, most scientists in the field believed that DNA synthesis was too complicated to be accurately reflected outside the living cell. PMID:16493419

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

    PubMed

    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. PMID:26289299

  20. Kinetics and thermodynamics of DNA polymerases with exonuclease proofreading

    NASA Astrophysics Data System (ADS)

    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.

  1. A meiotic DNA polymerase from a mushroom, Agaricus bisporus.

    PubMed Central

    Takami, K; Matsuda, S; Sono, A; Sakaguchi, K

    1994-01-01

    A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state. Images Figure 2 Figure 5 Figure 6 PMID:8172591

  2. Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein.

    PubMed Central

    Pavco, P A; Steege, D A

    1991-01-01

    As a means of generating homogeneous populations of elongation complexes with the RNA polymerases encoded by phages T7 and SP6, transcription has been carried out in vitro on templates associated with the Gln-111 mutant of EcoRI endonuclease. The Gln-111 protein, as a result of a single amino acid substitution at position 111, lacks cleavage function yet shows higher than wild-type affinity for the EcoRI recognition sequence GAATTC. On a series of linear and circular templates associated with Gln-111 protein, blockage of the phage RNA polymerase elongation complex is observed. The 3' endpoint of the major blocked-length RNA species, just 3 bp upstream from the GAATTC, reveals an extremely close approach of polymerase's leading edge to essential contacts between Gln-111 protein and its binding site. In contrast to E. coli RNA polymerase, which is blocked stably and quantitatively by Gln-111 protein (Pavco, P.A. and Steege, D. A. (1990) J. Biol. Chem. 265, 9960-9969), the phage polymerases show substantial levels of readthrough transcription beyond the protein block. Images PMID:1891355

  3. Kinetics and thermodynamics of exonuclease-deficient DNA polymerases

    NASA Astrophysics Data System (ADS)

    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.

  4. Formation of phosphonester bonds catalyzed by DNA polymerase.

    PubMed Central

    Victorova, L S; Dyatkina, N B; Mozzherin DJu; Atrazhev, A M; Krayevsky, A A; Kukhanova, M K

    1992-01-01

    3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma- diphosphate and 2'-deoxythymidine-5'-(alpha-methylphosphonyl)-beta, gamma- diphosphate have been synthesized. Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase beta from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E. coli DNA polymerase I, Klenow fragment. The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains. For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound. DNA polymerases alpha and epsilon from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned. The 3'----5'-exonuclease activity of DNA polymerase I, Klenow fragment, hydrolyzed DNA fragments containing phosphonomethyl internucleoside groups, while such DNA fragments were resistant to the E. coli exonuclease III. Images PMID:1371865

  5. A binding free energy decomposition approach for accurate calculations of the fidelity of DNA polymerases

    PubMed Central

    Rucker, Robert; Oelschlaeger, Peter; Warshel, Arieh

    2010-01-01

    DNA polymerase β (pol β) is a small eukaryotic enzyme with the ability to repair short single-stranded DNA gaps that has found use as a model system for larger replicative DNA polymerases. For all DNA polymerases, the factors determining their catalytic power and fidelity are the interactions between the bases of the base pair, amino acids near the active site, and the two magnesium ions. In this report, we study effects of all three aspects on human pol β transition state (TS) binding free energies by reproducing a consistent set of experimentally determined data for different structures. Our calculations comprise the combination of four different base pairs (incoming pyrimidine nucleotides incorporated opposite both matched and mismatched purines) with four different pol β structures (wild type and three separate mutations of ionized residues to alanine). We decompose the incoming deoxynucleoside 5′-triphosphate-TS, and run separate calculations for the neutral base part and the highly charged triphosphate part, using different dielectric constants in order to account for the specific electrostatic environments. This new approach improves our ability to predict the effect of matched and mismatched base pairing and of mutations in DNA polymerases on fidelity and may be a useful tool in studying the potential of DNA polymerase mutations in the development of cancer. It also supports our point of view with regards to the origin of the structural control of fidelity, allowing for a quantified description of the fidelity of DNA polymerases. PMID:19842163

  6. Human DNA polymerase θ grasps the primer terminus to mediate DNA repair.

    PubMed

    Zahn, Karl E; Averill, April M; Aller, Pierre; Wood, Richard D; Doublié, Sylvie

    2015-04-01

    DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break-inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining. PMID:25775267

  7. Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

    PubMed Central

    Zahn, Karl E.; Averill, April M.; Aller, Pierre; Wood, Richard D.; Doublié, Sylvie

    2015-01-01

    DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Breast, lung and oral cancers over-express polymerase θ, and reduction of its activity in mammalian cells increases sensitivity to double-strand break inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ employs a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction to the 3’-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions, or extend poorly annealed DNA termini to mediate end-joining. PMID:25775267

  8. Getting it Right: How DNA Polymerases Select the Right Nucleotide.

    PubMed

    Ludmann, Samra; Marx, Andreas

    2016-01-01

    All living organisms are defined by their genetic code encrypted in their DNA. DNA polymerases are the enzymes that are responsible for all DNA syntheses occurring in nature. For DNA replication, repair and recombination these enzymes have to read the parental DNA and recognize the complementary nucleotide out of a pool of four structurally similar deoxynucleotide triphosphates (dNTPs) for a given template. The selection of the nucleotide is in accordance with the Watson-Crick rule. In this process the accuracy of DNA synthesis is crucial for the maintenance of the genome stability. However, to spur evolution a certain degree of freedom must be allowed. This brief review highlights the mechanistic basis for selecting the right nucleotide by DNA polymerases. PMID:27052761

  9. Optimal numbers of residues in linkers of DNA polymerase I, T7 primase and DNA polymerase IV.

    PubMed

    Fu, Yi-Ben; Wang, Zhan-Feng; Wang, Peng-Ye; Xie, Ping

    2016-01-01

    DNA polymerase I (PolI), T7 primase and DNA polymerase IV (Dpo4) have a common feature in their structures that the two main domains are connected by an unstructured polypeptide linker. To perform their specific enzymatic activities, the enzymes are required to rearrange the position and orientation of one domain relative to the other into an active mode. Here, we show that the three enzymes share the same mechanism of the transition from the inert to active modes and use the minimum numbers of residues in their linkers to achieve the most efficient transitions. The transition time to the finally active mode is sensitively dependent on the stretched length of the linker in the finally active mode while is insensitive to the position and orientation in the initially inert state. Moreover, we find that for any enzyme whose two domains are connected by an unstructured flexible linker, the stretched length (L) of the linker in the finally active mode and the optimal number (Nopt) of the residues in the linker satisfy relation L ≈ αNopt, with α = 0.24-0.27 nm being a constant insensitive to the system. PMID:27364863

  10. Optimal numbers of residues in linkers of DNA polymerase I, T7 primase and DNA polymerase IV

    PubMed Central

    Fu, Yi-Ben; Wang, Zhan-Feng; Wang, Peng-Ye; Xie, Ping

    2016-01-01

    DNA polymerase I (PolI), T7 primase and DNA polymerase IV (Dpo4) have a common feature in their structures that the two main domains are connected by an unstructured polypeptide linker. To perform their specific enzymatic activities, the enzymes are required to rearrange the position and orientation of one domain relative to the other into an active mode. Here, we show that the three enzymes share the same mechanism of the transition from the inert to active modes and use the minimum numbers of residues in their linkers to achieve the most efficient transitions. The transition time to the finally active mode is sensitively dependent on the stretched length of the linker in the finally active mode while is insensitive to the position and orientation in the initially inert state. Moreover, we find that for any enzyme whose two domains are connected by an unstructured flexible linker, the stretched length (L) of the linker in the finally active mode and the optimal number (Nopt) of the residues in the linker satisfy relation L ≈ αNopt, with α = 0.24–0.27 nm being a constant insensitive to the system. PMID:27364863

  11. A phage-encoded inhibitor of Escherichia coli DNA replication targets the DNA polymerase clamp loader.

    PubMed

    Yano, Sho T; Rothman-Denes, Lucia B

    2011-03-01

    Coliphage N4 infection leads to shut-off of host DNA replication without inhibition of host transcription or translation. We report the identification and characterization of gp8, the N4 gene product responsible for this phenotype. N4 gp8 is an Escherichia coli bacteriostatic inhibitor that colocalizes with the E. coli replisome in a replication-dependent manner. Gp8 was purified and observed to cross-link to complexes containing the replicative DNA polymerase, DNAP III, in vivo. Purified gp8 inhibits DNA polymerization by DNA polymerase III holoenzyme in vitro by interfering with polymerase processivity. Gp8 specifically inhibits the clamp-loading activity of DNAP III by targeting the delta subunit of the DNAP III clamp loader; E. coli mutations conferring gp8 resistance were identified in the holA gene, encoding delta. Delta and gp8 interact in vitro; no interaction was detected between gp8 inactive mutants and wild-type delta or between delta gp8-resistant mutants and wild-type gp8. Therefore, this work identifies the DNAP III clamp loader as a new target for inhibition of bacterial growth. Finally, we show that gp8 is not essential in N4 development under laboratory conditions, but its activity contributes to phage yield. PMID:21205014

  12. [The biological effect of Y-family DNA polymerases on the translesion synthesis].

    PubMed

    Gong, Yi; Yang, Jin

    2013-02-01

    A common DNA polymerase can replicate DNA which functions normally. However, if DNA suffers damage, the genome can not be replicated by a common DNA polymerase because DNA lesions will block the replication apparatus. Another kind of DNA polymerases in organism, Y-family DNA polymerases which is also called translesion synthesis (TLS) polymerases, can deal with this problem. Their main functions are bypassing the lesions in DNA, replicating the genome and saving the dying cells. This thesis presents a historical review of the literature pertinent to the structure, functions and roles of Y-family DNA polymerases. PMID:23488167

  13. Mutational clusters generated by non-processive polymerases: A case study using DNA polymerase betain vitro.

    PubMed

    García-Villada, Libertad; Drake, John W

    2010-08-01

    Available DNA mutational spectra reveal that the number of mutants with multiple mutations ("multiples") is usually greater than expected from a random distribution of mutations among mutants. These overloads imply the occurrence of non-random clusters of mutations, probably generated during episodes of low-fidelity DNA synthesis. Excess multiples have been reported not only for viruses, bacteria, and eukaryotic cells but also for the DNA polymerases of phages T4 and RB69 in vitro. In the simplest case of a purified polymerase, non-random clusters may be generated by a subfraction of phenotypic variants able to introduce more errors per cycle of DNA synthesis than the normal enzyme. According to this hypothesis, excess multiples are not expected with non-processive polymerases even if they harbor rare mutator variants. DNA polymerase beta (Pol beta) is a mammalian DNA-repair polymerase with very low processivity. Although several Pol beta mutational spectra have been described, there is conflicting evidence on whether or not excess multiples occur, with spectra based on the HSV-tk system tending to show excess multiples. Excess multiples generated by Pol beta or any of its mutants might imply that the excesses of multiples observed in numerous other systems, especially those with processive polymerases, could be artifactual. Here, the distributions of mutations generated by native and recombinant rat Pol beta and by the Pol beta(Y265C) mutator were analyzed in the M13mp2 lacZalpha system. Our results present no evidence for a significant excess of multiples over the expected numbers with any of the Pol beta enzymes tested in this system. The reported excess of Pol beta-generated multiples in the HSV-tk system may reflect a reduced efficiency of detection of base substitutions that cause weak phenotypes, which in turn may artifactually increase the frequency of multiples. PMID:20627824

  14. Inhibition and site modification of human hepatitis B virus DNA polymerase by pyridoxal 5'-phosphate

    SciTech Connect

    Oh, S.H.; Park, Y.H.; Kim, I.S.; Woo, K.

    1987-05-01

    Pyridoxal 5'-phosphate(PLP) modification of human hepatitis B virus (H3V) DNA polymerase was attempted in order to characterize the nature of the enzyme. Dane particle cores isolated from serum of a chronic HBV carrier by sucrose density gradient centrifugation contained DNA polymerase activity, and the enzyme activity was inhibited specifically by PLP in noncompetitive fashion with respective to dNTP. Kinetic study indicates that HBV DNA polymerase has a Km of 0.31..mu..M for dTTP and an apparent Ki of 2mM for PLP. Sodium borohydride reduction of PLP-HEV core particles caused almost complete inhibition of HBV DNA polymerase activity. Reduction of PLP-HBV core particles by /sup 3/H labeled NaBH4 followed by SDS polyacrylamide gel electrophoresis was carried out, and the fluorography of the SDS polyacrylamide gel revealed 3 major bands corresponding to molecular weights of 21,000, 80,000 and > 100,000. Dane particle associated DNA polymerase inhibition by PLP is mediated through Schiff's base formation with a free amino group present at catalytic site of the enzyme. A core protein having an approximate molecular weight of 80,000 is considered as HBV DNA polymerase.

  15. Cooperation between catalytic and DNA binding domains enhances thermostability and supports DNA synthesis at higher temperatures by thermostable DNA polymerases.

    PubMed

    Pavlov, Andrey R; Pavlova, Nadejda V; Kozyavkin, Sergei A; Slesarev, Alexei I

    2012-03-13

    We have previously introduced a general kinetic approach for comparative study of processivity, thermostability, and resistance to inhibitors of DNA polymerases [Pavlov, A. R., et al. (2002) Proc. Natl. Acad. Sci. U.S.A.99, 13510-13515]. The proposed method was successfully applied to characterize hybrid DNA polymerases created by fusing catalytic DNA polymerase domains with various sequence-nonspecific DNA binding domains. Here we use the developed kinetic analysis to assess basic parameters of DNA elongation by DNA polymerases and to further study the interdomain interactions in both previously constructed and new chimeric DNA polymerases. We show that connecting helix-hairpin-helix (HhH) domains to catalytic polymerase domains can increase thermostability, not only of DNA polymerases from extremely thermophilic species but also of the enzyme from a faculatative thermophilic bacterium Bacillus stearothermophilus. We also demonstrate that addition of Topo V HhH domains extends efficient DNA synthesis by chimerical polymerases up to 105 °C by maintaining processivity of DNA synthesis at high temperatures. We found that reversible high-temperature structural transitions in DNA polymerases decrease the rates of binding of these enzymes to the templates. Furthermore, activation energies and pre-exponential factors of the Arrhenius equation suggest that the mechanism of electrostatic enhancement of diffusion-controlled association plays a minor role in binding of templates to DNA polymerases. PMID:22320201

  16. DNA sequencing using polymerase substrate-binding kinetics

    PubMed Central

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  17. DNA sequencing using polymerase substrate-binding kinetics.

    PubMed

    Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

    2015-01-01

    Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

  18. The roles of DNA polymerase ζ and the Y family DNA polymerases in promoting or preventing genome instability

    PubMed Central

    Sharma, Shilpy; Helchowski, Corey M.; Canman, Christine E.

    2012-01-01

    Cancer cells display numerous abnormal characteristics which are initiated and maintained by elevated mutation rates and genome instability. Chromosomal DNA is continuously surveyed for the presence of damage or blocked replication forks by the DNA Damage Response (DDR) network. The DDR is complex and includes activation of cell cycle checkpoints, DNA repair, gene transcription, and induction of apoptosis. Duplicating a damaged genome is associated with elevated risks to fork collapse and genome instability. Therefore, the DNA Damage Tolerance (DDT) pathway is also employed to enhance survival and involves the recruitment of translesion DNA synthesis (TLS) polymerases to sites of replication fork blockade or single stranded DNA gaps left after the completion of replication in order to restore DNA to its double stranded form before mitosis. TLS polymerases are specialized for inserting nucleotides opposite DNA adducts, abasic sites, or DNA crosslinks. By definition, the DDT pathway is not involved in the actual repair of damaged DNA, but provides a mechanism to tolerate DNA lesions during replication thereby increasing survival and lessening the chance for genome instability. However this may be associated with increased mutagenesis. In this review, we will describe the specialized functions of Y family polymerases (Rev1, Polη, Polι and Polκ) and DNA polymerase ζ in lesion bypass, mutagenesis, and prevention of genome instability, the latter due to newly appreciated roles in DNA repair. The recently described role of the Fanconi anemia pathway in regulating Rev1 and Polζ-dependent TLS is also discussed in terms of their involvement in TLS, interstrand crosslink repair, and homologous recombination. PMID:23195997

  19. Phosphorothioate primers improve the amplification of DNA sequences by DNA polymerases with proofreading activity.

    PubMed Central

    Skerra, A

    1992-01-01

    Two thermostable DNA polymerases with proofreading activity--Vent DNA polymerase and Pfu DNA polymerase--have attracted recent attention, mainly because of their enhanced fidelities during amplification of DNA sequences by the polymerase chain reaction. A severe disadvantage for their practical application, however, results from the observation that due to their 3' to 5' exonuclease activities these enzymes degrade the oligodeoxynucleotides serving as primers for the DNA synthesis. It is demonstrated that this exonucleolytic attack on the primer molecules can be efficiently prevented by the introduction of single phosphorothioate bonds at their 3' termini. This strategy, which can be easily accomplished using routine DNA synthesis methodology, may open the way to a widespread use of these novel enzymes in the polymerase chain reaction. Images PMID:1641322

  20. Efficient DNA sequencing on microtiter plates using dried reagents and Bst DNA polymerase.

    PubMed

    Earley, J J; Kuivaniemi, H; Prockop, D J; Tromp, G

    1993-01-01

    Sequenase, Taq DNA polymerase and Bst DNA polymerase were tested for sequencing of DNA on microtiter plates using dried down reagents. Several parameters were investigated to expedite the drying process while minimizing damage to the enzyme. Sequenase did not tolerate drying very well, and frequently generated sequences with weak signals and many sites of premature termination. With Taq DNA polymerase it was possible to obtain sequences of good quality. However, there was considerable variation of results between experiments and between batches of microtiter plates. Bst DNA polymerase generated sequences of excellent quality. It was stable for more than a week in dried-down state at -20 degrees C and at least overnight at room temperature. The method described here using Bst DNA polymerase is well suited for laboratory robots and workstations that typically employ 96-well microtiter plates. PMID:8173079

  1. Expression and Characterization of the RKOD DNA Polymerase in Pichia pastoris

    PubMed Central

    Wang, Fei; Li, Shuntang; Zhao, Hui; Bian, Lu; Chen, Liang; Zhang, Zhen; Zhong, Xing; Ma, Lixin; Yu, Xiaolan

    2015-01-01

    The present study assessed high-level expression of the KOD DNA polymerase in Pichia pastoris. Thermococcus kodakaraensis KOD1 is a DNA polymerase that is widely used in PCR. The DNA coding sequence of KOD was optimized based on the codon usage bias of P. pastoris and synthesized by overlapping PCR, and the nonspecific DNA-binding protein Sso7d from the crenarchaeon Sulfolobus solfataricus was fused to the C-terminus of KOD. The resulting novel gene was cloned into a pHBM905A vector and introduced into P. pastoris GS115 for secretory expression. The yield of the target protein reached approximately 250 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks. This yield is much higher than those of other DNA polymerases expressed heterologously in Escherichia coli. The recombinant enzyme was purified, and its enzymatic features were studied. Its specific activity was 19,384 U/mg. The recombinant KOD expressed in P. pastoris exhibited excellent thermostability, extension rate and fidelity. Thus, this report provides a simple, efficient and economic approach to realize the production of a high-performance thermostable DNA polymerase on a large scale. This is the first report of the expression in yeast of a DNA polymerase for use in PCR. PMID:26134129

  2. DNA Polymerases Divide the Labor of Genome Replication.

    PubMed

    Lujan, Scott A; Williams, Jessica S; Kunkel, Thomas A

    2016-09-01

    DNA polymerases synthesize DNA in only one direction, but large genomes require RNA priming and bidirectional replication from internal origins. We review here the physical, chemical, and evolutionary constraints underlying these requirements. We then consider the roles of the major eukaryotic replicases, DNA polymerases α, δ, and ɛ, in replicating the nuclear genome. Pol α has long been known to extend RNA primers at origins and on Okazaki fragments that give rise to the nascent lagging strand. Taken together, more recent results of mutation and ribonucleotide incorporation mapping, electron microscopy, and immunoprecipitation of nascent DNA now lead to a model wherein Pol ɛ and Pol δ, respectively, synthesize the majority of the nascent leading and lagging strands of undamaged DNA. PMID:27262731

  3. A solution to release twisted DNA during chromosome replication by coupled DNA polymerases

    PubMed Central

    Kurth, Isabel; Georgescu, Roxana E.; O’Donnell, Mike

    2013-01-01

    Chromosomal replication machines contain coupled DNA polymerases that simultaneously replicate the leading and lagging strands1. However, coupled replication presents a largely unrecognized topological problem. Since DNA polymerase must travel a helical path during synthesis, the physical connection between leading and lagging strand polymerases causes the daughter strands to entwine, or produces extensive buildup of negative supercoils in the newly synthesized DNA2–4. How DNA polymerases maintain their connection during coupled replication despite these topological challenges is a mystery. Here, we examine the dynamics of the E. coli replisome, by ensemble and single-molecule methods that may solve this topological problem independent of topoisomerases. We find that the lagging strand polymerase frequently releases from an Okazaki fragment before completion, leaving single-strand gaps behind. Dissociation of the polymerase does not result in loss from the replisome due to its contact with the leading-strand polymerase. This behavior, referred to as “signal release”, had been thought to require a protein, possibly primase, to pry polymerase from incompletely extended DNA fragments5–7. However, we observe that signal release is independent of primase and does not appear to require a protein trigger at all. Instead, the lagging-strand polymerase is simply less processive in the context of a replisome. Interestingly, when the lagging-strand polymerase is supplied with primed DNA in trans, uncoupling it from the fork, high processivity is restored. Hence, we propose that coupled polymerases introduce topological changes, possibly by accumulation of superhelical tension in the newly synthesized DNA, that cause lower processivity and transient lagging-strand polymerase dissociation from DNA. PMID:23535600

  4. EBV DNA polymerase inhibition of tannins from Eugenia uniflora.

    PubMed

    Lee, M H; Chiou, J F; Yen, K Y; Yang, L L

    2000-06-30

    Nasopharyngeal carcinoma (NPC) is one of the high population malignant tumors among Chinese in southern China and southeast Asia. Epstein-Barr virus (EBV) is a human B lymphotropic herpes virus which is known to be closely associated with NPC. EBV DNA polymerase is a key enzyme during EBV replication and is measured by its radioactivity. The addition of phorbol 12-myristate 13-acetate to Raji cell cultures led to a large increase in EBV DNA polymerase, which was purified by sequential DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography. Four tannins were isolated from the active fractions of Eugenia uniflora L., which were tested for the inhibition of EBV DNA polymerase. The results showed the 50% inhibitory concentration (IC(50)) values of gallocatechin, oenothein B, eugeniflorins D(1) and D(2) were 26.5 62.3, 3.0 and 3.5 microM, respectively. Furthermore, when compared with the positive control (phosphonoacetic acid), an inhibitor of EBV replication, the IC(50) value was 16.4 microM. In view of the results, eugeniflorins D(1) and D(2) are the potency principles in the inhibition of EBV DNA polymerase from E. uniflora. PMID:10806300

  5. Molecular basis for DNA strand displacement by NHEJ repair polymerases

    PubMed Central

    Bartlett, Edward J.; Brissett, Nigel C.; Plocinski, Przemyslaw; Carlberg, Tom; Doherty, Aidan J.

    2016-01-01

    The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal (Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair. PMID:26405198

  6. Molecular basis for DNA strand displacement by NHEJ repair polymerases.

    PubMed

    Bartlett, Edward J; Brissett, Nigel C; Plocinski, Przemyslaw; Carlberg, Tom; Doherty, Aidan J

    2016-03-18

    The non-homologous end-joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) in all domains of life. Archaea and bacteria utilize a conserved set of multifunctional proteins in a pathway termed Archaeo-Prokaryotic (AP) NHEJ that facilitates DSB repair. Archaeal NHEJ polymerases (Pol) are capable of strand displacement synthesis, whilst filling DNA gaps or partially annealed DNA ends, which can give rise to unligatable intermediates. However, an associated NHEJ phosphoesterase (PE) resects these products to ensure that efficient ligation occurs. Here, we describe the crystal structures of these archaeal (Methanocella paludicola) NHEJ nuclease and polymerase enzymes, demonstrating their strict structural conservation with their bacterial NHEJ counterparts. Structural analysis, in conjunction with biochemical studies, has uncovered the molecular basis for DNA strand displacement synthesis in AP-NHEJ, revealing the mechanisms that enable Pol and PE to displace annealed bases to facilitate their respective roles in DSB repair. PMID:26405198

  7. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies

    PubMed Central

    Macao, Bertil; Grönke, Sebastian; Siibak, Triinu; Stewart, James B; Baggio, Francesca; Dols, Jacqueline; Partridge, Linda; Falkenberg, Maria; Wredenberg, Anna; Larsson, Nils-Göran

    2016-01-01

    Replication errors are the main cause of mtDNA mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineered the tamas locus, encoding fly POLγA, and introduced alleles expressing exonuclease- (exo-) and polymerase-deficient (pol-) POLγA versions. The exo- mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol- mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. PMID:26554610

  8. Mapping DNA polymerase errors by single-molecule sequencing.

    PubMed

    Lee, David F; Lu, Jenny; Chang, Seungwoo; Loparo, Joseph J; Xie, Xiaoliang S

    2016-07-27

    Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replication product is tagged with a unique nucleotide sequence before amplification. This allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases. PMID:27185891

  9. Plastid DNA polymerases from higher plants, Arabidopsis thaliana

    SciTech Connect

    Mori, Yoko; Kimura, Seisuke; Saotome, Ai; Kasai, Nobuyuki; Sakaguchi, Norihiro; Uchiyama, Yukinobu; Ishibashi, Toyotaka; Yamamoto, Taichi; Chiku, Hiroyuki; Sakaguchi, Kengo . E-mail: kengo@rs.noda.sut.ac.jp

    2005-08-19

    Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H{sub 2}O{sub 2}. These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.

  10. Insertion of oxidized nucleotide triggers rapid DNA polymerase opening

    PubMed Central

    Kim, Taejin; Freudenthal, Bret D.; Beard, William A.; Wilson, Samuel H.; Schlick, Tamar

    2016-01-01

    A novel mechanism is unveiled to explain why a pro-mutagenic nucleotide lesion (oxidized guanine, 8-oxoG) causes the mammalian DNA repair polymerase-β (pol-β) to rapidly transition to an inactive open conformation. The mechanism involves unexpected features revealed recently in time-lapse crystallography. Specifically, a delicate water network associated with a lesion-stabilizing auxilliary product ion Mg(p) triggers a cascade of events that leads to poor active site geometry and the rupture of crucial molecular interactions between key residues in both the anti(8-oxoG:C) and syn(8-oxoG:A) systems. Once the base pairs in these lesioned systems are broken, dislocation of both Asp192 (a metal coordinating ligand) and the oxoG phosphate group (PO4) interfere with the hydrogen bonding between Asp192 and Arg258, whose rotation toward Asp192 is crucial to the closed-to-open enzyme transition. Energetically, the lesioned open states are similar in energy to those of the corresponding closed complexes after chemistry, in marked contrast to the unlesioned pol-β anti(G:C) system, whose open state is energetically higher than the closed state. The delicate surveillance system offers a fundamental protective mechanism in the cell that triggers DNA repair events which help deter insertion of oxidized lesions. PMID:27034465

  11. Insertion of oxidized nucleotide triggers rapid DNA polymerase opening.

    PubMed

    Kim, Taejin; Freudenthal, Bret D; Beard, William A; Wilson, Samuel H; Schlick, Tamar

    2016-05-19

    A novel mechanism is unveiled to explain why a pro-mutagenic nucleotide lesion (oxidized guanine, 8-oxoG) causes the mammalian DNA repair polymerase-β (pol-β) to rapidly transition to an inactive open conformation. The mechanism involves unexpected features revealed recently in time-lapse crystallography. Specifically, a delicate water network associated with a lesion-stabilizing auxilliary product ion Mg(p) triggers a cascade of events that leads to poor active site geometry and the rupture of crucial molecular interactions between key residues in both the anti(8-oxoG:C) and syn(8-oxoG:A) systems. Once the base pairs in these lesioned systems are broken, dislocation of both Asp192 (a metal coordinating ligand) and the oxoG phosphate group (PO4) interfere with the hydrogen bonding between Asp192 and Arg258, whose rotation toward Asp192 is crucial to the closed-to-open enzyme transition. Energetically, the lesioned open states are similar in energy to those of the corresponding closed complexes after chemistry, in marked contrast to the unlesioned pol-β anti(G:C) system, whose open state is energetically higher than the closed state. The delicate surveillance system offers a fundamental protective mechanism in the cell that triggers DNA repair events which help deter insertion of oxidized lesions. PMID:27034465

  12. Monitoring DNA polymerase with nanotube-based nanocircuits

    NASA Astrophysics Data System (ADS)

    Li, Yan; Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry; Collins, Philip

    DNA polymerases play an important role in the process of life by accurately and efficiently replicating our genetic information. They use a single-stranded DNA as a template and incorporate nucleotides to create the full, double-stranded DNA. Recent experiments have successfully monitored this process by attaching a Klenow fragment of polymerase I to a carbon nanotube and measuring the current along the tube. Follow-up experiments have shown promise for distinguishing between DNA base pairs when nucleotide analogs are used, thus opening a new avenue for DNA sequencing. In this talk, we present results from computational studies on DNA polymerase I nanocircuits. The enzyme was first equilibrated in molecular dynamics and then density functional theory and Keldysh non-equilibrium Green's function methods were used to calculate the ballistic transmission coefficients and currents for different enzymatic states. Our results show significant change in current when the enzyme alternates between open (idle) and closed (synthesizing) states. We can also differentiate between some template bases when modified nucleotides and gate scanning are used.

  13. Computational investigation of CNT-based DNA polymerase nanocircuits

    NASA Astrophysics Data System (ADS)

    Li, Yan; Hodak, Miroslav; Lu, Wenchang; Bernholc, Jerry; Collins, Philip

    2015-03-01

    DNA polymerases are important enzymes that replicate DNA molecules with very low error rates - about one error in 105 bases. Recently, it was found that the replication process can be electrically monitored by attaching a Klenow fragment of polymerase I to the surface of a carbon nanotube and monitoring the current along the tube [1]. In this talk, we report results from computational studies on DNA polymerase nanocircuits. We have first performed classical molecular dynamics (MD) calculations to get snapshots of different enzymatic stages, particularly the open state (no DNA binding) and the closed state (DNA double helix binding). We then used density functional theory (DFT) and Keldysh non-equilibrium Green's function (NEGF) formalism to calculate transmission coefficients and currents for each enzymatic state. Our results show that the transmission spectrum and the currents change significantly when the enzyme moves from the open to the closed state. While the initial experiments did not show signal differences between dissimilar bases, the theoretical work in progress is investigating conditions where bases might have distinct signatures, which would allow for DNA sequencing.

  14. Nucleotide excision repair DNA synthesis by excess DNA polymerase beta: a potential source of genetic instability in cancer cells.

    PubMed

    Canitrot, Y; Hoffmann, J S; Calsou, P; Hayakawa, H; Salles, B; Cazaux, C

    2000-09-01

    The nucleotide excision repair pathway contributes to genetic stability by removing a wide range of DNA damage through an error-free reaction. When the lesion is located, the altered strand is incised on both sides of the lesion and a damaged oligonucleotide excised. A repair patch is then synthesized and the repaired strand is ligated. It is assumed that only DNA polymerases delta and/or epsilon participate to the repair DNA synthesis step. Using UV and cisplatin-modified DNA templates, we measured in vitro that extracts from cells overexpressing the error-prone DNA polymerase beta exhibited a five- to sixfold increase of the ultimate DNA synthesis activity compared with control extracts and demonstrated the specific involvement of Pol beta in this step. By using a 28 nt gapped, double-stranded DNA substrate mimicking the product of the incision step, we showed that Pol beta is able to catalyze strand displacement downstream of the gap. We discuss these data within the scope of a hypothesis previously presented proposing that excess error-prone Pol beta in cancer cells could perturb the well-defined specific functions of DNA polymerases during error-free DNA transactions. PMID:10973926

  15. Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing. Final report, June 1, 1988--January 31, 1996

    SciTech Connect

    Richardson, C.C.

    1996-08-01

    This project has focused on the DNA polymerase of phage T7 for use in DNA sequencing. A complex of T7 DNA polymerase and E. coli thioredoxin form a highly processive DNA polymerase. The exonuclease activity of the enzyme can be reduced by chemical or genetic modifications resulting in an enzyme that has several properties useful in sequencing including high processivity and lack of discrimination against dideoxynucleotides. Manganese ion eliminates all discrimination against ddNTPs allowing sequence determination based on band intensity. A single tyrosine residue in the active site of T7 DNA polymerase is responsible for the efficient incorporation of ddNMPs. Replacement of the phenylalanine at this position in Klenow or Taq DNA polymerase with tyrosine eliminates discrimination against ddNTPs, a property that has advantages for cycle sequencing. Pyrophosphorolysis catalyzed by a polymerase results in the hydrolysis of specific fragments in DNA sequencing reactions, a problem that is eliminated by the addition of pyrophosphatase. The thioredoxin domain of gene 5 protein has been identified and transferred to Klenow DNA polymerase to make it processive. We have crystallized a complex of T7 DNA polymerase/thioredoxin bound to a primer-template in the presence of a dNTP.

  16. Mutants affecting nucleotide recognition by T7 DNA polymerase.

    PubMed

    Donlin, M J; Johnson, K A

    1994-12-13

    Analysis of two mutations affecting nucleotide selection by the DNA polymerase from bacteriophage T7 is reported here. Two conserved residues (Glu480 and Tyr530) in the polymerase active site of an exonuclease deficient (exo-) T7 DNA polymerase were mutated using site-directed mutagenesis (Glu480-Asp and Tyr530-Phe). The kinetic and equilibrium constants governing DNA binding, nucleotide incorporation, and pyrophosphorolysis were measured with the mutants E480D(exo-) and Y530F(exo-) in single-turnover experiments using rapid chemical quench-flow methods. Both mutants have slightly lower Kd values for DNA binding compared to that of wild-type(exo-). With Y530F(exo-) the ground state nucleotide binding affinity was unchanged from wild-type for dGTP and dCTP, was 2-fold lower for dATP and 8-10-fold lower for dTTP binding. With E480D(exo-), the binding constants were 5-6-fold lower for dATP, dGTP, and dCTP and 40-fold lower for dTTP binding compared to those constants for wild-type(exo-). The significance of a specific destabilization of dTTP binding by these amino acids was examined using a dGTP analog, deoxyinosine triphosphate, which mimics the placement and number of hydrogen bonds of an A:T base pair. The Kd for dCTP opposite inosine was unchanged with wild-type(exo-) (197 microM) but higher with Y530F(exo-) (454 microM) and with E480D(exo-) (1 mM). The Kd for dITP was the same with wild-type(exo-) (180 microM) and Y530F(exo-) (229 microM), but significantly higher with E480D(exo-) (3.2 mM). These data support the suggestion that E480 selectively stabilizes dTTP in the wild-type enzyme, perhaps by hydrogen bonding to the unbonded carbonyl. Data on the incorporation of dideoxynucleotide analogs were consistent with the observation of a selective stabilization of dTTP by both residues. Pyrophosphorolysis experiments revealed that neither mutation had a significant effect on the chemistry of polymerization. The fidelity of the mutants were examined in

  17. Effect of nitroso-chloramphenicol on mitochondrial DNA polymerase activity

    SciTech Connect

    Lim, L.O.; Abou-Khalil, W.H.; Yunis, A.A.; Abou-Khalil, S.

    1984-08-01

    A study was made of the effects of nitroso-chloramphenicol, chloramphenicol, amino-chloramphenicol, and thiamphenicol on the activity of mitochondrial DNA polymerase of rat liver. /sup 3/H-thymidine triphosphate incorporation into DNA was used to measure the DNA polymerase activity in the mitochondrial matrix fraction. This fraction was in the supernatant of sonicated mitochondria obtained by ultracentrifugation. Under standard experimental conditions, thymidine triphosphate incorporation was time dependent up to 10 minutes. This activity was enhanced by ..beta..-mercaptoethanol and was blocked by the known polymerase inhibitors ethidium bromide and 2',3'-dideoxythymidine 5'-triphosphate. Chloramphenicol and its analogues, amino-chloramphenicol and thiamphenicol, did not have a significant effect on the polymerase activity, whereas nitroso-chloramphenicol was inhibitory. The degree of inhibition was dependent on the experimental conditions. Thus, in the absence of ..beta..-mercaptoethanol, nitroso-chloramphenicol was inhibitory. The degree of inhibition was dependent on the experimental conditions. Under similar conditions, the addition of dithiothreitol also provided partial protection. On the other hand, the inhibition by nitroso-chloramphenicol was significantly enhanced with its preincubation in the mitochondrial matrix fraction before the addition of nucleotides and DNA; thus after 40 minutes of preincubation, nitroso-chloramphenicol at a concentration of 200 ..mu..mol/L gave 53% inhibition, and produced total inhibition at 600 ..mu..mol/L. The addition of NADH or NADPH to the preincubation medium produced substantial protection against nitroso-chloramphenicol, whereas nicotinamide-adenine dinucleotide had no effect. These results suggest that mitochondrial DNA polymerase may be a target for nitroso-chloramphenicol action.

  18. Function of DNA polymerase I in RNA-primed synthesis of bacteriophage M-13 duplex DNA.

    PubMed Central

    Schneck, P K; Staudenbauer, W L; Hofschneider, P H

    1976-01-01

    Cell-free extracts from Escherichia coli contain a DNA polymerase activity resistant to SH-blocking agents, which is capable of synthesizing complementary strand DNA on a circular M-13 DNA template by extension of RNA primers. This activity is considered to be identical with DNA polymerase I (or some altered form of this enzyme) since it is missing in extracts from po1A- cells. DNA synthesis in the presence of SH-blocking agents occurs at a reduced rate as compared to untreated controls and leads to the formation of DNA chains of defined size (0.4-0.5 genome's length). It is concluded that efficient M-13 duplex DNA synthesis requires the cooperation of both DNA polymerase I and III. PMID:1272793

  19. The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase.

    PubMed

    Dohrmann, Paul R; Correa, Raul; Frisch, Ryan L; Rosenberg, Susan M; McHenry, Charles S

    2016-02-18

    There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ'χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-Ctag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-Ctag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ'χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F' episome. PMID:26786318

  20. The DNA polymerase III holoenzyme contains γ and is not a trimeric polymerase

    PubMed Central

    Dohrmann, Paul R.; Correa, Raul; Frisch, Ryan L.; Rosenberg, Susan M.; McHenry, Charles S.

    2016-01-01

    There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-Ctag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-Ctag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome. PMID:26786318

  1. Purification and properties of a pea chloroplast DNA polymerase

    PubMed Central

    McKown, R. L.; Tewari, K. K.

    1984-01-01

    A DNA polymerase has been purified >3,000-fold from the chloroplasts of pea plants by chromatography on DEAE-cellulose, phosphocellulose, single-stranded DNA-agarose, and sedimentation in a glycerol gradient. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate indicates that the final fraction contained a single discernible protein band of 90,000 daltons. Gel filtration on Sephacryl S-200 and glycerol gradient sedimentation under nondenaturing conditions demonstrate that the chloroplast DNA polymerase has a native molecular mass of approximately 87,000 daltons. The purified polymerase lacks any associated nuclease activity. The enzyme activity is inhibited by N-ethylmaleimide (74% at 1.0 mM) and ethidium bromide (90% at 0.23 mM) and is resistant to aphidicolin. The purified enzyme is totally dependent on the presence of added DNA, has an absolute requirement for Mg2+ (12 mM optimal), is stimulated by K+ (120 mM optimal), and requires all four deoxynucleoside triphosphates for maximum activity. Native DNA which has been degraded to a limited extent with DNase I is the most efficient template. Images PMID:16593454

  2. Comparison of six commercially-available DNA polymerases for direct PCR.

    PubMed

    Miura, Masashi; Tanigawa, Chihiro; Fujii, Yoshito; Kaneko, Satoshi

    2013-01-01

    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. PMID:24213192

  3. Functional interplay of DnaE polymerase, DnaG primase and DnaC helicase within a ternary complex, and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis

    PubMed Central

    Rannou, Olivier; Le Chatelier, Emmanuelle; Larson, Marilynn A.; Nouri, Hamid; Dalmais, Bérengère; Laughton, Charles; Jannière, Laurent; Soultanas, Panos

    2013-01-01

    Bacillus subtilis has two replicative DNA polymerases. PolC is a processive high-fidelity replicative polymerase, while the error-prone DnaEBs extends RNA primers before hand-off to PolC at the lagging strand. We show that DnaEBs interacts with the replicative helicase DnaC and primase DnaG in a ternary complex. We characterize their activities and analyse the functional significance of their interactions using primase, helicase and primer extension assays, and a ‘stripped down’ reconstituted coupled assay to investigate the coordinated displacement of the parental duplex DNA at a replication fork, synthesis of RNA primers along the lagging strand and hand-off to DnaEBs. The DnaG–DnaEBs hand-off takes place after de novo polymerization of only two ribonucleotides by DnaG, and does not require other replication proteins. Furthermore, the fidelity of DnaEBs is improved by DnaC and DnaG, likely via allosteric effects induced by direct protein–protein interactions that lower the efficiency of nucleotide mis-incorporations and/or the efficiency of extension of mis-aligned primers in the catalytic site of DnaEBs. We conclude that de novo RNA primer synthesis by DnaG and initial primer extension by DnaEBs are carried out by a lagging strand–specific subcomplex comprising DnaG, DnaEBs and DnaC, which stimulates chromosomal replication with enhanced fidelity. PMID:23563155

  4. DNA replication: polymerase epsilon as a non-catalytic converter of the helicase.

    PubMed

    Zegerman, Philip

    2013-04-01

    In eukaryotes DNA polymerase epsilon (ε) synthesises the leading DNA strand during replication. A new study provides insight into how this polymerase also functions independently of its enzyme activity to assemble and activate the replicative helicase. PMID:23578873

  5. Computational investigation of locked nucleic acid (LNA) nucleotides in the active sites of DNA polymerases by molecular docking simulations.

    PubMed

    Poongavanam, Vasanthanathan; Madala, Praveen K; Højland, Torben; Veedu, Rakesh N

    2014-01-01

    Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5'-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3'-endo conformation which in fact helps better orienting within the active site. PMID:25036012

  6. Biochemical evidence for the requirement of Hoogsteen base pairing for replication by human DNA polymerase iota.

    PubMed

    Johnson, Robert E; Prakash, Louise; Prakash, Satya

    2005-07-26

    Because of the near geometric identity of Watson-Crick (W-C) GxC and AxT base pairs, a given DNA polymerase forms the four possible correct base pairs with nearly identical catalytic efficiencies. However, human DNA polymerase iota (Pol iota), a member of the Y family of DNA polymerases, exhibits a marked template specificity, being more efficient at incorporating the correct nucleotide opposite template purines than opposite pyrimidines. By using 7-deazaadenine and 7-deazaguanine as the templating residues, which disrupt Hoogsteen base pair formation, we show that, unlike the other DNA polymerases belonging to the A, B, or Y family, DNA synthesis by Pol iota is severely inhibited by these N7-modified bases. These observations provide biochemical evidence that, during normal DNA synthesis, template purines adopt a syn conformation in the Pol iota active site, enabling the formation of a Hoogsteen base pair with the incoming pyrimidine nucleotide. Additionally, mutational studies with Leu-62, which lies in close proximity to the templating residue in the Pol iota ternary complex, have indicated that both factors, steric constraints within the active site and the stability provided by the hydrogen bonds in the Hoogsteen base pair, contribute to the efficiency of correct nucleotide incorporation opposite template purines by Pol iota. PMID:16014707

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

    NASA Technical Reports Server (NTRS)

    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.

  8. Priming DNA replication from triple helix oligonucleotides: possible threestranded DNA in DNA polymerases.

    PubMed

    Lestienne, Patrick P

    2011-01-01

    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. PMID:22229092

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

    PubMed Central

    Lestienne, Patrick P.

    2011-01-01

    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. PMID:22229092

  10. Crystal Structure of Yeast DNA Polymerase ε Catalytic Domain

    PubMed Central

    Jain, Rinku; Rajashankar, Kanagalaghatta R.; Buku, Angeliki; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2014-01-01

    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. PMID:24733111

  11. The direct application of the polymerase chain reaction to DNA extracted from foods.

    PubMed

    Dickinson, J H; Kroll, R G; Grant, K A

    1995-04-01

    Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods. PMID:7766115

  12. Substrate-dependent millisecond domain motions in DNA polymerase β

    PubMed Central

    Berlow, Rebecca B.; Swain, Monalisa; Dalal, Shibani; Sweasy, Joann B.; Loria, J. Patrick

    2012-01-01

    DNA polymerase β (Pol β) is a 39 kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers and these mutations further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s–1. Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped, DNA substrate bound form. Upon binding DNA, these lyase domain motions are significantly quenched whereas evidence for conformational motions in the polymerase domain become apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation, lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity. PMID:22446382

  13. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

    PubMed Central

    2011-01-01

    Background The DNA repair protein O6-Methylguanine-DNA methyltransferase (MGMT) confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP) the most commonly used for promoter methylation study, while immunohistochemistry (IHC) has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. Methods A computer-aided search of MEDLINE (1950-October 2009), EBSCO (1966-October 2009) and EMBASE (1974-October 2009) was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Results Of 254 studies identified as eligible for full-text review, 52 (20.5%) met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others) was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Conclusions Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably. PMID:21269507

  14. Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase C Terminus Is Required for Nuclear Localization and Viral DNA Replication

    PubMed Central

    Feng, Guozhong

    2014-01-01

    ABSTRACT The DNA polymerase (DNApol) of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is essential for viral DNA replication. The DNApol exonuclease and polymerase domains are highly conserved and are considered functional in DNA replication. However, the role of the DNApol C terminus has not yet been characterized. To identify whether only the exonuclease and polymerase domains are sufficient for viral DNA replication, several DNApol C-terminal truncations were cloned into a dnapol-null AcMNPV bacmid with a green fluorescent protein (GFP) reporter. Surprisingly, most of the truncation constructs, despite containing both exonuclease and polymerase domains, could not rescue viral DNA replication and viral production in bacmid-transfected Sf21 cells. Moreover, GFP fusions of these same truncations failed to localize to the nucleus. Truncation of the C-terminal amino acids 950 to 984 showed nuclear localization but allowed for only limited and delayed viral spread. The C terminus contains a typical bipartite nuclear localization signal (NLS) motif at residues 804 to 827 and a monopartite NLS motif at residues 939 to 948. Each NLS, as a GFP fusion peptide, localized to the nucleus, but both NLSs were required for nuclear localization of DNApol. Alanine substitutions in a highly conserved baculovirus DNApol sequence at AcMNPV DNApol amino acids 972 to 981 demonstrated its importance for virus production and DNA replication. Collectively, the data indicated that the C terminus of AcMNPV DNApol contains two NLSs and a conserved motif, all of which are required for nuclear localization of DNApol, viral DNA synthesis, and virus production. IMPORTANCE The baculovirus DNA polymerase (DNApol) is a highly specific polymerase that allows viral DNA synthesis and hence virus replication in infected insect cells. We demonstrated that the exonuclease and polymerase domains of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) alone are

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

    PubMed

    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; Takeda, Shunichi

    2015-02-18

    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 normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases. PMID:25628356

  16. Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases α, δ, and ε in Saccharomyces cerevisiae.

    PubMed

    Dubarry, Marion; Lawless, Conor; Banks, A Peter; Cockell, Simon; Lydall, David

    2015-10-01

    Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase α-primase (Pol α) and DNA polymerase δ (Pol δ) replicate the lagging-strand and Pol α and DNA polymerase ε (Pol ε) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We combined temperature-sensitive mutations affecting the three replicative polymerases, Pol α, Pol δ, and Pol ε with genome-wide collections of null and reduced function mutations. We identify large numbers of genetic interactions that inform about the roles that specific genes play to help Pol α, Pol δ, and Pol ε function. Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not represent the majority of the genetic interactions identified. Instead our data support a model for division of labor between the different DNA polymerases during DNA replication. For example, our genetic interaction data are consistent with biochemical data showing that Pol ε is more important to the Pre-Loading complex than either Pol α or Pol δ. We also observed distinct patterns of genetic interactions between leading- and lagging-strand DNA polymerases, with particular genes being important for coupling proliferating cell nuclear antigen loading/unloading (Ctf18, Elg1) with nucleosome assembly (chromatin assembly factor 1, histone regulatory HIR complex). Overall our data reveal specialized genetic networks that affect different aspects of leading- and lagging-strand DNA replication. To help others to engage with these data we have generated two novel, interactive visualization tools, DIXY and Profilyzer. PMID:26297725

  17. Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases α, δ, and ε in Saccharomyces cerevisiae

    PubMed Central

    Dubarry, Marion; Lawless, Conor; Banks, A. Peter; Cockell, Simon; Lydall, David

    2015-01-01

    Three major DNA polymerases replicate the linear eukaryotic chromosomes. DNA polymerase α-primase (Pol α) and DNA polymerase δ (Pol δ) replicate the lagging-strand and Pol α and DNA polymerase ε (Pol ε) the leading-strand. To identify factors affecting coordination of DNA replication, we have performed genome-wide quantitative fitness analyses of budding yeast cells containing defective polymerases. We combined temperature-sensitive mutations affecting the three replicative polymerases, Pol α, Pol δ, and Pol ε with genome-wide collections of null and reduced function mutations. We identify large numbers of genetic interactions that inform about the roles that specific genes play to help Pol α, Pol δ, and Pol ε function. Surprisingly, the overlap between the genetic networks affecting the three DNA polymerases does not represent the majority of the genetic interactions identified. Instead our data support a model for division of labor between the different DNA polymerases during DNA replication. For example, our genetic interaction data are consistent with biochemical data showing that Pol ε is more important to the Pre-Loading complex than either Pol α or Pol δ. We also observed distinct patterns of genetic interactions between leading- and lagging-strand DNA polymerases, with particular genes being important for coupling proliferating cell nuclear antigen loading/unloading (Ctf18, Elg1) with nucleosome assembly (chromatin assembly factor 1, histone regulatory HIR complex). Overall our data reveal specialized genetic networks that affect different aspects of leading- and lagging-strand DNA replication. To help others to engage with these data we have generated two novel, interactive visualization tools, DIXY and Profilyzer. PMID:26297725

  18. Replication across Regioisomeric Ethylated Thymidine Lesions by Purified DNA Polymerases

    PubMed Central

    Andersen, Nisana; Wang, Pengcheng; Wang, Yinsheng

    2013-01-01

    Causal links exist between smoking cigarettes and cancer development. Some genotoxic agents in cigarette smoke are capable of alkylating nucleobases in DNA and higher levels of ethylated DNA lesions were observed in smokers than non-smokers. In this study, we examined comprehensively how the regioisomeric O2-, N3- and O4-ethylthymidine (O2-, N3- and O4-EtdT) perturb DNA replication mediated by purified human DNA polymerases (hPol) η, κ, and ι, yeast DNA polymerase ζ (yPol ζ), and the exonuclease-free Klenow fragment (Kf−) of Escherichia coli DNA polymerase I. Our results showed that hPol η and Kf− could bypass all three lesions and generate full-length replication products, whereas hPol ι stalled after inserting a single nucleotide opposite the lesions. Bypass carried out by hPol κ and yPol ζ differed markedly amongst the three lesions: Consistent with its known capability in bypassing efficiently the minor-groove N2-substituted 2′-deoxyguanosine lesions, hPol κ was able to bypass O2-EtdT, though it experienced great difficulty in bypassing N3-EtdT and O4-EtdT; yPol ζ was only modestly blocked by O4-EtdT, but the polymerase was highly hindered by O2-EtdT and N3-EtdT. LC-MS/MS analysis of the replication products revealed that DNA synthesis opposite O4-EtdT was highly error-prone, with dGMP being preferentially inserted, while the presence of O2-EtdT and N3-EtdT in template DNA directed substantial frequencies of misincorporation of dGMP and, for hPol ι and Kf−, dTMP. Thus, our results suggested that O2-EtdT and N3-EtdT may also contribute to the AT→TA and AT→GC mutations observed in cells and tissues of animals exposed to ethylating agents. PMID:24134187

  19. Target-triggered three-way junction structure and polymerase/nicking enzyme synergetic isothermal quadratic DNA machine for highly specific, one-step, and rapid microRNA detection at attomolar level.

    PubMed

    Zhang, Qing; Chen, Feng; Xu, Feng; Zhao, Yongxi; Fan, Chunhai

    2014-08-19

    MicroRNAs (miRNAs) play important roles in many biological processes and are regarded as promising cancer biomarkers. Herein, a highly specific, one-step, and rapid miRNAs detection strategy with attomolar sensitivity has been developed on the basis of a target-triggered three-way junction (3-WJ) structure and polymerase/nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). To this end, 3-WJ probes (primer and template) are designed to selectively recognize target miRNA and form the stable 3-WJ structure to trigger ESQM, resulting in a high quadratic amplified signal. A high specificity is demonstrated by the excellent discrimination of even single-base mismatched homologous sequences with mismatched bases in varied locations (close to the 3'-end, the 5'-end, and the middle). In addition, a low detection limit down to 2 amol was achieved within 30 min. This sensitivity is much higher than those of most linear amplification-based approaches and is even comparable to those of some exponential amplification-based methods. Furthermore, the applicability of this method in complex samples was demonstrated by the analysis of cancer cell small RNA extracts, results of which were in good agreement with those obtained by a commercial miRNA kit and previously published data. The miRNA with a 3' end modification (2'-O-methylation), such as plant miRNA, was also successfully detected, confirming the good universality of the proposed strategy. It is worthwhile to point out that several well-established methods using miRNA as primer for polymerization reaction are of relatively poor performance in the analysis of these modified miRNA. Therefore, these merits endow the developed strategy with powerful implications for biological research and an effective diagnostic assay. PMID:25072308

  20. Recent Insight into the Kinetic Mechanisms and Conformational Dynamics of Y-Family DNA Polymerases

    PubMed Central

    2015-01-01

    The kinetic mechanisms by which DNA polymerases catalyze DNA replication and repair have long been areas of active research. Recently discovered Y-family DNA polymerases catalyze the bypass of damaged DNA bases that would otherwise block replicative DNA polymerases and stall replication forks. Unlike DNA polymerases from the five other families, the Y-family DNA polymerases have flexible, solvent-accessible active sites that are able to tolerate various types of damaged template bases and allow for efficient lesion bypass. Their promiscuous active sites, however, also lead to fidelities that are much lower than those observed for other DNA polymerases and give rise to interesting mechanistic properties. Additionally, the Y-family DNA polymerases have several other unique structural features and undergo a set of conformational changes during substrate binding and catalysis different from those observed for replicative DNA polymerases. In recent years, pre-steady-state kinetic methods have been extensively employed to reveal a wealth of information about the catalytic properties of these fascinating noncanonical DNA polymerases. Here, we review many of the recent findings on the kinetic mechanisms of DNA polymerization with undamaged and damaged DNA substrates by the Y-family DNA polymerases, and the conformational dynamics employed by these error-prone enzymes during catalysis. PMID:24716482

  1. DNA polymerase γ and disease: what we have learned from yeast

    PubMed Central

    Lodi, Tiziana; Dallabona, Cristina; Nolli, Cecilia; Goffrini, Paola; Donnini, Claudia; Baruffini, Enrico

    2015-01-01

    Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (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 γ holoenzyme is expressed in yeast and complements the loss of Mip1. PMID:25852747

  2. Assays for Hepatitis B Virus DNA-and RNA-Dependent DNA Polymerase Activities.

    PubMed

    Shaw, T; Locarnini, S A

    2000-01-01

    Genomes of the hepatitis B viruses (HBVs) consist of approx 3.2 kb of partly double-stranded DNA containing three or four overlapping open reading frames, the largest of which encodes the viral polymerase (Pol) protein. After entry into the cell and uncoating, the viral genome is transported to the nucleus where it is converted into a covalently closed circular (CCC) or supercoiled molecule by cellular repair mechanisms. The viral CCC DNA is transcribed, presumably by host cell RNA polymerase II, into unspliced, capped polyadenylated mRNA species from which viral proteins are transcribed. In addition, terminally redundant 3.5-kb RNA transcripts, which function as pregenomes, are produced and exported to the cytoplasm where they are packaged into viral core particles in which reverse transcription, pregenome degradation, and duplication occurs, reproducing the partly double-stranded HBV genome (for recent review, see ref. 1). Besides its essential role in HBV genome replication, HBV Pol is also involved in virus assembly, and because hepadnaviruses do not encode enzymes functionally equivalent to deoxynucleoside kinases (2), functions associated with HBV Pol are probably the only virus-specific targets for antiviral activity of nucleoside analogs. In vitro assays for inhibition of HBV Pol functions by deoxynucleoside triphosphate (dNTP) analogs are useful indicators but, because of restrictions imposed by hepatocyte enzymology, provide no guarantee of potential anti-HBV activity of the parent (deoxy)nucleoside analogs in intact cells (2). PMID:21331902

  3. Analysis of ancient DNA from coprolites: a perspective with random amplified polymorphic DNA-polymerase chain reaction approach.

    PubMed

    Iñiguez, Alena M; Araújo, Adauto; Ferreira, Luiz Fernando; Vicente, Ana Carolina P

    2003-01-01

    The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA) present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pretreatment was essential to overcome the DNA degradation and the serial dilutions helped with to prevent polymerase chain reaction (PCR) inhibitors. Moreover, the random amplified polymorphic DNA-PCR has been shown to be a reliable technique for further experiments to recover specific aDNA sequences. PMID:12687765

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

    NASA Astrophysics Data System (ADS)

    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.

  5. Structural Determinant for Switching between the Polymerase and Exonuclease Modes in the PCNA-Replicative DNA Polymerase Complex

    NASA Astrophysics Data System (ADS)

    Nishida, Hirokazu; Mayanagi, Kouta; Ishino, Yoshizumi; Morikawa, Kosuke

    Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with a cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction. Electron microscopy analyses confirmed that this complex structure exists among the multiple functional configurations in solution. Together with data from mutational analyses, this structural study indicated that the novel interaction between a stretched loop of PCNA and the PfuPol Thumb domain is quite important, in addition to the authentic PCNA-polymerase recognition site (PIP box). A comparison of the present structures with the previously reported structures of polymerases complexed with DNA suggested that the second interaction site plays a crucial role in switching between the polymerase and exonuclease modes, by stabilizing only the polymerase mode. This proposed mechanism of fidelity control of replicative DNA polymerases was supported by experiments, in which a mutation within the second interaction site caused an enhancement in the exonuclease activity in the presence of PCNA.

  6. Interaction of Human DNA Polymerase α and DNA Polymerase I from Bacillus stearothermophilus with Hypoxanthine and 8-Oxoguanine Nucleotides †

    PubMed Central

    Patro, Jennifer N.; Urban, Milan; Kuchta, Robert D.

    2009-01-01

    To better understand how DNA polymerases interact with mutagenic bases, we examined how human DNA polymerase α (pol α), a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF), an A family enzyme, generate adenine:hypoxanthine and adenine:8-oxo-7,8-dihydroguanine (8-oxoG) base pairs. Pol α strongly discriminated against polymerizing dATP opposite 8-oxoG, and removing N1, N6, or N7 further inhibited incorporation, whereas removing N3 from dATP dramatically increased incorporation (32-fold). Eliminating N6 from 3-deaza-dATP now greatly reduced incorporation, suggesting that incorporation of dATP (analogues) opposite 8-oxoguanine proceeds via a Hoogsteen base-pair and that pol α uses N3 of a purine dNTP to block this incorporation. Pol α also polymerized 8-oxo-dGTP across from a templating A, and removing N6 from the template adenine inhibited incorporation of 8-oxoG. The effects of N1, N6, and N7 demonstrated a strong interdependence during formation of adenine:hypoxanthine base-pairs by pol α and N3 of dATP again helps prevent polymerization opposite a templating hypoxanthine. BF very efficiently polymerized 8-oxo-dGTP opposite adenine, and N1 and N7 of adenine appear to play important roles. BF incorporates dATP opposite 8-oxoG less efficiently, and modifying N1, N6, or N7 greatly inhibits incorporation. N6, and to a lesser extent N1, help drive hypoxanthine:adenine base pair formation by BF. The mechanistic implications of these results showing that different polymerases interact very differently with base lesions are discussed. PMID:19642651

  7. Structural Basis of High-Fidelity DNA Synthesis by Yeast DNA Polymerase δ

    SciTech Connect

    Swan, M.; Johnson, R; Prakash, L; Prakash, S; Aggarwal, A

    2009-01-01

    DNA polymerase ? (Pol ?) has a crucial role in eukaryotic replication. Now the crystal structure of the yeast DNA Pol ? catalytic subunit in complex with template primer and incoming nucleotide is presented at 2.0-A resolution, providing insight into its high fidelity and a framework to understand the effects of mutations involved in tumorigenesis.

  8. Molecular cytogenetics by polymerase catalyzed amplification or in situ labelling of specific nucleic acid sequences

    SciTech Connect

    Bolund, L.; Brandt, C.; Hindkjaer, J.; Koch, J.; Koelvraa, S.; Pedersen, S. )

    1993-01-01

    The Polymerase Chain Reaction (PCR) can be performed on isolated cells or chromosomes and the product can be analyzed by DNA technology or by FISH to test metaphases. The authors have good experiences analyzing aberrant chromosomes by FACS sorting, PCR with degenerated primers and painting of test metaphases with the PCR product. They also utilize polymerases for PRimed IN Situ labelling (PRINS) of specific nucleic acid sequences. In PRINS oligonucleotides are hybridized to their target sequences and labeled nucleotides are incorporated at the site of hybridization with the oligonucleotide as primer. PRINS may eventually allow the study of individual genes, gene expression and even somatic mutations (in mRNA) in single cells.

  9. The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

    PubMed

    Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P; Lucas, Thomas P; R-Bhojappa, Ramanagouda; Tang, Guo-Qing; Raney, Kevin; Yin, Y Whitney; Patel, Smita S

    2016-08-01

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA. PMID:27311715

  10. Human DNA Polymerase Kappa Encircles DNA: Implicatins for Mismatch Extension and Lesion Bypass

    SciTech Connect

    Lone,S.; Townson, S.; Uljon, S.; Johnson, R.; Brahma, A.; Nair, D.; Prakash, S.; Prakash, L.; Aggarwal, A.

    2007-01-01

    Human DNA polymerase (Pol ) is a proficient extender of mispaired primer termini on undamaged DNAs and is implicated in the extension step of lesion bypass. We present here the structure of Pol catalytic core in ternary complex with DNA and an incoming nucleotide. The structure reveals encirclement of the DNA by a unique 'N-clasp' at the N terminus of Pol , which augments the conventional right-handed grip on the DNA by the palm, fingers, and thumb domains and the PAD and provides additional thermodynamic stability. The structure also reveals an active-site cleft that is constrained by the close apposition of the N-clasp and the fingers domain, and therefore can accommodate only a single Watson-Crick base pair. Together, DNA encirclement and other structural features help explain Pol 's ability to extend mismatches and to promote replication through various minor groove DNA lesions, by extending from the nucleotide incorporated opposite the lesion by another polymerase.

  11. Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus

    PubMed Central

    Kane, Shawn D.; Bullard, James M.

    2015-01-01

    DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX (τ and γ), δ and δ′ components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions. PMID:25688300

  12. HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase β

    PubMed Central

    Fang, Qingming; Inanc, Burcu; Schamus, Sandy; Wang, Xiao-hong; Wei, Leizhen; Brown, Ashley R.; Svilar, David; Sugrue, Kelsey F.; Goellner, Eva M.; Zeng, Xuemei; Yates, Nathan A.; Lan, Li; Vens, Conchita; Sobol, Robert W.

    2014-01-01

    Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase β (Polβ) and XRCC1 is thought to facilitate repair by recruiting Polβ to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polβ. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polβ and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance. PMID:25423885

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

    PubMed Central

    Koonin, Eugene V

    2006-01-01

    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 help of the archaeal

  14. DNA Polymerase λ Inactivation by Oxidized Abasic Sites&

    PubMed Central

    Stevens, Adam J.; Guan, Lirui; Bebenek, Katarzyna; Kunkel, Thomas A.; Greenberg, Marc M.

    2013-01-01

    Base excision repair plays a vital role in maintaining genomic integrity in mammalian cells. DNA polymerase λ is believed to play a backup role to DNA polymerase β in base excision repair. Two oxidized abasic lesions that are produced by a variety of DNA damaging agents, including several antitumor antibiotics, the C4′-oxidized abasic site following Ape1 incision (pC4-AP) and 5′-(2-phosphoryl-1,4-dioxobutane) (DOB), irreversibly inactivate Pol β and Pol λ. The interactions of DOB and pC4-AP with Pol λ are examined in detail using DNA substrates containing these lesions at defined sites. Single turnover kinetic experiments show that Pol λ excises DOB almost 13-times more slowly than a 5′-phosphorylated 2-deoxyribose (dRP). pC4-AP is excised approximately twice as fast as DOB. The absolute rate constants are considerably slower than those reported for Pol β at the respective reactions, suggesting that Pol λ may be an inefficient backup in BER. DOB inactivates Pol λ approximately 3-fold less efficiently than it does Pol β and the difference is attributable to a higher KI (33 ± 7 nM). Inactivation of Pol λ’s lyase activity by DOB also prevents the enzyme from carrying out polymerization following preincubation of the protein and DNA. Mass spectral analysis of GluC digested Pol λ inactivated by DOB shows that Lys324 is modified. There is inferential support that Lys312 may also be modified. Both residues are within the Pol λ lyase active site. Protein modification involves reaction with released but-2-ene-1,4-dial. When acting on pC4-AP, Pol λ achieves approximately 4 turnovers on average before being inactivated. Lyase inactivation by pC4-AP is also accompanied by loss of polymerase activity and mass spectrometry indicates that Lys312 and Lys324 are modified by the lesion. The ability of DOB and pC4-AP to inactivate Pol λ provides additional evidence that these lesions are significant sources of the cytotoxicity of DNA damaging agents that

  15. Evolution of thermophilic DNA polymerases for the recognition and amplification of C2ʹ-modified DNA

    NASA Astrophysics Data System (ADS)

    Chen, Tingjian; Hongdilokkul, Narupat; Liu, Zhixia; Adhikary, Ramkrishna; Tsuen, Shujian S.; Romesberg, Floyd E.

    2016-06-01

    The PCR amplification of oligonucleotides enables the evolution of sequences called aptamers that bind specific targets with antibody-like affinity. However, in many applications the use of these aptamers is limited by nuclease-mediated degradation. In contrast, oligonucleotides that are modified at their sugar C2ʹ positions with methoxy or fluorine substituents are stable to nucleases, but they cannot be synthesized by natural polymerases. Here we report the development of a polymerase-evolution system and its use to evolve thermostable polymerases that efficiently interconvert C2ʹ-OMe-modified oligonucleotides and their DNA counterparts via ‘transcription’ and ‘reverse transcription’ or, more importantly, that PCR-amplify partially C2ʹ-OMe- or C2ʹ-F-modified oligonucleotides. A mechanistic analysis demonstrates that the ability to amplify the modified oligonucleotides evolved by optimizing interdomain interactions that stabilize the catalytically competent closed conformation of the polymerase. The evolved polymerases should find practical applications and the developed evolution system should be a powerful tool for tailoring polymerases to have other types of novel function.

  16. A novel in vitro assay to study the mechanism by which DNA polymerases bypass blocking lesions to DNA replication

    SciTech Connect

    Randall, S.K.

    1989-01-01

    We devised a simple gel assay to measure insertion kinetics for any dNTP substrate opposite a target site. Our ability to synthesize an abasic lesion and place it at a single site in synthetic oligonucleotides allows for an in vitro analysis of the mechanism by which DNA polymerases bypass blocking lesions to DNA replication and to identify E. coli polymerases and accessory proteins that allow for insertion and bypass of such lesions. Using this assay we examine the preferred insertion of dATP by Drosophila DNA polymerase {alpha} opposite the abasic lesion compared to dGTP, dCTP, and dTTP for all different nearest-neighbors. The preferred insertion of dATP is governed by a V{sub max} discrimination little affected by nearest-neighbors. A DNA polymerase activity was purified from E coli, deleted for DNA polymerase I, that appears to be part of the SOS response of E. coli since it cannot be induced in lexA(Ind{sup {minus}}) strains. This inducible polymerase is DNA polymerase II. In contrast to DNA polymerase III, DNA polymerase II efficiently incorporates nucleotides opposite the abasic lesion and continues DNA synthesis. We addressed the role of E. coli DNA polymerase I targeted SOS mutagenesis.

  17. DNA-dependent DNA polymerase from yeast mitochondria. Dependence of enzyme activity on conditions of cell growth, and properties of the highly purified polymerase.

    PubMed

    Wintersberger, U; Blutsch, H

    1976-09-01

    The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on the carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondria DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacylamide gel electrophoresis revealed the following structure: a monomer of molecular weight around 60 000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120 000 which under low salt concentration (0.2 M Tris-HCl buffer) formed higher aggregates. For optimal activity an Mg2+ ion concentration of 50 mM was found necessary, Mn ions did not promote activity at any concentration tested (0.5--50 mM). Indeed, if added to Mg2+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2+ ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the Km concentration. The enzyme preferred a gapped template primer, poly(dA) - (dT)10, over nicked DNA and was unable to use a polyribonucleotide template, poly(rA) - (dT)10. In the purest preparations no exonuclease activity could be detected. PMID:786635

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

    PubMed

    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. PMID:23543747

  19. The Functions of Serine 687 Phosphorylation of Human DNA Polymerase η in UV Damage Tolerance.

    PubMed

    Dai, Xiaoxia; You, Changjun; Wang, Yinsheng

    2016-06-01

    DNA polymerase η (polη) is a Y-family translesion synthesis polymerase that plays a key role in the cellular tolerance toward UV irradiation-induced DNA damage. Here, we identified, for the first time, the phosphorylation of serine 687 (Ser(687)), which is located in the highly conserved nuclear localization signal (NLS) region of human polη and is mediated by cyclin-dependent kinase 2 (CDK2). We also showed that this phosphorylation is stimulated in human cells upon UV light exposure and results in diminished interaction of polη with proliferating cell nuclear antigen (PCNA). Furthermore, we demonstrated that the phosphorylation of Ser(687) in polη confers cellular protection from UV irradiation and increases the efficiency in replication across a site-specifically incorporated cyclobutane pyrimidine dimer in human cells. Based on these results, we proposed a mechanistic model where Ser(687) phosphorylation functions in the reverse polymerase switching step of translesion synthesis: The phosphorylation brings negative charges to the NLS of polη, which facilitates its departure from PCNA, thereby resetting the replication fork for highly accurate and processive DNA replication. Thus, our study, together with previous findings, supported that the posttranslational modifications of NLS of polη played a dual role in polymerase switching, where Lys(682) deubiquitination promotes the recruitment of polη to PCNA immediately prior to lesion bypass and Ser(687) phosphorylation stimulates its departure from the replication fork immediately after lesion bypass. PMID:26988343

  20. Functional association of poly(ADP-ribose) polymerase with DNA polymerase alpha-primase complex: a link between DNA strand break detection and DNA replication.

    PubMed Central

    Dantzer, F; Nasheuer, H P; Vonesch, J L; de Murcia, G; Ménissier-de Murcia, J

    1998-01-01

    Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template. PMID:9518481

  1. The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

    PubMed Central

    Roy, Upasana; Mukherjee, Shivam; Sharma, Anjali; Frank, Ekaterina G.; Schärer, Orlando D.

    2016-01-01

    Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases. PMID:27257072

  2. The 29 DNA Polymerase: Protein-Primer Structure Suggests a Model of the Initiation to Elongation Transition

    SciTech Connect

    Kamtekar,S.; Berman, A.; Wang, J.; Lazaro, J.; Vega, M.; Blanco, L.; Salas, M.; Steitz, T.

    2006-01-01

    The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage {phi}29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 Angstroms resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.

  3. The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient.

    PubMed

    Dalal, Shibani; Chikova, Anna; Jaeger, Joachim; Sweasy, Joann B

    2008-02-01

    Approximately 30% of human tumors characterized to date express DNA polymerase beta (pol beta) variant proteins. Two of the polymerase beta cancer-associated variants are sequence-specific mutators, and one of them binds to DNA but has no polymerase activity. The Leu22Pro (L22P) DNA polymerase beta variant was identified in a gastric carcinoma. Leu22 resides within the 8 kDa amino terminal domain of DNA polymerase beta, which exhibits dRP lyase activity. This domain catalyzes the removal of deoxyribose phosphate during short patch base excision repair. We show that this cancer-associated variant has very little dRP lyase activity but retains its polymerase activity. Although residue 22 has no direct contact with the DNA, we report here that the L22P variant has reduced DNA-binding affinity. The L22P variant protein is deficient in base excision repair. Molecular dynamics calculations suggest that alteration of Leu22 to Pro changes the local packing, the loop connecting helices 1 and 2 and the overall juxtaposition of the helices within the N-terminal domain. This in turn affects the shape of the binding pocket that is required for efficient dRP lyase catalysis. PMID:18039710

  4. Pseudomonas aeruginosa phage PaP1 DNA polymerase is an A-family DNA polymerase demonstrating ssDNA and dsDNA 3'-5' exonuclease activity.

    PubMed

    Liu, Binyan; Gu, Shiling; Liang, Nengsong; Xiong, Mei; Xue, Qizhen; Lu, Shuguang; Hu, Fuquan; Zhang, Huidong

    2016-08-01

    Most phages contain DNA polymerases, which are essential for DNA replication and propagation in infected host bacteria. However, our knowledge on phage-encoded DNA polymerases remains limited. This study investigated the function of a novel DNA polymerase of PaP1, which is the lytic phage of Pseudomonas aeruginosa. PaP1 encodes its sole DNA polymerase called Gp90 that was predicted as an A-family DNA polymerase with polymerase and 3'-5' exonuclease activities. The sequence of Gp90 is homologous but not identical to that of other A-family DNA polymerases, such as T7 DNA polymerases (Pol) and DNA Pol I. The purified Gp90 demonstrated a polymerase activity. The processivity of Gp90 in DNA replication and its efficiency in single-dNTP incorporation are similar to those of T7 Pol with processive thioredoxin (T7 Pol/trx). Gp90 can degrade ssDNA and dsDNA in 3'-5' direction at a similar rate, which is considerably lower than that of T7 Pol/trx. The optimized conditions for polymerization were a temperature of 37 °C and a buffer consisting of 40 mM Tris-HCl (pH 8.0), 30 mM MgCl2, and 200 mM NaCl. These studies on DNA polymerase encoded by PaP1 help advance our knowledge on phage-encoded DNA polymerases and elucidate PaP1 propagation in infected P. aeruginosa. PMID:27052734

  5. RB69 DNA Polymerase Structure, Kinetics, and Fidelity

    PubMed Central

    2015-01-01

    This review will summarize our structural and kinetic studies of RB69 DNA polymerase (RB69pol) as well as selected variants of the wild-type enzyme that were undertaken to obtain a deeper understanding of the exquisitely high fidelity of B family replicative DNA polymerases. We discuss how the structures of the various RB69pol ternary complexes can be used to rationalize the results obtained from pre-steady-state kinetic assays. Our main findings can be summarized as follows. (i) Interbase hydrogen bond interactions can increase catalytic efficiency by 5000-fold; meanwhile, base selectivity is not solely determined by the number of hydrogen bonds between the incoming dNTP and the templating base. (ii) Minor-groove hydrogen bond interactions at positions n – 1 and n – 2 of the primer strand and position n – 1 of the template strand in RB69pol ternary complexes are essential for efficient primer extension and base selectivity. (iii) Partial charge interactions among the incoming dNTP, the penultimate base pair, and the hydration shell surrounding the incoming dNTP modulate nucleotide insertion efficiency and base selectivity. (iv) Steric clashes between mismatched incoming dNTPs and templating bases with amino acid side chains in the nascent base pair binding pocket (NBP) as well as weak interactions and large gaps between the incoming dNTPs and the templating base are some of the reasons that incorrect dNTPs are incorporated so inefficiently by wild-type RB69pol. In addition, we developed a tC°–tCnitro Förster resonance energy transfer assay to monitor partitioning of the primer terminus between the polymerase and exonuclease subdomains. PMID:24720884

  6. Single-Molecule Electronic Monitoring of DNA Polymerase Activity

    NASA Astrophysics Data System (ADS)

    Marushchak, Denys O.; Pugliese, Kaitlin M.; Turvey, Mackenzie W.; Choi, Yongki; Gul, O. Tolga; Olsen, Tivoli J.; Rajapakse, Arith J.; Weiss, Gregory A.; Collins, Philip G.

    Single-molecule techniques can reveal new spatial and kinetic details of the conformational changes occurring during enzymatic catalysis. Here, we investigate the activity of DNA polymerases using an electronic single-molecule technique based on carbon nanotube transistors. Single molecules of the Klenow fragment (KF) of polymerase I were conjugated to the transistors and then monitored via fluctuations in electrical conductance. Continuous, long-term monitoring recorded single KF molecules incorporating up to 10,000 new bases into single-stranded DNA templates. The duration of individual incorporation events was invariant across all analog and native nucleotides, indicating that the precise structure of different base pairs has no impact on the timing of incorporation. Despite similar timings, however, the signal magnitudes generated by certain analogs reveal alternate conformational states that do not occur with native nucleotides. The differences induced by these analogs suggest that the electronic technique is sensing KF's O-helix as it tests the stability of nascent base pairs.

  7. Detection of Neospora caninum DNA by the polymerase chain reaction.

    PubMed

    Payne, S; Ellis, J

    1996-04-01

    Neospora caninum is a cyst-forming coccidian parasite which is now recognised as a major cause of abortion and neonatal mortality in cattle and other livestock. This study describes the primary DNA structure of the transcribed spacer region of the rDNA of N. caninum. Of importance is that the sequence data generated have been used to develop a species-specific PCR test for N. caninum DNA, which will prove valuable in epidemiology studies on neosporosis. PMID:8773521

  8. DNA polymerases alpha and gamma during pre-emergence and early larval development of Artemia.

    PubMed

    Slater, J M; McLennan, A G

    1982-12-15

    DNA polymerases alpha and gamma have been studied in cryptobiotic cysts and developing embryos and larvae of the brine shrimp Artemia. The two enzymes readily separate on Cibacron blue 3-GA Matrex gel. Assay requirements with activated DNA as primer-template are pH 8.0, 1 mM Mg2+, 50 mM K+ for DNA polymerase alpha and pH 8.4, 10 mM Mg2+, 80 mM K+ for DNA polymerase gamma. DNA polymerase alpha is inhibited by N-ethylmaleimide (94% and 100% at 1 mM and 10 mM respectively) and aphidicolin (96% at 60 microM). DNA polymerase gamma is also sensitive to N-ethylmaleimide (83% and 100% inhibition at 1 mM and 10 mM respectively) but is resistant to aphidicolin. 2',3'-Dideoxythymidine 5'-triphosphate (ddTTP) inhibits the gamma polymerase by 88% when in fivefold excess over dTTP whereas the alpha polymerase is unaffected by this compound. DNA polymerase alpha has a sedimentation coefficient of 7.6 S which is reduced to 6.2 S by a phenylmethylsulphonyl fluoride-sensitive proteinase. The gamma polymerase sediments at 8.3 S. No DNA polymerase beta activity could be detected. After the reinitiation of development both activities increased twofold up to 8 h (gamma polymerase) and 16 h (alpha polymerase), then declined before the onset of nuclear DNA replication after hatching. Thymidine kinase activity increased over 200-fold up to the time of replication. Analysis on Percoll density gradients of the intracellular distribution of both polymerases during development suggests that the changes in their activities may be due to migration from storage sites to replication complexes in the nuclei and mitochondria. The content of the mitochondrial DNA polymerase gamma in different batches of cysts may reflect the relative viabilities of the cysts. PMID:7151804

  9. Mechanistic Investigation of the Bypass of a Bulky Aromatic DNA Adduct Catalyzed by a Y-family DNA Polymerase

    PubMed Central

    Gadkari, Varun V.; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

    2014-01-01

    3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2’-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dGC8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dGC8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dGC8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dGC8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dGC8-N-ABA bypass catalyzed by Dpo4. PMID:25048879

  10. Nested methylation-specific polymerase chain reaction cancer detection method

    DOEpatents

    Belinsky, Steven A.; Palmisano, William A.

    2007-05-08

    A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

  11. A read-ahead function in archaeal DNA polymerases detects promutagenic template-strand uracil

    PubMed Central

    Greagg, Martin A.; Fogg, Mark J.; Panayotou, George; Evans, Steven J.; Connolly, Bernard A.; Pearl, Laurence H.

    1999-01-01

    Deamination of cytosine to uracil is the most common promutagenic change in DNA, and it is greatly increased at the elevated growth temperatures of hyperthermophilic archaea. If not repaired to cytosine prior to replication, uracil in a template strand directs incorporation of adenine, generating a G⋅C → A⋅U transition mutation in half the progeny. Surprisingly, genomic analysis of archaea has so far failed to reveal any homologues of either of the known families of uracil-DNA glycosylases responsible for initiating the base-excision repair of uracil in DNA, which is otherwise universal. Here we show that DNA polymerases from several hyperthermophilic archaea (including Vent and Pfu) specifically recognize the presence of uracil in a template strand and stall DNA synthesis before mutagenic misincorporation of adenine. A specific template-checking function in a DNA polymerase has not been observed previously, and it may represent the first step in a pathway for the repair of cytosine deamination in archaea. PMID:10430892

  12. Transcription-coupled Hypernegative Supercoiling of Plasmid DNA by T7 RNA Polymerase in Escherichia coli Topoisomerase I Deficient Strains

    PubMed Central

    Samul, Rebecca; Leng, Fenfei

    2007-01-01

    Summary Transcription by RNA polymerase can stimulate negative DNA supercoiling in Escherichia coli topA strains. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription in which positive DNA supercoils are generated in front of a translocating RNA polymerase and negative supercoils behind it. However, since a specific system is lacking to study the factors governing this biologically important process, the parameters regulating transcription-coupled DNA supercoiling (TCDS) in Escherichia coli still remain elusive. In this paper, we describe our efforts to study TCDS in Escherichia coli using a newly developed system. This system consists of a topA strain, VS111(DE3) or DM800(DE3), in which a λDE3 prophage containing a T7 RNA polymerase gene under control of the lacUV5 promoter has been integrated into the cell chromosome, along with a set of plasmids producing RNA transcripts of various lengths by T7 RNA polymerase. Using this system, we found that transcription by T7 RNA polymerase strikingly induced formation of hypernegatively supercoiled plasmid DNA. We also discovered, for the first time, that TCDS was dependent on the length of RNA transcripts in vivo, precisely predicted by the “twin-supercoiled-domain” model of transcription. Furthermore, our results demonstrated that hypernegative supercoiling of plasmid DNA by T7 RNA polymerase did not require anchoring of DNA to the bacterial cytoplasmic membrane. These results indicate that a transcribing RNA polymerase alone is sufficient to cause change of local DNA superhelicity, which can have a powerful impact on the conformation and function of critical DNA sequence elements, such as promoters and DNA replication origins. PMID:17980389

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

    PubMed Central

    Iyer, Lakshminarayan M; Koonin, Eugene V; Aravind, L

    2003-01-01

    Background The eukaryotic RNA-dependent RNA polymerase (RDRP) is involved in the amplification of regulatory microRNAs during post-transcriptional gene silencing. This enzyme is highly conserved in most eukaryotes but is missing in archaea and bacteria. No evolutionary relationship between RDRP and other polymerases has been reported so far, hence the origin of this eukaryote-specific polymerase remains a mystery. Results Using extensive sequence profile searches, we identified bacteriophage homologs of the eukaryotic RDRP. The comparison of the eukaryotic RDRP and their homologs from bacteriophages led to the delineation of the conserved portion of these enzymes, which is predicted to harbor the catalytic site. Further, detailed sequence comparison, aided by examination of the crystal structure of the DNA-dependent RNA polymerase (DDRP), showed that the RDRP and the β' subunit of DDRP (and its orthologs in archaea and eukaryotes) contain a conserved double-psi β-barrel (DPBB) domain. This DPBB domain contains the signature motif DbDGD (b is a bulky residue), which is conserved in all RDRPs and DDRPs and contributes to catalysis via a coordinated divalent cation. Apart from the DPBB domain, no similarity was detected between RDRP and DDRP, which leaves open two scenarios for the origin of RDRP: i) RDRP evolved at the onset of the evolution of eukaryotes via a duplication of the DDRP β' subunit followed by dramatic divergence that obliterated the sequence similarity outside the core catalytic domain and ii) the primordial RDRP, which consisted primarily of the DPBB domain, evolved from a common ancestor with the DDRP at a very early stage of evolution, during the RNA world era. The latter hypothesis implies that RDRP had been subsequently eliminated from cellular life forms and might have been reintroduced into the eukaryotic genomes through a bacteriophage. Sequence and structure analysis of the DDRP led to further insights into the evolution of RNA polymerases

  14. Altered immunoglobulin hypermutation pattern and frequency in complementary mouse models of DNA polymerase ζ activity

    PubMed Central

    Daly, Janssen; Bebenek, Katarzyna; Watt, Danielle L.; Richter, Kathleen; Jiang, Chuancang; Zhao, Ming-Lang; Ray, Madhumita; McGregor, W. Glenn; Kunkel, Thomas A.; Diaz, Marilyn

    2012-01-01

    To test the hypothesis that DNA polymerase ζ participates in immunoglobulin hypermutation, we generated two mouse models of Pol ζ function: a B-cell specific conditional knock-out and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B-cells had a reduction in mutation frequency at immunoglobulin loci in the spleen and in Peyer’s Patches, while knock-in mice with a mutagenic Pol ζ, displayed a marked increase in mutation frequency in Peyer’s Patches revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in immunoglobulin hypermutation. PMID:22547703

  15. Stopped in its tracks: the RNA polymerase molecular motor as a robust sensor of DNA damage.

    PubMed

    Howan, K; Monnet, J; Fan, J; Strick, T R

    2014-08-01

    DNA repair is often a complex, multi-component, multi-step process; this makes detailed kinetic analysis of the different steps of repair a challenging task using standard biochemical methods. At the same time, single-molecule methods are well-suited for extracting kinetic information despite time-averaging due to diffusion of biochemical components and stochasticity of chemical reaction steps. Here we discuss recent experiments using DNA nanomanipulation in a magnetic trap to study the initiation of transcription-coupled repair in a model bacterial system comprising the canonical Escherichia coli RNA polymerase and the Mfd translocase which specifically binds to it. These experiments provide kinetic insight into the reaction process, helping to explain how Mfd discriminates between transcribing RNAP and stalled RNAP. They also identify a reliably long-lived intermediate containing Mfd translocase and, potentially, RNA polymerase. This intermediate presumably serves as a platform for assembly of downstream repair components UvrAB(C). PMID:24685770

  16. Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR

    PubMed Central

    Kermekchiev, Milko B.; Tzekov, Anatoly; Barnes, Wayne M.

    2003-01-01

    Although the thermophilic bacterium Thermus aquaticus grows optimally at 70°C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20–37°C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37°C yet retain apparently normal activity at 68°C and resistance at 95°C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity. PMID:14576300

  17. Conserved Overlapping Gene Arrangement, Restricted Expression, and Biochemical Activities of DNA Polymerase ν (POLN)*

    PubMed Central

    Takata, Kei-ichi; Tomida, Junya; Reh, Shelley; Swanhart, Lisa M.; Takata, Minoru; Hukriede, Neil A.; Wood, Richard D.

    2015-01-01

    DNA polymerase ν (POLN) is one of 16 DNA polymerases encoded in vertebrate genomes. It is important to determine its gene expression patterns, biological roles, and biochemical activities. By quantitative analysis of mRNA expression, we found that POLN from the zebrafish Danio rerio is expressed predominantly in testis. POLN is not detectably expressed in zebrafish embryos or in mouse embryonic stem cells. Consistent with this, injection of POLN-specific morpholino antisense oligonucleotides did not interfere with zebrafish embryonic development. Analysis of transcripts revealed that vertebrate POLN has an unusual gene expression arrangement, sharing a first exon with HAUS3, the gene encoding augmin-like complex subunit 3. HAUS3 is broadly expressed in embryonic and adult tissues, in contrast to POLN. Differential expression of POLN and HAUS3 appears to arise by alternate splicing of transcripts in mammalian cells and zebrafish. When POLN was ectopically overexpressed in human cells, it specifically coimmunoprecipitated with the homologous recombination factors BRCA1 and FANCJ, but not with previously suggested interaction partners (HELQ and members of the Fanconi anemia core complex). Purified zebrafish POLN protein is capable of thymine glycol bypass and strand displacement, with activity dependent on a basic amino acid residue known to stabilize the primer-template. These properties are conserved with the human enzyme. Although the physiological function of pol ν remains to be clarified, this study uncovers distinctive aspects of its expression control and evolutionarily conserved properties of this DNA polymerase. PMID:26269593

  18. Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate.

    PubMed Central

    Focher, F; Maga, G; Bendiscioli, A; Capobianco, M; Colonna, F; Garbesi, A; Spadari, S

    1995-01-01

    L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides. Images PMID:7544886

  19. Stereospecificity of human DNA polymerases alpha, beta, gamma, delta and epsilon, HIV-reverse transcriptase, HSV-1 DNA polymerase, calf thymus terminal transferase and Escherichia coli DNA polymerase I in recognizing D- and L-thymidine 5'-triphosphate as substrate.

    PubMed

    Focher, F; Maga, G; Bendiscioli, A; Capobianco, M; Colonna, F; Garbesi, A; Spadari, S

    1995-08-11

    L-beta-Deoxythymidine (L-dT), the optical enantiomer of D-beta-deoxythymidine (D-dT), and L-enantiomers of nucleoside analogs, such as 5-iodo-2'-deoxy-L-uridine (L-IdU) and E-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymidine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase in vitro, but also human DNA polymerase alpha, gamma, delta and epsilon, human immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coli DNA polymerase 1 and calf thymus terminal transferase, although DNA polymerase beta was resistant; (iii) whereas DNA polymerase beta, gamma, delta and epsilon are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly incorporate at least one L-dTMP residue, with DNA polymerase alpha and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3'-OH terminus make DNA more resistant to 3'-->5' exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides. PMID:7544886

  20. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase [delta

    SciTech Connect

    Swan, Michael K.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2009-09-25

    DNA polymerase {delta} (Pol {delta}) is a high-fidelity polymerase that has a central role in replication from yeast to humans. We present the crystal structure of the catalytic subunit of yeast Pol {delta} in ternary complex with a template primer and an incoming nucleotide. The structure, determined at 2.0-{angstrom} resolution, catches the enzyme in the act of replication, revealing how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the 'sensing' interactions near the primer terminus, which signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of cancer-causing mutations in Pol {delta}.

  1. Replication of linear duplex DNA in vitro with bacteriophage T5 DNA polymerase

    SciTech Connect

    Fujimura, R. K.; Das, S. K.; Allison, D. P.; Roop, B. C.

    1980-01-01

    Two sets of experiments are presented that attempt to contribute to understanding the mechanisms of DNA replication. The specific areas discussed are fidelity of DNA replication and initiation of replication of duplex DNA. (ACR)

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

    PubMed Central

    Mallik, Sarita; Popodi, Ellen M.; Hanson, Andrew J.

    2015-01-01

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

  3. Structure of the SSB-DNA polymerase III interface and its role in DNA replication

    SciTech Connect

    Marceau, Aimee H; Bahng, Soon; Massoni, Shawn C; George, Nicholas P; Sandler, Steven J; Marians, Kenneth J; Keck, James L

    2012-05-22

    Interactions between single-stranded DNA-binding proteins (SSBs) and the DNA replication machinery are found in all organisms, but the roles of these contacts remain poorly defined. In Escherichia coli, SSB's association with the χ subunit of the DNA polymerase III holoenzyme has been proposed to confer stability to the replisome and to aid delivery of primers to the lagging-strand DNA polymerase. Here, the SSB-binding site on χ is identified crystallographically and biochemical and cellular studies delineate the consequences of destabilizing the χ/SSB interface. An essential role for the χ/SSB interaction in lagging-strand primer utilization is not supported. However, sequence changes in χ that block complex formation with SSB lead to salt-dependent uncoupling of leading- and lagging-strand DNA synthesis and to a surprising obstruction of the leading-strand DNA polymerase in vitro, pointing to roles for the χ/SSB complex in replisome establishment and maintenance. Destabilization of the χ/SSB complex in vivo produces cells with temperature-dependent cell cycle defects that appear to arise from replisome instability.

  4. Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton

    PubMed Central

    Schmidt, Helen F; Sakowski, Eric G; Williamson, Shannon J; Polson, Shawn W; Wommack, KEric

    2014-01-01

    Virioplankton have a significant role in marine ecosystems, yet we know little of the predominant biological characteristics of aquatic viruses that influence the flow of nutrients and energy through microbial communities. Family A DNA polymerases, critical to DNA replication and repair in prokaryotes, are found in many tailed bacteriophages. The essential role of DNA polymerase in viral replication makes it a useful target for connecting viral diversity with an important biological feature of viruses. Capturing the full diversity of this polymorphic gene by targeted approaches has been difficult; thus, full-length DNA polymerase genes were assembled out of virioplankton shotgun metagenomic sequence libraries (viromes). Within the viromes novel DNA polymerases were common and found in both double-stranded (ds) DNA and single-stranded (ss) DNA libraries. Finding DNA polymerase genes in ssDNA viral libraries was unexpected, as no such genes have been previously reported from ssDNA phage. Surprisingly, the most common virioplankton DNA polymerases were related to a siphovirus infecting an α-proteobacterial symbiont of a marine sponge and not the podoviral T7-like polymerases seen in many other studies. Amino acids predictive of catalytic efficiency and fidelity linked perfectly to the environmental clades, indicating that most DNA polymerase-carrying virioplankton utilize a lower efficiency, higher fidelity enzyme. Comparisons with previously reported, PCR-amplified DNA polymerase sequences indicated that the most common virioplankton metagenomic DNA polymerases formed a new group that included siphoviruses. These data indicate that slower-replicating, lytic or lysogenic phage populations rather than fast-replicating, highly lytic phages may predominate within the virioplankton. PMID:23985748

  5. Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton.

    PubMed

    Schmidt, Helen F; Sakowski, Eric G; Williamson, Shannon J; Polson, Shawn W; Wommack, K Eric

    2014-01-01

    Virioplankton have a significant role in marine ecosystems, yet we know little of the predominant biological characteristics of aquatic viruses that influence the flow of nutrients and energy through microbial communities. Family A DNA polymerases, critical to DNA replication and repair in prokaryotes, are found in many tailed bacteriophages. The essential role of DNA polymerase in viral replication makes it a useful target for connecting viral diversity with an important biological feature of viruses. Capturing the full diversity of this polymorphic gene by targeted approaches has been difficult; thus, full-length DNA polymerase genes were assembled out of virioplankton shotgun metagenomic sequence libraries (viromes). Within the viromes novel DNA polymerases were common and found in both double-stranded (ds) DNA and single-stranded (ss) DNA libraries. Finding DNA polymerase genes in ssDNA viral libraries was unexpected, as no such genes have been previously reported from ssDNA phage. Surprisingly, the most common virioplankton DNA polymerases were related to a siphovirus infecting an α-proteobacterial symbiont of a marine sponge and not the podoviral T7-like polymerases seen in many other studies. Amino acids predictive of catalytic efficiency and fidelity linked perfectly to the environmental clades, indicating that most DNA polymerase-carrying virioplankton utilize a lower efficiency, higher fidelity enzyme. Comparisons with previously reported, PCR-amplified DNA polymerase sequences indicated that the most common virioplankton metagenomic DNA polymerases formed a new group that included siphoviruses. These data indicate that slower-replicating, lytic or lysogenic phage populations rather than fast-replicating, highly lytic phages may predominate within the virioplankton. PMID:23985748

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

    PubMed

    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; <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

  7. Characterization of novel hepadnaviral RNA species accumulated in hepatoma cells treated with viral DNA polymerase inhibitors.

    PubMed

    Zhang, Pinghu; Liu, Fei; Guo, Fang; Zhao, Qiong; Chang, Jinhong; Guo, Ju-Tao

    2016-07-01

    Inhibitors of hepadnaviral DNA polymerases are predicted to inhibit both minus and plus strand of viral DNA synthesis and arrest viral DNA replication at the stage of pregenomic (pg) RNA-containing nucleocapsids. However, analyses of the RNA species of human and duck hepatitis B viruses (HBV and DHBV, respectively) in hepatoma cells treated with viral DNA polymerase inhibitors revealed the genesis of novel RNA species migrating slightly faster than the full-length pgRNA. The DNA polymerase inhibitor-induced accumulation of these RNA species were abolished in the presence of alpha-interferon or HBV nucleocapsid assembly inhibitors. Moreover, they were protected from microccocal nuclease digestion and devoid of a poly-A tail. These characteristics suggest that the novel RNA species are most likely generated from RNase H cleavage of encapsidated pgRNA, after primer translocation and synthesis of the 5' terminal portion of minus strand DNA. In support of this hypothesis, DNA polymerase inhibitor treatment of chicken hepatoma cells transfected with a DHBV genome encoding an RNase H inactive DNA polymerase (E696H) failed to produce such RNA species. Our results thus suggest that the currently available DNA polymerase inhibitors do not efficiently arrest minus strand DNA synthesis at the early stage in hepatocytes. Hence, development of novel antiviral agents that more potently suppress viral DNA synthesis or viral nucleocapsid assembly inhibitors that are mechanistically complementary to the currently available DNA polymerase inhibitors are warranted. PMID:27083116

  8. Adenovirus type 2 VAI RNA transcription by polymerase III is blocked by sequence-specific methylation.

    PubMed Central

    Jüttermann, R; Hosokawa, K; Kochanek, S; Doerfler, W

    1991-01-01

    Sequence-specific methylation of the promoter and adjacent regions in mammalian genes transcribed by RNA polymerase II leads to the inhibition of these genes. So far, RNA polymerase III-transcribed genes have not been investigated in depth. We therefore studied methylation effects on the RNA polymerase III-transcribed VAI gene of adenovirus type 2 DNA. The VAI gene contains 20 5'-CG-3' dinucleotides, of which 4 (20%) can be methylated by HpaII (5'-CCGG-3') and HhaI (5'-GCGC-3'). Three of these 5'-CG-3' sequences are located close to the internal regulatory region of the VAI segment. An unmethylated, a 5'-CCGG-3'- and 5'-GCGC-3'-methylated, and a 5'-CG-3'-methylated pUC18 construct containing the VAI and VAII regions were transfected into mammalian cells. In many experiments, an inactivating effect of 5'-CCGG-3' and 5'-GCGC-3' DNA methylation on the VAI region was not observed. In contrast, methylation of all 20 5'-CG-3' sequences in the VAI region by a CpG-specific DNA methyltransferase from Spiroplasma species did interfere with VAI transcription. Transcription of the VAI- and VAII- and of the VAI-containing constructs was also shown to be inhibited in an in vitro cell-free transcription system after the constructs had been methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences or at all 5'-CG-3' sequences. When an oligodeoxyribonucleotide which carried the internal control block A of the VAI region was methylated at three 5'-CG-3' sequences, the formation of a complex with HeLa nuclear proteins was abrogated. The results presented support the notion that the VAI gene transcribed by the DNA-dependent RNA polymerase III is also inactivated by methylation of the decisive 5'-CG-3' sequences. Images PMID:2002541

  9. Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting

    SciTech Connect

    Lee, M.Y.W.T.; Toomey, N.L.

    1987-02-24

    DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and n-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 A and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase delta preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptides component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase delta with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase ..cap alpha.. inhibited placental polymerase ..cap alpha.. but did not inhibit DNA polymerase delta, while the murine polyclonal antisera to polymerase delta inhibited delta but not ..cap alpha... These findings establish the existence of DNA polymerase delta in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.

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

    PubMed Central

    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

  11. DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae

    SciTech Connect

    Budd, M.E.; Wittrup, K.D.; Bailey, J.E.; Campbell, J.L.

    1989-02-01

    We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

  12. Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase.

    PubMed

    Indiani, Chiara; Langston, Lance D; Yurieva, Olga; Goodman, Myron F; O'Donnell, Mike

    2009-04-14

    All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with DnaB helicase and regulate its rate of unwinding, slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely exchange with the polymerase III (Pol III) replicase on the beta-clamp and function with DnaB helicase to form alternative replisomes, even before Pol III stalls at a lesion. DNA damage-induced levels of Pol II and Pol IV dominate the clamp, slowing the helicase and stably maintaining the architecture of the replication machinery while keeping the fork moving. We propose that these dynamic actions provide additional time for normal excision repair of lesions before the replication fork reaches them and also enable the appropriate translesion polymerase to sample each lesion as it is encountered. PMID:19279203

  13. Translesion DNA polymerases remodel the replisome and alter the speed of the replicative helicase

    PubMed Central

    Indiani, Chiara; Langston, Lance D.; Yurieva, Olga; Goodman, Myron F.; O'Donnell, Mike

    2009-01-01

    All cells contain specialized translesion DNA polymerases that replicate past sites of DNA damage. We find that Escherichia coli translesion DNA polymerase II (Pol II) and polymerase IV (Pol IV) function with DnaB helicase and regulate its rate of unwinding, slowing it to as little as 1 bp/s. Furthermore, Pol II and Pol IV freely exchange with the polymerase III (Pol III) replicase on the β-clamp and function with DnaB helicase to form alternative replisomes, even before Pol III stalls at a lesion. DNA damage-induced levels of Pol II and Pol IV dominate the clamp, slowing the helicase and stably maintaining the architecture of the replication machinery while keeping the fork moving. We propose that these dynamic actions provide additional time for normal excision repair of lesions before the replication fork reaches them and also enable the appropriate translesion polymerase to sample each lesion as it is encountered. PMID:19279203

  14. Involvement of DNA polymerase alpha in host cell reactivation of UV-irradiated herpes simplex virus

    SciTech Connect

    Nishiyama, Y.; Yoshida, S.; Maeno, K.

    1984-02-01

    Aphidicolin is a potent inhibitor of both host cell DNA polymerase alpha and herpes simplex virus (HSV)-induced DNA polymerase but has no effect on DNA polymerases beta and gamma of host cells. By using an aphidicolin-resistant mutant (Aphr) of HSV, a possible involvement of DNA polymerase alpha in host cell reactivation of UV-damaged HSV was studied. Plaque formation by UV-irradiated Aphr was markedly inhibited by 1 microgram of aphidicolin per ml, which did not affect the plating efficiency of nonirradiated Aphr. Aphidicolin added before 12 h postinfection inhibited plaque formation by irradiated Aphr, which became aphidicolin insensitive after 36 h postinfection. The results strongly suggest that host cell DNA polymerase alpha is involved in the repair of UV-irradiated HSV DNA.

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

    DOEpatents

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

    2013-06-25

    A method 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 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.

  16. DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase.

    PubMed

    Khairnar, Nivedita P; Misra, Hari S

    2009-09-01

    The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase beta. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5'-deoxyribose phosphate (5'-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5'-dRP lyase and a truncated polymerase domain was comparatively less sensitive to gamma-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair. PMID:19542005

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

    PubMed Central

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

    2015-01-01

    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. PMID:26519465

  18. Structural Basis for DNA-Hairpin Promoter Recognition by the Bacteriophage N4 Virion RNA Polymerase

    SciTech Connect

    Gleghorn, M.; Davydova, E; Rothman-Denes, L; Murakami, K

    2008-01-01

    Coliphage N4 virion-encapsidated RNA polymerase (vRNAP) is a member of the phage T7-like single-subunit RNA polymerase (RNAP) family. Its central domain (mini-vRNAP) contains all RNAP functions of the full-length vRNAP, which recognizes a 5 to 7 base pair stem and 3 nucleotide loop hairpin DNA promoter. Here, we report the X-ray crystal structures of mini-vRNAP bound to promoters. Mini-vRNAP uses four structural motifs to recognize DNA sequences at the hairpin loop and stem and to unwind DNA. Despite their low sequence similarity, three out of four motifs are shared with T7 RNAP that recognizes a double-stranded DNA promoter. The binary complex structure and results of engineered disulfide linkage experiments reveal that the plug and motif B loop, which block the access of template DNA to the active site in the apo-form mini-vRNAP, undergo a large-scale conformational change upon promoter binding, explaining the restricted promoter specificity that is critical for N4 phage early transcription.

  19. Targeted radiosensitization of cells expressing truncated DNA polymerase {beta}.

    PubMed

    Neijenhuis, Sari; Verwijs-Janssen, Manon; van den Broek, Lenie J; Begg, Adrian C; Vens, Conchita

    2010-11-01

    Ionizing radiation (IR) is an effective anticancer treatment, although failures still occur. To improve radiotherapy, tumor-targeted strategies are needed to increase radiosensitivity of tumor cells, without influencing normal tissue radiosensitivity. Base excision repair (BER) and single-strand break repair (SSBR) contribute to the determination of sensitivity to IR. A crucial protein in BER/SSBR is DNA polymerase β (polβ). Aberrant polβ expression is commonly found in human tumors and leads to inhibition of BER. Here, we show that truncated polβ variant (polβ-Δ)-expressing cells depend on homologous recombination (HR) for survival after IR, indicating that a considerable fraction of polβ-Δ-induced lesions are subject to repair by HR. Increased sensitization was found not to result from involvement in DNA-dependent protein kinase-dependent nonhomologous end joining, the other major double-strand break repair pathway. Caffeine and the ATM inhibitor Ku55933 cause polβ-Δ-dependent radiosensitization. Consistent with the observed HR dependence and the known HR-modulating activity of ATM, polβ-Δ-expressing cells showed increased radiosensitization after BRCA2 knockdown that is absent under ATM-inhibited conditions. Our data suggest that treatment with HR modulators is a promising therapeutic strategy for exploiting defects in the BER/SSBR pathway in human tumors. PMID:20978197

  20. Functional analysis of Drosophila DNA polymerase ε p58 subunit

    PubMed Central

    Sahashi, Ritsuko; Matsuda, Risa; Suyari, Osamu; Kawai, Mieko; Yoshida, Hideki; Cotterill, Sue; Yamaguchi, Masamitsu

    2013-01-01

    DNA polymerase ε (polε) plays a central role in DNA replication in eukaryotic cells, and has been suggested to the main synthetic polymerase on the leading strand. It is a hetero-tetrameric enzyme, comprising a large catalytic subunit (the A subunit ~250 kDa), a B subunit of ~60 kDa in most species (~80 kDa in budding yeast) and two smaller subunits (each ~20 kDa). In Drosophila, two subunits of polε (dpolε) have been identified. One is the 255 kDa catalytic subunit (dpolεp255), and the other is the 58 kDa subunit (dpolεp58). The functions of the B subunit have been mainly studied in budding yeast and mammalian cell culture, few studies have been performed in the context of an intact multicellular organism and therefore its functions in this context remain poorly understood. To address this we examined the in vivo role of dpolεp58 in Drosophila. A homozygous dpolεp58 mutant is pupal lethal, and the imaginal discs are less developed in the third instar larvae. In the eye discs of this mutant S phases, as measured by BrdU incorporation assays, were significantly reduced. In addition staining with an anti-phospho histone H3 (PH3) antibody, (a marker of M phase), was increased in the posterior region of eye discs, where usually cells stop replicating and start differentiation. These results indicate that dpolεp58 is essential for Drosophila development and plays an important role in progression of S phase in mitotic cell cycles. We also observed that the size of nuclei in salivary gland cells were decreased in dpolεp58 mutant, indicating that dpolεp58 also plays a role in endoreplication. Furthermore we detect a putative functional interaction between dpolε and ORC2 in discs suggesting that polε plays a role in the initiation of DNA replication in Drosophila. PMID:24224125

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

    PubMed Central

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

    2014-01-01

    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. PMID:24614752

  2. Sensitive and specific polymerase chain reaction detection of Toxoplasma gondii for veterinary and medical diagnosis.

    PubMed Central

    MacPherson, J M; Gajadhar, A A

    1993-01-01

    A polymerase chain reaction (PCR) method was developed for the detection of Toxoplasma gondii. A universal- and a T. gondii-specific primer was used to amplify a region of the small subunit ribosomal RNA gene. This approach allows for a theoretical detection limit of 0.01 zoite of T. gondii per sample assayed. Experiments showed that this PCR method could detect 0.1 pg of T. gondii DNA, which represents about one organism. Polymerase chain reaction tests using DNAs of cat, dog, swine, cattle, human, Sarcocystis cruzi, Eimeria ahsata, E. vermiformis, and Escherichia coli indicated no cross-reaction with nucleic acids of hosts, related coccidia, or bacteria. Data on the sensitivity and specificity suggest that this PCR assay could be extremely useful for the diagnosis of toxoplasmosis in human and veterinary medicine, as well as for food safety surveys. Images Fig. 1. Fig. 2. PMID:8431804

  3. PDIP46 (DNA polymerase δ interacting protein 46) is an activating factor for human DNA polymerase δ

    PubMed Central

    Zheng, Rong; Yue, Fu; Lin, Szu Hua Sharon; Rahmeh, Amal A.; Lee, Ernest Y. C.; Zhang, Zhongtao; Lee, Marietta Y. W. T.

    2016-01-01

    PDIP46 (SKAR, POLDIP3) was discovered through its interaction with the p50 subunit of human DNA polymerase δ (Pol δ). Its functions in DNA replication are unknown. PDIP46 associates with Pol δ in cell extracts both by immunochemical and protein separation methods, as well as by ChIP analyses. PDIP46 also interacts with PCNA via multiple copies of a novel PCNA binding motif, the APIMs (AlkB homologue-2 PCNA-Interacting Motif). Sites for both p50 and PCNA binding were mapped to the N-terminal region containing the APIMs. Functional assays for the effects of PDIP46 on Pol δ activity on singly primed ssM13 DNA templates revealed that it is a novel and potent activator of Pol δ. The effects of PDIP46 on Pol δ in primer extension, strand displacement and synthesis through simple hairpin structures reveal a mechanism where PDIP46 facilitates Pol δ4 synthesis through regions of secondary structure on complex templates. In addition, evidence was obtained that PDIP46 is also capable of exerting its effects by a direct interaction with Pol δ, independent of PCNA. Mutation of the Pol δ and PCNA binding region resulted in a loss of PDIP46 functions. These studies support the view that PDIP46 is a novel accessory protein for Pol δ that is involved in cellular DNA replication. This raises the possibility that altered expression of PDIP46 or its mutation may affect Pol δ functions in vivo, and thereby be a nexus for altered genomic stability. PMID:26819372

  4. Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

    PubMed Central

    Jones, D H; Winistorfer, S C

    1992-01-01

    We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information. Images PMID:1371352

  5. Triplex DNA: A new platform for polymerase chain reaction – based biosensor

    PubMed Central

    Li, Yubin; Miao, Xiangmin; Ling, Liansheng

    2015-01-01

    Non - specific PCR amplification and DNA contamination usually accompany with PCR process, to overcome these problems, here we establish a sensor for thrombin by sequence - specific recognition of the PCR product with molecular beacon through triplex formation. Probe A and probe B were designed for the sensor, upon addition of thrombin, two probes hybridized to each other and the probe B was extended in the presence of Klenow Fragment polymerase and dNTPs. The PCR amplification occurred with further addition of Taq DNA Polymerase and two primers, the PCR product was recognized by molecular beacon through triplex formation. The fluorescence intensity increased with the logarithm of the concentration of thrombin over the range from 1.0 × 10−12 M to 1.0 × 10−7 M, with a detection limit of 261 fM. Moreover, the effect of DNA contamination and non - specific amplification could be ignored completely in the proposed strategy. PMID:26268575

  6. Triplex DNA: A new platform for polymerase chain reaction-based biosensor.

    PubMed

    Li, Yubin; Miao, Xiangmin; Ling, Liansheng

    2015-01-01

    Non-specific PCR amplification and DNA contamination usually accompany with PCR process, to overcome these problems, here we establish a sensor for thrombin by sequence-specific recognition of the PCR product with molecular beacon through triplex formation. Probe A and probe B were designed for the sensor, upon addition of thrombin, two probes hybridized to each other and the probe B was extended in the presence of Klenow Fragment polymerase and dNTPs. The PCR amplification occurred with further addition of Taq DNA Polymerase and two primers, the PCR product was recognized by molecular beacon through triplex formation. The fluorescence intensity increased with the logarithm of the concentration of thrombin over the range from 1.0 × 10(-12) M to 1.0 × 10(-7) M, with a detection limit of 261 fM. Moreover, the effect of DNA contamination and non - specific amplification could be ignored completely in the proposed strategy. PMID:26268575

  7. Structure-function studies of the herpes simplex virus type 1 DNA polymerase.

    PubMed Central

    Haffey, M L; Novotny, J; Bruccoleri, R E; Carroll, R D; Stevens, J T; Matthews, J T

    1990-01-01

    The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity. Images PMID:2168983

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

    SciTech Connect

    Aller, Pierre; Ye, Yu; Wallace, Susan S.; Burrows, Cynthia J.; Doubli, Sylvie

    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.

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

    PubMed

    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. PMID:27333783

  10. Detection of varicella-zoster virus (VZV) DNA in clinical samples from patients with VZV by the polymerase chain reaction.

    PubMed Central

    Kido, S; Ozaki, T; Asada, H; Higashi, K; Kondo, K; Hayakawa, Y; Morishima, T; Takahashi, M; Yamanishi, K

    1991-01-01

    A polymerase chain reaction system for the detection of varicella-zoster virus was established. Of 25 nucleotides, 4 oligonucleotide pairs (regions of thymidine kinase, thymidylate synthetase, glycoprotein I, and immediate early gene) were synthesized. The first three oligonucleotide pairs could be used as primers on the basis of specific DNA amplification. Varicella-zoster virus DNA was amplified by this polymerase chain reaction system in 20 of 20 vesicle samples, 5 of 6 crusts, and 12 of 13 throat swabs collected from patients with clinical varicella. Images PMID:1847154

  11. Specific detection of banana residue in processed foods using polymerase chain reaction.

    PubMed

    Sakai, Yumiko; Ishihata, Kimie; Nakano, Shigeru; Yamada, Toshihiro; Yano, Takeo; Uchida, Kouji; Nakao, Yoshiki; Urisu, Atsuo; Adachi, Reiko; Teshima, Reiko; Akiyama, Hiroshi

    2010-07-28

    Specific polymerase chain reaction (PCR) methods were developed for the detection of banana residue in processed foods. For high banana specificity, the primer set BAN-F/BAN-R was designed on the basis of the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) genes of chloroplasts and used to obtain amplified products specific to banana by both conventional and real-time PCR. To confirm the specificity of these methods, genomic DNA samples from 31 other species were examined; no amplification products were detected. Subsequently, eight kinds of processed foods containing banana were investigated using these methods to confirm the presence of banana DNA. Conventional PCR had a detection limit of 1 ppm (w/w) banana DNA spiked in 50 ng of salmon testis DNA, whereas SYBR Green I real-time semiquantitative PCR had a detection limit as low as 10 ppm banana DNA. Thus, both methods show high sensitivity and may be applicable as specific tools for the detection of trace amounts of banana in commercial food products. PMID:20604506

  12. Down syndrome as a model of DNA polymerase beta haploinsufficiency and accelerated aging.

    PubMed

    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. PMID:22019846

  13. The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts.

    PubMed

    Wyss, Laura A; Nilforoushan, Arman; Williams, David M; Marx, Andreas; Sturla, Shana J

    2016-08-19

    Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O(6)-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O(6)-methylguanine (O(6)-MeG) and O(6)-carboxymethylguanine (O(6)-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates ( BENZI: TP and BIM: TP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide BENZI: opposite adducts, with up to 150-fold higher catalytic efficiency for O(6)-MeG over guanine in the template. Furthermore, addition of artificial nucleotide BENZI: was required for full-length DNA synthesis during bypass of O(6)-CMG. Selective incorporation of the artificial nucleotide opposite an O(6)-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BENZI: TP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BENZI: TP opposite biologically relevant O(6)-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies. PMID:27378785

  14. Base-by-Base Counting of Nucleotide Incorporations by DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Turvey, Mackenzie W.; Gul, O. Tolga; Pugliese, Kaitlin M.; Marushchak, Denys O.; Rajapakse, Arith J.; Weiss, Gregory A.; Collins, Phillip G.

    Previously, the catalytic cycle of DNA polymerase has been recorded by tethering single polymerase molecules to single-walled carbon nanotube field effect transistors (FETs). As the polymerase incorporates nucleotides into a single-stranded DNA template, it generates electrical signals in the SWCNT-FET. Here, we investigate the accuracy of this electronic method by using low concentrations (<10 nM) of DNA template, such that the signal consists of long, diffusion-limited pauses interrupted by template binding and a burst of nucleotide incorporation events. By counting the events generated by as few as 10 template molecules, template length has been correctly determined with <1 base pair resolution. Furthermore, differing template lengths can be identified and correctly enumerated in solutions containing mixtures of templates. Processivity of the Klenow Fragment of DNA polymerase currently limits read lengths to 50-100 base pairs, but the FET technique should work equally well with longer-processivity polymerases.

  15. Gastrointestinal Hyperplasia with Altered Expression of DNA Polymerase β

    PubMed Central

    Yoshizawa, Katsuhiko; Jelezcova, Elena; Brown, Ashley R.; Foley, Julie F.; Nyska, Abraham; Cui, Xiangli; Hofseth, Lorne J.; Maronpot, Robert M.; Wilson, Samuel H.; Sepulveda, Antonia R.; Sobol, Robert W.

    2009-01-01

    Background Altered expression of DNA polymerase β (Pol β) has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol β 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 β. 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 β over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol β expression. We observed elevated expression of Pol β in stomach adenomas and thyroid follicular carcinomas, but reduced Pol β 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. PMID:19654874

  16. Structural basis for cisplatin DNA damage tolerance by human polymerase η during cancer chemotherapy

    PubMed Central

    Ummat, Ajay; Rechkoblit, Olga; Jain, Rinku; Choudhary, Jayati R.; Johnson, Robert E.; Silverstein, Timothy D.; Buku, Angeliki; Lone, Samer; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2012-01-01

    A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase η (Polη) is a key polymerase that allows cancer cells to cope with cisplatin–DNA adducts formed during chemotherapy. We present here a structure of human Polη inserting dCTP opposite a cisplatin intrastrand cross-link (PtGpG). We show that specificity of human Polη for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG•dCTP base pair and to accommodate the lesion without steric hindrance. The specificity is augmented by residues Gln38 and Ser62 that interact with PtGpG, and Arg61 that interacts with incoming dCTP. Collectively, the structure provides a basis for understanding how Polη in human cells can tolerate DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy. PMID:22562137

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

    PubMed Central

    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

  18. Unexpected Role for Helicobacter pylori DNA Polymerase I As a Source of Genetic Variability

    PubMed Central

    Arana, Mercedes E.; Gasparutto, Didier; Guérois, Raphaël; Kunkel, Thomas A.; Radicella, J. Pablo

    2011-01-01

    Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity. PMID:21731507

  19. Detection of Pneumocystis carinii DNA in sputum and bronchoalveolar lavage samples by polymerase chain reaction.

    PubMed Central

    Olsson, M; Elvin, K; Löfdahl, S; Linder, E

    1993-01-01

    A polymerase chain reaction (PCR)-based assay was developed for the detection of Pneumocystis carinii DNA in induced sputum and bronchoscopic alveolar lavage samples. The primer pair was selected from the published sequence of the thymidylate synthase gene of P. carinii derived from infected rats. The amplified DNA fragment of 403 bp was detected by agarose gel electrophoresis and by Southern and slot blot hybridization. No positive reaction was seen with DNA from different microorganisms typically found in the respiratory tract. P. carinii DNA was demonstrated in 30 of 42 sputum samples from immunosuppressed patients, whereas 21 of 42 sputum samples were positive by indirect immunofluorescence (IFL). Among the 42 patients, 14 were receiving prophylactic chemotherapy. In that group, PCR detected P. carinii in nine sputum samples, whereas IFL detected P. carinii in only four sputum samples. A positive PCR result was also seen in 5 of 43 IFL-negative bronchoscopic alveolar lavage samples from patients with respiratory symptoms. The PCR assay detected 10 copies of the target DNA, which corresponds to 10(-18) g of the specific P. carinii sequence. The results indicate that PCR amplification in combination with DNA hybridization is specific and is a more sensitive diagnostic method than IFL for the detection of P. carinii. Images PMID:8432806

  20. Structural analysis of isosteviol and related compounds as DNA polymerase and DNA topoisomerase inhibitors.

    PubMed

    Mizushina, Yoshiyuki; Akihisa, Toshihiro; Ukiya, Motohiko; Hamasaki, Yusuke; Murakami-Nakai, Chikako; Kuriyama, Isoko; Takeuchi, Toshifumi; Sugawara, Fumio; Yoshida, Hiromi

    2005-09-01

    Isosteviol (ent-16-ketobeyeran-19-oic acid) is a hydrolysis product of stevioside, which is a natural sweetener produced in the leaves of Stevia rebaudiana (Bertoni) Bertoni. In this report, we prepared isosteviol and related compounds from stevioside by microbial transformation and chemical conversion and assayed the inhibitory activities toward DNA metabolic enzymes and human cancer cell growth. Among twelve compounds obtained, only isosteviol (compound 3) potently inhibited both mammalian DNA polymerases (pols) and human DNA topoisomerase II (topo II), and IC50 value for pol alpha was 64.0 microM. This compound had no inhibitory effect on higher plant (cauliflower) pols, prokaryotic pols, human topo I, and DNA metabolic enzymes such as human telomerase, T7 RNA polymerase, and bovine deoxyribonuclease I. With pol alpha, isosteviol acted non-competitively with the DNA template-primer and nucleotide substrate. Isosteviol prevented the growth of human cancer cells, with LD50 values of 84-167 microM, and 500 microg of the compound caused a marked reduction in TPA (12-O-tetradecanoylphorbol-13-acetate)-induced inflammation (inhibitory effect, 53.0%). The relationship between the structure of stevioside-based compounds and these activities were discussed. PMID:15935396

  1. Computational Investigation of Locked Nucleic Acid (LNA) Nucleotides in the Active Sites of DNA Polymerases by Molecular Docking Simulations

    PubMed Central

    Poongavanam, Vasanthanathan; Madala, Praveen K.; Højland, Torben; Veedu, Rakesh N.

    2014-01-01

    Aptamers constitute a potential class of therapeutic molecules typically selected from a large pool of oligonucleotides against a specific target. With a scope of developing unique shorter aptamers with very high biostability and affinity, locked nucleic acid (LNA) nucleotides have been investigated as a substrate for various polymerases. Various reports showed that some thermophilic B-family DNA polymerases, particularly KOD and Phusion DNA polymerases, accepted LNA-nucleoside 5′-triphosphates as substrates. In this study, we investigated the docking of LNA nucleotides in the active sites of RB69 and KOD DNA polymerases by molecular docking simulations. The study revealed that the incoming LNA-TTP is bound in the active site of the RB69 and KOD DNA polymerases in a manner similar to that seen in the case of dTTP, and with LNA structure, there is no other option than the locked C3′-endo conformation which in fact helps better orienting within the active site. PMID:25036012

  2. Detection and characterization of mammalian DNA polymerase beta mutants by functional complementation in Escherichia coli.

    PubMed Central

    Sweasy, J B; Loeb, L A

    1993-01-01

    We have designed and utilized a bacterial complementation system to identify and characterize mammalian DNA polymerase beta mutants. In this complementation system, wild-type rat DNA polymerase beta replaces both the replicative and repair functions of DNA polymerase I in the Escherichia coli recA718 polA12 double mutant; our 263 DNA polymerase beta mutants replace E. coli polymerase I less efficiently or not at all. Of the 10 mutants that have been shown to contain DNA sequence alterations, 2 exhibit a split phenotype with respect to complementation of the growth defect and methylmethanesulfonate sensitivity of the double mutant; one is a null mutant. The mutants possessing a split phenotype contain amino acid residue alterations within a putative nucleotide binding site of DNA polymerase beta. This approach for the isolation and evaluation of mutants of a mammalian DNA polymerase in E. coli may ultimately lead to a better understanding of the mechanism of action of this enzyme and to precisely defining its role in vertebrate cells. Images Fig. 2 PMID:8506308

  3. Oligonucleotides labeled with single fluorophores as sensors for deoxynucleotide triphosphate binding by DNA polymerases.

    PubMed

    Nikiforov, Theo T

    2014-01-01

    Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr²⁺ as the divalent metal cation, the formation of ternary DNA-polymerase-dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr²⁺ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2',3'-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg²⁺ as the metal ion. PMID:24096197

  4. Molecular basis of RNA polymerase promoter specificity switch revealed through studies of Thermus bacteriophage transcription regulator

    PubMed Central

    Severinov, Konstantin; Minakhin, Leonid; Sekine, Shun-ichi; Lopatina, Anna; Yokoyama, Shigeyuki

    2014-01-01

    Transcription initiation is the central point of gene expression regulation. Understanding of molecular mechanism of transcription regulation requires, ultimately, the structural understanding of consequences of transcription factors binding to DNA-dependent RNA polymerase (RNAP), the enzyme of transcription. We recently determined a structure of a complex between transcription factor gp39 encoded by a Thermus bacteriophage and Thermus RNAP holoenzyme. In this addendum to the original publication, we highlight structural insights that explain the ability of gp39 to act as an RNAP specificity switch which inhibits transcription initiation from a major class of bacterial promoters, while allowing transcription from a minor promoter class to continue. PMID:25105059

  5. An Oligonucleotide Affinity Column for RNA-Dependent DNA Polymerase from RNA Tumor Viruses

    PubMed Central

    Gerwin, Brenda I.; Milstien, Julie B.

    1972-01-01

    Columns of (dT)12-18-cellulose provide a one-step enrichment procedure for RNA-dependent DNA polymerase. The enzyme of the virus from RD-114 cells, as well as that from Rauscher murine leukemia virus, have been purified in this way. The preference of viral as compared to cellular DNA polymerases for (dT)12-18 as a primer is reflected in the fact that the DNA polymerases of uninfected cells do not bind to this column. Viral enzymes have been purified and identified from crude cellular extracts. PMID:4506781

  6. Polymerase ribozyme efficiency increased by G/T-rich DNA oligonucleotides

    PubMed Central

    Yao, Chengguo; Müller, Ulrich F.

    2011-01-01

    The RNA world hypothesis states that the early evolution of life went through a stage where RNA served as genome and as catalyst. The replication of RNA world organisms would have been facilitated by ribozymes that catalyze RNA polymerization. To recapitulate an RNA world in the laboratory, a series of RNA polymerase ribozymes was developed previously. However, these ribozymes have a polymerization efficiency that is too low for self-replication, and the most efficient ribozymes prefer one specific template sequence. The limiting factor for polymerization efficiency is the weak sequence-independent binding to its primer/template substrate. Most of the known polymerase ribozymes bind an RNA heptanucleotide to form the P2 duplex on the ribozyme. By modifying this heptanucleotide, we were able to significantly increase polymerization efficiency. Truncations at the 3′-terminus of this heptanucleotide increased full-length primer extension by 10-fold, on a specific template sequence. In contrast, polymerization on several different template sequences was improved dramatically by replacing the RNA heptanucleotide with DNA oligomers containing randomized sequences of 15 nt. The presence of G and T in the random sequences was sufficient for this effect, with an optimal composition of 60% G and 40% T. Our results indicate that these DNA sequences function by establishing many weak and nonspecific base-pairing interactions to the single-stranded portion of the template. Such low-specificity interactions could have had important functions in an RNA world. PMID:21622900

  7. Stable interactions between DNA polymerase δ catalytic and structural subunits are essential for efficient DNA repair.

    PubMed

    Brocas, Clémentine; Charbonnier, Jean-Baptiste; Dhérin, Claudine; Gangloff, Serge; Maloisel, Laurent

    2010-10-01

    Eukaryotic DNA polymerase δ (Pol δ) activity is crucial for chromosome replication and DNA repair and thus, plays an essential role in genome stability. In Saccharomyces cerevisiae, Pol δ is a heterotrimeric complex composed of the catalytic subunit Pol3, the structural B subunit Pol31, and Pol32, an additional auxiliary subunit. Pol3 interacts with Pol31 thanks to its C-terminal domain (CTD) and this interaction is of functional importance both in DNA replication and DNA repair. Interestingly, deletion of the last four C-terminal Pol3 residues, LSKW, in the pol3-ct mutant does not affect DNA replication but leads to defects in homologous recombination and in break-induced replication (BIR) repair pathways. The defect associated with pol3-ct could result from a defective interaction between Pol δ and a protein involved in recombination. However, we show that the LSKW motif is required for the interaction between Pol3 C-terminal end and Pol31. This loss of interaction is relevant in vivo since we found that pol3-ct confers HU sensitivity on its own and synthetic lethality with a POL32 deletion. Moreover, pol3-ct shows genetic interactions, both suppression and synthetic lethality, with POL31 mutant alleles. Structural analyses indicate that the B subunit of Pol δ displays a major conserved region at its surface and that pol31 alleles interacting with pol3-ct, correspond to substitutions of Pol31 amino acids that are situated in this particular region. Superimposition of our Pol31 model on the 3D architecture of the phylogenetically related DNA polymerase α (Pol α) suggests that Pol3 CTD interacts with the conserved region of Pol31, thus providing a molecular basis to understand the defects associated with pol3-ct. Taken together, our data highlight a stringent dependence on Pol δ complex stability in DNA repair. PMID:20813592

  8. Elevated DNA polymerase alpha, DNA polymerase beta, and DNA topoisomerase II in a melphalan-resistant rhabdomyosarcoma xenograft that is cross-resistant to nitrosoureas and topotecan.

    PubMed

    Friedman, H S; Dolan, M E; Kaufmann, S H; Colvin, O M; Griffith, O W; Moschel, R C; Schold, S C; Bigner, D D; Ali-Osman, F

    1994-07-01

    Previous investigations have revealed that the human TE-671 MR human rhabdomyosarcoma xenograft selected in vivo for melphalan resistance (M. C. Rosenberg, et al., Cancer Res., 49: 6917-6922, 1989) is cross-resistant to a wide variety of alkylating agents and to bleomycin, but is collaterally sensitive to etoposide. Although glutathione levels were noted to be elevated in TE-671 MR compared to the melphalan-sensitive parental TE-671 xenograft, treatment with buthionine sulfoximine to deplete glutathione levels did not fully restore melphalan sensitivity in the TE-671 MR xenograft. The present studies were undertaken to search for additional mechanisms of resistance in the TE-671 MR xenograft. Drug sensitivity testing performed at the dose of agents that was lethal to 10% of the animals revealed that the TE-671 MR xenograft maintained resistance to the bifunctional cross-linking agent 1,3-bis(2-chloroethyl)-1-nitrosourea and was cross-resistant to the topoisomerase I poison topotecan. Treatment with buthionine sulfoximine did not sensitize the TE-671 MR xenograft to 1,3-bis(2-chloroethyl)-1-nitrosourea. Further, even though O6-alkylguanine-DNA alkyltransferase levels were high in both the TE-671 and TE-671 MR xenografts, depletion of O6-alkylguanine-DNA alkyltransferase activity by treatment with O6-benzylguanine substantially sensitized the TE-671 xenografts but not the TE-671 MR xenografts, suggesting an additional mechanism of resistance. Measurement of additional enzyme activities that might be involved in DNA repair revealed significant elevations in DNA polymerase alpha (46 +/- 8 (SD) units/mg protein in TE-671, 69 +/- 6 units/mg protein in TE-671 MR, P < 0.05) and DNA polymerase beta (0.43 +/- 0.01 units/mg protein in TE-671, 0.78 +/- 0.12 units/mg protein in TE-671 MR, P < 0.05) but not DNA polymerase delta or total DNA ligase. Examination of topoisomerases by activity assays and Western blotting revealed a 2-fold increase in topoisomerase II and a 2-fold

  9. Replication by a single DNA polymerase of a stretched single-stranded DNA

    PubMed Central

    Maier, Berenike; Bensimon, David; Croquette, Vincent

    2000-01-01

    A new approach to the study of DNA/protein interactions has been opened through the recent advances in the manipulation of single DNA molecules. These allow the behavior of individual molecular motors to be studied under load and compared with bulk measurements. One example of such a motor is the DNA polymerase, which replicates DNA. We measured the replication rate by a single enzyme of a stretched single strand of DNA. The marked difference between the elasticity of single- and double-stranded DNA allows for the monitoring of replication in real time. We have found that the rate of replication depends strongly on the stretching force applied to the template. In particular, by varying the load we determined that the biochemical steps limiting replication are coupled to movement. The replication rate increases at low forces, decreases at forces greater than 4 pN, and ceases when the single-stranded DNA substrate is under a load greater than ≈20 pN. The decay of the replication rate follows an Arrhenius law and indicates that multiple bases on the template strand are involved in the rate-limiting step of each cycle. This observation is consistent with the induced-fit mechanism for error detection during replication. PMID:11050232

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

    DOEpatents

    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.

  11. Virtual screening reveals a viral-like polymerase inhibitor that complexes with the DNA polymerase of Moniliophthora perniciosa.

    PubMed

    Andrade, B S; Souza, C S; Santos, G; Góes-Neto, A

    2016-01-01

    The filamentous fungus Moniliophthora perniciosa is a basidiomycota that causes the witches' broom disease in cocoa trees (Theobroma cacao L.). The mitochondrial DNA polymerase of M. perniciosa (MpmitDNApol) is classified within the B family of DNA polymerases, which can be found in viruses and cellular organelles. Using virtual screening processes, accessing KEGG, PubChem, and ZINC databases, we selected the 27 best putative nucleoside viral-like polymerase inhibitors to test against MpmitDNApol. We used Autodock Vina to perform docking simulations of the selected molecules and to return energy values in several ligand conformations. Then, we used Pymol v1.7.4.4 to check the stereochemistry of chiral carbons, hydrogen bonding receptors, absence or presence of hydrogen, sub and superstructure, numbers of rings, rotatable bonds, and donor groups. We selected the Entecavir Hydrate, a drug used to control hepatitis B; subsequently AMBER 14 was used to describe the behavior of polymerase-entecavir complex after setting up 3500 ps of simulation in water at a temperature of 300 K. From the simulation, a graph of Potential Energy was generated revealing that the ligand remains in the catalytic site after 3500 ps with a final energy of -612,587.4214 kcal/mol. PMID:27323084

  12. Repeated tertiary fold of RNA polymerase II and implications for DNA binding.

    PubMed

    Fu, J; Gerstein, M; David, P R; Gnatt, A L; Bushnell, D A; Edwards, A M; Kornberg, R D

    1998-07-17

    X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein. PMID:9665838

  13. In Silico Screening for Novel Inhibitors of DNA Polymerase III Alpha Subunit of Mycobacterium tuberculosis (MtbDnaE2, H37Rv)

    PubMed Central

    Jadaun, Alka; Sudhakar D, Raja; Subbarao, N.; Dixit, Aparna

    2015-01-01

    Tuberculosis, a pandemic disease is caused by Mycobacterium tuberculosis (Mtb). DNA polymerase III encoded by DnaE2 of Mtb is specifically required for its survival in vivo, and hence can be considered to be a potential drug target. Amino acid sequence analysis of the MtbDnaE2 and its human counterpart does not show any significant similarity. Therefore, a 3D model of the MtbDnaE2 was generated using Modeller 9v10 with the template structure of E. Coli DNA polymerase III alpha subunit (2HNH_A). The generated models were validated using a number of programmes such as RAMPAGE/PROCHECK, VERIFY_3D, and ProSA. MtbDnaE2 has few conserved residues and four conserved domains similar to that present in DNA polymerase III of E. coli. In silico screening was performed with bioactive anti-tuberculosis compounds and 6-AU (a known inhibitor of DNA polymerase III of Bacillus subtilis) and its analogues against the modeled MtbDnaE2 structure. Docking was performed using GOLD v5.2 software which resulted in the identification of top ten compounds with high GOLD fitness scores and binding affinity (X-Score). To further evaluate the efficacy of these compounds, in silico ADMET analysis was performed using MedChem Designer v3. Given their high binding affinity to the targeted MtbDnaE2, which is essential for DNA replication in the Mtb and good ADMET properties, these compounds are promising candidates for further evaluation and development as anti-tubercular agents. PMID:25811866

  14. Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

    SciTech Connect

    Xia, Shuangluo; Christian, Thomas D.; Wang, Jimin; Konigsberg, William H.

    2012-09-17

    Minor groove hydrogen bonding (HB) interactions between DNA polymerases (pols) and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer extension. Because there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2'-deoxyadenosine (3DA), an analogue of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady-state kinetic parameters for the insertion of dAMP opposite dT using primer/templates (P/T)-containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 10{sup 2}-10{sup 3}-fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base pair geometry of 3DA/dT is exactly the same as those of dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 10{sup 2}-10{sup 3}-fold decrease in the rate of nucleotide incorporation. The minor groove HB interactions between position n-2 of the primer strand and RB69pol fix the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands, so that they are in the optinal orientation for DNA synthesis.

  15. Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

    SciTech Connect

    Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.; Dasgupta, A.

    1987-11-01

    The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

  16. Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

    PubMed Central

    Guilliam, Thomas A.; Jozwiakowski, Stanislaw K.; Ehlinger, Aaron; Barnes, Ryan P.; Rudd, Sean G.; Bailey, Laura J.; Skehel, J. Mark; Eckert, Kristin A.; Chazin, Walter J.; Doherty, Aidan J.

    2015-01-01

    PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication. PMID:25550423

  17. Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase*

    PubMed Central

    Kasiviswanathan, Rajesh; Copeland, William C.

    2011-01-01

    During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2′-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3–14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3′-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed. PMID:21778232

  18. Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA Polymerase

    PubMed Central

    Genna, Vito; Gaspari, Roberto; Dal Peraro, Matteo; De Vivo, Marco

    2016-01-01

    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. PMID:26935581

  19. Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA Polymerase-η.

    PubMed

    Genna, Vito; Gaspari, Roberto; Dal Peraro, Matteo; De Vivo, Marco

    2016-04-01

    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. PMID:26935581

  20. DNA Polymerase III, but Not Polymerase IV, Must Be Bound to a τ-Containing DnaX Complex to Enable Exchange into Replication Forks.

    PubMed

    Yuan, Quan; Dohrmann, Paul R; Sutton, Mark D; McHenry, Charles S

    2016-05-27

    Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III α that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a τ-containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of τ or the alternative shorter dnaX translation product γ showed that τ-, τ2-, or τ3-DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one τ. Previously, it was thought that at least two τs in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed. PMID:27056333

  1. Role of DNA polymerase κ in the maintenance of genomic stability

    PubMed Central

    Pillaire, Marie-Jeanne; Bétous, Rémy; Hoffmann, Jean-Sébastien

    2014-01-01

    To ensure high cell viability and genomic stability, cells have evolved two major mechanisms to deal with the constant challenge of DNA replication fork arrest during S phase of the cell cycle: (1) induction of the ataxia telangiectasia and Rad3-related (ATR) replication checkpoint mechanism, and (2) activation of a pathway that bypasses DNA damage and DNA with abnormal structure and is mediated by translesion synthesis (TLS) Y-family DNA polymerases. This review focuses on how DNA polymerase kappa (Pol κ), one of the most highly conserved TLS DNA polymerases, is involved in each of these pathways and thereby coordinates them to choreograph the response to a stalled replication fork. We also describe how loss of Pol κ regulation, which occurs frequently in human cancers, affects genomic stability and contributes to cancer development. PMID:27308312

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

    SciTech Connect

    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.

  3. Wanderings of a DNA enzymologist: from DNA polymerase to viral latency.

    PubMed

    Lehman, I Robert

    2006-01-01

    I am a member of what has been called, perhaps too grandiosely, "The Greatest Generation." I grew up during the Great Depression and served in the U.S. Army during World War II. Because of my military service and the benefits of the GI Bill, I was able to attend college and, later, graduate school. Early in my graduate studies, I became fascinated with enzymes and the biochemical reactions that they catalyze. This fascination has never left me during the 50 years I have been a "DNA enzymologist." I was fortunate to have had as a mentor Arthur Kornberg, one of the great biochemists of the twentieth century, and a splendid group of postdocs and graduate students. I have studied DNA polymerases, DNA nucleases, DNA ligases, and DNA recombinases, enzymes that are critical to our understanding of DNA replication, repair, and recombination. Most recently, I have been studying herpes virus replication and inadvertently wandered into an entirely new area-viral latency. PMID:16756482

  4. Replication of N[superscript 2],3-Ethenoguanine by DNA Polymerases

    SciTech Connect

    Zhao, Linlin; Christov, Plamen P.; Kozekov, Ivan D.; Pence, Matthew G.; Pallan, Pradeep S.; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter

    2014-10-02

    The unstable DNA adduct N2,3-ethenoguanine, a product of both exposure to the carcinogen vinyl chloride and of oxidative stress, was built into an oligonucleotide, using an isostere strategy to stabilize the glycosidic bond. This modification was then used to examine the cause of mutations by DNA polymerases, in terms of both the biochemistry of the lesion and a structure of the lesion within a polymerase.

  5. A RecA Protein Surface Required for Activation of DNA Polymerase V

    PubMed Central

    Gruber, Angela J.; Erdem, Aysen L.; Sabat, Grzegorz; Karata, Kiyonobu; Jaszczur, Malgorzata M.; Vo, Dan D.; Olsen, Tayla M.; Woodgate, Roger; Goodman, Myron F.; Cox, Michael M.

    2015-01-01

    DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis. PMID:25811184

  6. Mitochondrial DNA polymerase from embryos of Drosophila melanogaster: purification, subunit structure, and partial characterization

    SciTech Connect

    Wernette, C.M.; Kaguni, L.S.

    1986-11-05

    The mitochondrial DNA polymerase has been purified to near-homogeneity from early embryos of Drosophila melanogaster. Sodium dodecyl sulfate gel electrophoresis of the highly purified enzyme reveals two polypeptides with molecular masses of 125,000 and 35,000 daltons, in a ratio of 1:1. The enzyme has a sedimentation coefficient of 7.6 S and a stokes radius of 51 A. Taken together, the data suggest that the D. melanogaster DNA polymerase ..gamma.. is a heterodimer. DNA polymerase activity gel analysis has allowed the assignment of the DNA polymerization function to the large subunit. The DNA polymerase exhibits a remarkable ability to utilize efficiently a variety of template-primers including gapped DNA, poly(rA).oligo(dT) and singly primed phiX174 DNA. Both the crude and the highly purified enzymes are stimulated by KCl, and inhibited by dideoxythymidine triphosphate and by N-ethylmaleimide. Thus, the catalytic properties of the near-homogeneous Drosophila enzyme are consistent with those of DNA polymerase ..gamma.. as partially purified from several vertebrates.

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

    PubMed

    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. PMID:26894757

  8. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I.

    PubMed

    Dass, Randall A; Sarshad, Aishe A; Carson, Brittany B; Feenstra, Jennifer M; Kaur, Amanpreet; Obrdlik, Ales; Parks, Matthew M; Prakash, Varsha; Love, Damon K; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C; Percipalle, Piergiorgio; Brown, Anthony M C; Vincent, C Theresa

    2016-08-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  9. Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I

    PubMed Central

    Dass, Randall A.; Sarshad, Aishe A.; Feenstra, Jennifer M.; Kaur, Amanpreet; Pietras, Kristian; Serra, Rosa; Blanchard, Scott C.; Percipalle, Piergiorgio; Brown, Anthony M. C.; Vincent, C. Theresa

    2016-01-01

    Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo. PMID:27500936

  10. Characterization of the mammalian DNA polymerase gene(s) and enzyme(s). Annual progress report

    SciTech Connect

    Mishra, N.C.

    1994-01-01

    Consistent with the long term goal of our research to understand the nature of the key enzymes in eukaryotic DNA replication we have characterized the properties of the wild type DNA polymerases of the {alpha}-family and their mutants. We have also provided evidence for the role of aphidicolin in the elongation process of the in vivo DNA replication in eukaryotic cells. We also developed a technology for planned prep from a large numbers of clones for direct screening by size or restriction digestion in order to facilitate our goals to clone the DNA polymerase gene.

  11. Aberrant DNA Polymerase α Is Excluded from the Nucleus by Defective Import and Degradation in the Nucleus*

    PubMed Central

    Eichinger, Christian S.; Mizuno, Takeshi; Mizuno, Keiko; Miyake, Yasuyuki; Yanagi, Ken-ichiro; Imamoto, Naoko; Hanaoka, Fumio

    2009-01-01

    DNA polymerase α is essential for the onset of eukaryotic DNA replication. Its correct folding and assembly within the nuclear replication pre-initiation complex is crucial for normal cell cycle progression and genome maintenance. Due to a single point mutation in the largest DNA polymerase α subunit, p180, the temperature-sensitive mouse cell line tsFT20 exhibits heat-labile DNA polymerase α activity and S phase arrest at restrictive temperature. In this study, we show that an aberrant form of endogenous p180 in tsFT20 cells (p180tsFT20) is strictly localized in the cytoplasm while its wild-type counterpart enters the nucleus. Time-lapse fluorescence microscopy with enhanced green fluorescent protein-tagged or photoactivatable green fluorescent protein-tagged p180tsFT20 variants and inhibitor analysis revealed that the exclusion of aberrant p180tsFT20 from the nucleus is due to two distinct mechanisms: first, the inability of newly synthesized (cytoplasmic) p180tsFT20 to enter the nucleus and second, proteasome-dependent degradation of nuclear-localized protein. The nuclear import defect seems to result from an impaired association of aberrant de novo synthesized p180tsFT20 with the second subunit of DNA polymerase α, p68. In accordance, we show that RNA interference of p68 results in a decrease of the overall p180 protein level and in a specific increase of cytoplasmic localized p180 in NIH3T3 cells. Taken together, our data suggest two mechanisms that prevent the nuclear expression of aberrant DNA polymerase α. PMID:19726690

  12. Stopped-flow DNA polymerase assay by continuous monitoring of dNTP incorporation by fluorescence.

    PubMed

    Montgomery, Jesse L; Rejali, Nick; Wittwer, Carl T

    2013-10-15

    DNA polymerase activity was measured by a stopped-flow assay that monitors polymerase extension using an intercalating dye. Double-stranded DNA formation during extension of a hairpin substrate was monitored at 75°C for 2 min. Rates were determined in nucleotides per second per molecule of polymerase (nt/s) and were linear with time and polymerase concentration from 1 to 50 nM. The concentrations of 15 available polymerases were quantified and their extension rates determined in 50 mM Tris, pH 8.3, 0.5 mg/ml BSA, 2 mM MgCl₂, and 200 μM each dNTP as well as their commercially recommended buffers. Native Taq polymerases had similar extension rates of 10-45 nt/s. Three alternative polymerases showed faster speeds, including KOD (76 nt/s), Klentaq I (101 nt/s), and KAPA2G (155 nt/s). Fusion polymerases including Herculase II and Phusion were relatively slow (3-13 nt/s). The pH optimum for Klentaq extension was between 8.5 and 8.7 with no effect of Tris concentration. Activity was directly correlated to the MgCl2 concentration and inversely correlated to the KCl concentration. This continuous assay is relevant to PCR and provides accurate measurement of polymerase activity using a defined template without the need of radiolabeled substrates. PMID:23872003

  13. Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

    NASA Astrophysics Data System (ADS)

    Qin, Yangzhong; Zhong, Dongping

    2014-03-01

    Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined in nanospace but mostly highly mobile. Here, we report our studies of interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local sidechain fluctuations in the apo, binary and ternary states of Dpo4, we observed that the DNA binding interface and active site is dynamically solvent accessible and the interfacial water dynamics are slightly slow but similar to the surface hydration water fluctuations on picosecond time scales. Our MD simulations also show the binding interface full of water molecules and nonspecific weak interactions with protein and DNA. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation while the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase.

  14. Detection of Mycobacterium leprae DNA from Archaeological Skeletal Remains in Japan Using Whole Genome Amplification and Polymerase Chain Reaction

    PubMed Central

    Suzuki, Koichi; Takigawa, Wataru; Tanigawa, Kazunari; Nakamura, Kazuaki; Ishido, Yuko; Kawashima, Akira; Wu, Huhehasi; Akama, Takeshi; Sue, Mariko; Yoshihara, Aya; Mori, Shuichi; Ishii, Norihisa

    2010-01-01

    Background Identification of pathogen DNA from archaeological human remains is a powerful tool in demonstrating that the infectious disease existed in the past. However, it is very difficult to detect trace amounts of DNA remnants attached to the human skeleton, especially from those buried in a humid atmosphere with a relatively high environmental temperature such as in Asia. Methodology/Principal Findings Here we demonstrate Mycobacterium leprae DNA from archaeological skeletal remains in Japan by polymerase chain reaction, DNA sequencing and single nucleotide polymorphism (SNP) analysis. In addition, we have established a highly sensitive method of detecting DNA using a combination of whole genome amplification and polymerase chain reaction, or WGA-PCR, which provides superior sensitivity and specificity in detecting DNA from trace amounts of skeletal materials. Conclusion/Significance We have detected M. leprae DNA in archaeological skeletal remains for the first time in the Far East. Its SNP genotype corresponded to type 1; the first detected case worldwide of ancient M. leprae DNA. We also developed a highly sensitive method to detect ancient DNA by utilizing whole genome amplification. PMID:20865042

  15. Absence of a role for DNA polymerase II in SOS-induced translesion bypass of phi X174.

    PubMed Central

    Kow, Y W; Faundez, G; Hays, S; Bonner, C A; Goodman, M F; Wallace, S S

    1993-01-01

    In order to examine the possible role of Escherichia coli DNA polymerase II in SOS-induced translesion bypass, Weigle reactivation and mutation induction were measured with single-stranded phi X174 transfecting DNA containing individual lesions. No decrease in bypass of thymine glycol or cyclobutane pyrimidine dimers in the absence of DNA polymerase II was observed. Furthermore, DNA polymerase II did not affect bypass of abasic sites when either survival or mutagenesis was the endpoint. Lastly, repair of gapped DNA molecules, intermediates in methyl-directed mismatch repair, was also unaffected by the presence or absence of DNA polymerase II. PMID:8419305

  16. Pushing the limits for amplifying BrdU-labeled DNA encoding 16S rRNA: DNA polymerase as the determining factor.

    PubMed

    Roux-Michollet, Dad D; Schimel, Joshua P; Holden, Patricia A

    2010-12-01

    Identifying microorganisms that are active under specific conditions in ecosystems is a challenge in microbial ecology. Recently, the bromodeoxyuridine (BrdU) technique was developed to label actively growing cells. BrdU, a thymidine analog, is incorporated into newly synthesized DNA, and the BrdU-labeled DNA is then isolated from total extractable DNA by immunocapture using a BrdU-specific antibody. Analyzing the BrdU-labeled DNA allows for assessing the actively growing community, which can then be compared to the unlabeled DNA that represents the total community. However, applying the BrdU approach to study soils has been problematic due to low DNA amounts and soil contaminants. To address these challenges, we developed a protocol, optimizing specificity and reproducibility, to amplify BrdU-labeled gene fragments encoding 16S rRNA. We found that the determining factor was the DNA polymerase: among the 13 different polymerases we tested, only 3 provided adequate yields with minimal contamination, and only two of those three produced similar amplification patterns of community DNA. PMID:20883730

  17. Mitochondrial DNA replication and disease: insights from DNA polymerase γ mutations

    PubMed Central

    Stumpf, Jeffrey D.

    2011-01-01

    DNA polymerase γ (pol γ), encoded by POLG, is responsible for replicating human mitochondrial DNA. About 150 mutations in the human POLG have been identified in patients with mitochondrial diseases such as Alpers syndrome, progressive external ophthalmoplegia, and ataxia-neuropathy syndromes. Because many of the mutations are described in single citations with no genotypic family history, it is important to ascertain which mutations cause or contribute to mitochondrial disease. The vast majority of data about POLG mutations has been generated from biochemical characterizations of recombinant pol γ. However, recently, the study of mitochondrial dysfunction in Saccharomyces cerevisiae and mouse models provides important in vivo evidence for the role of POLG mutations in disease. Also, the published 3D-structure of the human pol γ assists in explaining some of the biochemical and genetic properties of the mutants. This review summarizes the current evidence that identifies and explains disease-causing POLG mutations. PMID:20927567

  18. A putative Leishmania DNA polymerase theta protects the parasite against oxidative damage

    PubMed Central

    Fernández-Orgiler, Abel; Martínez-Jiménez, María I.; Alonso, Ana; Alcolea, Pedro J.; Requena, Jose M.; Thomas, María C.; Blanco, Luis; Larraga, Vicente

    2016-01-01

    Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression. PMID:27131366

  19. DNA polymerase beta reveals enhanced activity and processivity in reverse micelles.

    PubMed

    Anarbaev, Rashid O; Rogozina, Anastasia L; Lavrik, Olga I

    2009-04-01

    Water is essential for the stability and functions of proteins and DNA. Reverse micelles are simple model systems where the structure and dynamics of water are controlled. We have estimated the size of complex reverse micelles by light scattering technique and examined the local microenvironment using fluorescein as molecular probe. The micelle size and water polarity inside reverse micelles depend on water volume fraction. We have investigated the different hydration and confinement effects on activity, processivity, and stability of mammalian DNA polymerase beta in reverse micelles. The enzyme displays high processivity on primed single-stranded M13mp19 DNA with maximal activity at 10% of water content. The processivity and activity of DNA polymerase strongly depend on the protein concentration. The enzyme reveals also the enhanced stability in the presence of template-primer and at high protein concentration. The data provide direct evidence for strong influence of microenvironment on DNA polymerase activity. PMID:19138815

  20. A putative Leishmania DNA polymerase theta protects the parasite against oxidative damage.

    PubMed

    Fernández-Orgiler, Abel; Martínez-Jiménez, María I; Alonso, Ana; Alcolea, Pedro J; Requena, Jose M; Thomas, María C; Blanco, Luis; Larraga, Vicente

    2016-06-01

    Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression. PMID:27131366

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

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    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.

  3. Molecular events during translocation and proofreading extracted from 200 static structures of DNA polymerase

    PubMed Central

    Ren, Zhong

    2016-01-01

    DNA polymerases in family B are workhorses of DNA replication that carry out the bulk of the job at a high speed with high accuracy. A polymerase in this family relies on a built-in exonuclease for proofreading. It has not been observed at the atomic resolution how the polymerase advances one nucleotide space on the DNA template strand after a correct nucleotide is incorporated, that is, a process known as translocation. It is even more puzzling how translocation is avoided after the primer strand is excised by the exonuclease and returned back to the polymerase active site once an error occurs. The structural events along the bifurcate pathways of translocation and proofreading have been unwittingly captured by hundreds of structures in Protein Data Bank. This study analyzes all available structures of a representative member in family B and reveals the orchestrated event sequence during translocation and proofreading. PMID:27325739

  4. Replication of single-stranded DNA templates by primase-polymerase complexes of the yeast, Saccharomyces cerevisiae.

    PubMed Central

    Biswas, E E; Biswas, S B

    1988-01-01

    A partially purified primase-polymerase complex from the yeast, Saccharomyces cerevisiae, was capable of replicating a single stranded circular phage DNA into a replicative form with high efficiency. The primase-polymerase complex exhibited primase activity and polymerase activity on singly primed circular ssDNA as well as on gapped DNA. In addition, it was able to replicate an unprimed, single-stranded, circular phage DNA through a coupled primase-polymerase action. On Biogel A-O.5m filtration the primase-polymerase activities appeared in the void volume, demonstrating a mass of greater than 500 kilodaltons. Primase and various primase-polymerase complexes synthesized unique primers on single stranded DNA templates and the size distribution of primers was dependent on the structure of the DNA and the nature of the primase-polymerase assembly. Images PMID:3041377

  5. Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

    PubMed Central

    Flood, Carrie L.; Rodriguez, Gina P.; Bao, Gaobin; Shockley, Arthur H.; Kow, Yoke Wah; Crouse, Gray F.

    2015-01-01

    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. PMID:25742645

  6. Chromosome specific repetitive DNA sequences

    DOEpatents

    Moyzis, Robert K.; Meyne, Julianne

    1991-01-01

    A method is provided for determining specific nucleotide sequences useful in forming a probe which can identify specific chromosomes, preferably through in situ hybridization within the cell itself. In one embodiment, chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family me This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).

  7. Activation of an Mg2+-dependent DNA endonuclease of avian myeloblastosis virus alpha beta DNA polymerase by in vitro proteolytic cleavage.

    PubMed Central

    Grandgenett, D P; Golomb, M; Vora, A C

    1980-01-01

    Partial chymotryptic digestion of purified avian myeloblastosis virus alpha beta DNA polymerase resulted in the activation of a Mg2+-dependent DNA endonuclease activity. Incubation of the polymerase-protease mixture in the presence of super-coiled DNA and Mg2+ permitted detection of the cleaved polymerase fragment possessing DNA nicking activity. Protease digestion conditions were established permitting selective cleavage of beta to alpha, which contained DNA polymerase and RNase H activity and to a family of polypeptides ranging in size from 30,000 to 34,000 daltons. These latter beta-unique fragments were purified by polyuridylate-Sepharose 4B chromatography and were shown to contain both DNA binding and DNA endonuclease activities. We have demonstrated that this group of polymerase fragments derived by chymotryptic digestion of alpha beta DNA polymerase is similar to the in vivo-isolated avian myeloblastosis virus p32pol in size, sequence, and DNA endonuclease activity. Images PMID:6154149

  8. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain.

    PubMed

    Morrill, Summer A; Exner, Alexandra E; Babokhov, Michael; Reinfeld, Bradley I; Fuchs, Stephen M

    2016-05-27

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24-26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1 In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  9. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain*

    PubMed Central

    Morrill, Summer A.; Exner, Alexandra E.; Babokhov, Michael; Reinfeld, Bradley I.

    2016-01-01

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  10. Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene.

    PubMed Central

    Heilbronn, R; Jahn, G; Bürkle, A; Freese, U K; Fleckenstein, B; zur Hausen, H

    1987-01-01

    The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSV-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at Tm - 25 degrees C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Epstein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein. Images PMID:3023689

  11. Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene

    SciTech Connect

    Heilbronn, T.; Jahn, G.; Buerkle, A.; Freese, U.K.; Fleckenstein, B.; Zur Hausen, H.

    1987-01-01

    The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSF-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at T/sub m/ - 25/degrees/C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Esptein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein.

  12. Eukaryotic TLS polymerases.

    PubMed

    Tomczyk, Przemysław; Synowiec, Ewelina; Wysokiński, Daniel; Woźniak, Katarzyna

    2016-01-01

    TLS polymerases are able to replicate damaged DNA (called translesion DNA synthesis, TLS). Their presence prevents cell death as a result of violating the integrity of the genome. In vitro, they are mutator, but in vivo are recruited by specific types of DNA damage and usually replicate them in a correct manner. The best-known TLS polymerases belong to the Y family, such as Rev1, κ, η, ι, and polymerase ζ from the B family. There are two mechanisms of TLS polymerases action: polymerase-switching model and the gap-filling model. Selection of the mechanism primarily depends on the phase of the cell cycle. The regulation of these polymerases may take place at the transcriptional level and at level of recruitment to the sites of DNA damage. In the latter case post-translational modification of proteins - ubiquitination and sumoylation, and protein-protein interactions are crucial. PMID:27333922

  13. Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription

    SciTech Connect

    Bogenhagen, D.F.; Insdorf, N.F.

    1988-07-01

    The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor eluted from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA/sup Phe/ and the displacement loop.

  14. Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription.

    PubMed Central

    Bogenhagen, D F; Insdorf, N F

    1988-01-01

    The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor eluted from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA(Phe) and the displacement loop. Images PMID:2457154

  15. The Mechanism of the Translocation Step in DNA Replication by DNA Polymerase I: A Computer Simulation Analysis

    SciTech Connect

    Golosov, Andrei A.; Warren, Joshua J.; Beese, Lorena S.; Karplus, Martin

    2010-11-03

    High-fidelity DNA polymerases copy DNA rapidly and accurately by adding correct deoxynucleotide triphosphates to a growing primer strand of DNA. Following nucleotide incorporation, a series of conformational changes translocate the DNA substrate by one base pair step, readying the polymerase for the next round of incorporation. Molecular dynamics simulations indicate that the translocation consists globally of a polymerase fingers-opening transition, followed by the DNA displacement and the insertion of the template base into the preinsertion site. They also show that the pyrophosphate release facilitates the opening transition and that the universally conserved Y714 plays a key role in coupling polymerase opening to DNA translocation. The transition involves several metastable intermediates in one of which the O helix is bent in the vicinity of G711. Completion of the translocation appears to require a gating motion of the O1 helix, perhaps facilitated by the presence of G715. These roles are consistent with the high level of conservation of Y714 and the two glycine residues at these positions. It is likely that a corresponding mechanism is applicable to other polymerases.

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

    PubMed

    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. PMID:27372838

  17. Evolution of replicative DNA polymerases in archaea and their contributions to the eukaryotic replication machinery

    PubMed Central

    Makarova, Kira S.; Krupovic, Mart; Koonin, Eugene V.

    2014-01-01

    The elaborate eukaryotic DNA replication machinery evolved from the archaeal ancestors that themselves show considerable complexity. Here we discuss the comparative genomic and phylogenetic analysis of the core replication enzymes, the DNA polymerases, in archaea and their relationships with the eukaryotic polymerases. In archaea, there are three groups of family B DNA polymerases, historically known as PolB1, PolB2 and PolB3. All three groups appear to descend from the last common ancestors of the extant archaea but their subsequent evolutionary trajectories seem to have been widely different. Although PolB3 is present in all archaea, with the exception of Thaumarchaeota, and appears to be directly involved in lagging strand replication, the evolution of this gene does not follow the archaeal phylogeny, conceivably due to multiple horizontal transfers and/or dramatic differences in evolutionary rates. In contrast, PolB1 is missing in Euryarchaeota but otherwise seems to have evolved vertically. The third archaeal group of family B polymerases, PolB2, includes primarily proteins in which the catalytic centers of the polymerase and exonuclease domains are disrupted and accordingly the enzymes appear to be inactivated. The members of the PolB2 group are scattered across archaea and might be involved in repair or regulation of replication along with inactivated members of the RadA family ATPases and an additional, uncharacterized protein that are encoded within the same predicted operon. In addition to the family B polymerases, all archaea, with the exception of the Crenarchaeota, encode enzymes of a distinct family D the origin of which is unclear. We examine multiple considerations that appear compatible with the possibility that family D polymerases are highly derived homologs of family B. The eukaryotic DNA polymerases show a highly complex relationship with their archaeal ancestors including contributions of proteins and domains from both the family B and the

  18. Direct Probing of Solvent Accessibility and Mobility at the Binding Interface of Polymerase (Dpo4)-DNA Complex

    PubMed Central

    Qin, Yangzhong; Yang, Yi; Zhang, Luyuan; Fowler, Jason D.; Qiu, Weihong; Wang, Lijuan; Suo, Zucai; Zhong, Dongping

    2014-01-01

    Water plays essential structural and dynamical roles in protein-DNA recognition through contributing to enthalpic or entropic stabilization of binding complex and by mediating intermolecular interactions and fluctuations for biological function. These interfacial water molecules are confined by the binding partners in nanospace but in many cases they are highly mobile and exchange with outside bulk solution. Here, we report our studies of the interfacial water dynamics in the binary and ternary complexes of a polymerase (Dpo4) with DNA and an incoming nucleotide using a site-specific tryptophan probe with femtosecond resolution. By systematic comparison of the interfacial water motions and local sidechain fluctuations in the apo, binary and ternary states of Dpo4, we observed that the DNA binding interface and active site is dynamically solvent accessible and the interfacial water dynamics are similar to the surface hydration water fluctuations on picosecond time scales. Our molecular dynamics simulations also show the binding interface full of water molecules and nonspecific weak interactions. Such a fluid binding interface facilitates the polymerase sliding on DNA for fast translocation while the spacious and mobile hydrated active site contributes to the low fidelity of the lesion-bypass Y-family DNA polymerase. PMID:24308461

  19. Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay.

    PubMed Central

    Oberhaus, S M; Newbold, J E

    1993-01-01

    Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host. Images PMID:8411359

  20. Kinetics of Mismatch Formation opposite Lesions by the Replicative DNA Polymerase from Bacteriophage RB69

    SciTech Connect

    Hogg, Matthew; Rudnicki, Jean; Midkiff, John; Reha-Krantz, Linda; Doubli, Sylvie; Wallace, Susan S.

    2010-04-12

    The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analogue by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k{sub pol}) were generally higher for the variant, the presence of a lesion in the templating position reduced the ability of both the wild-type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead, the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG {center_dot} dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.

  1. Evidence for the involvement of human DNA polymerase N in the repair of DNA interstrand cross-links

    PubMed Central

    Zietlow, Laura; Smith, Leigh Anne; Bessho, Mika; Bessho, Tadayoshi

    2009-01-01

    Human DNA polymerase N (PolN) is an A-family nuclear DNA polymerase whose function is unknown. This study examines the possible role of PolN in DNA repair in human cells treated with PolN-targeted siRNA. HeLa cells with siRNA-mediated knockdown of PolN were more sensitive than control cells to DNA cross-linking agent mitomycin C (MMC), but were not hyper-sensitive to UV irradiation. The MMC hyper-sensitivity of PolN knockdown cells was rescued by the overexpression of DNA polymerase-proficient PolN but not by DNA polymerase-deficient PolN. Furthermore, in vitro experiments showed that purified PolN conducts low efficiency non-mutagenic bypass of a psoralen DNA interstrand cross-link (ICL), whose structure resembles an intermediate in the proposed pathway of ICL repair. These results suggest that PolN might play a role in translesion DNA synthesis during ICL repair in human cells. PMID:19908865

  2. EFFECTIVE METHOD TO EXTRACT DNA FROM ENVIRONMENTAL SAMPLES FOR POLYMERASE CHAIN REACTION AMPLIFICATION AND DNA FINGERPRINT ANALYSIS

    EPA Science Inventory

    A rapid direct-extraction method was used to obtain DNA from environmental soil samples. eat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. he DNA was purified by agarose gel electrophoresis, amplified with 16S based primers by use of the polymerase chain rea...

  3. DNA vector-based RNAi approach for stable depletion of poly(ADP-ribose) polymerase-1

    SciTech Connect

    Shah, Rashmi G.; Ghodgaonkar, Medini M.; Affar, El Bachir; Shah, Girish M. . E-mail: girish.shah@crchul.ulaval.ca

    2005-05-27

    RNA-mediated interference (RNAi) is a powerful technique that is now being used in mammalian cells to specifically silence a gene. Some recent studies have used this technique to achieve variable extent of depletion of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These studies reported either transient silencing of PARP-1 using double-stranded RNA or stable silencing of PARP-1 with a DNA vector which was introduced by a viral delivery system. In contrast, here we report that a simple RNAi approach which utilizes a pBS-U6-based DNA vector containing strategically selected PARP-1 targeting sequence, introduced in the cells by conventional CaPO{sub 4} protocol, can be used to achieve stable and specific silencing of PARP-1 in different types of cells. We also provide a detailed strategy for selection and cloning of PARP-1-targeting sequences for the DNA vector, and demonstrate that this technique does not affect expression of its closest functional homolog PARP-2.

  4. Molecular dynamics study of the opening mechanism for DNA polymerase I.

    PubMed

    Miller, Bill R; Parish, Carol A; Wu, Eugene Y

    2014-12-01

    During DNA replication, DNA polymerases follow an induced fit mechanism in order to rapidly distinguish between correct and incorrect dNTP substrates. The dynamics of this process are crucial to the overall effectiveness of catalysis. Although X-ray crystal structures of DNA polymerase I with substrate dNTPs have revealed key structural states along the catalytic pathway, solution fluorescence studies indicate that those key states are populated in the absence of substrate. Herein, we report the first atomistic simulations showing the conformational changes between the closed, open, and ajar conformations of DNA polymerase I in the binary (enzyme:DNA) state to better understand its dynamics. We have applied long time-scale, unbiased molecular dynamics to investigate the opening process of the fingers domain in the absence of substrate for B. stearothermophilis DNA polymerase in silico. These simulations are biologically and/or physiologically relevant as they shed light on the transitions between states in this important enzyme. All closed and ajar simulations successfully transitioned into the fully open conformation, which is known to be the dominant binary enzyme-DNA conformation from solution and crystallographic studies. Furthermore, we have detailed the key stages in the opening process starting from the open and ajar crystal structures, including the observation of a previously unknown key intermediate structure. Four backbone dihedrals were identified as important during the opening process, and their movements provide insight into the recognition of dNTP substrate molecules by the polymerase binary state. In addition to revealing the opening mechanism, this study also demonstrates our ability to study biological events of DNA polymerase using current computational methods without biasing the dynamics. PMID:25474643

  5. Quantification of the 35S promoter in DNA extracts from genetically modified organisms using real-time polymerase chain reaction and specificity assessment on various genetically modified organisms, part I: operating procedure.

    PubMed

    Fernandez, Sophie; Charles-Delobel, Chrystèle; Geldreich, Angèle; Berthier, Georges; Boyer, Francine; Collonnier, Cécile; Coué-Philippe, Géraldine; Diolez, Annick; Duplan, Marie-Noëlle; Kebdani, Naïma; Romaniuk, Marcel; Feinberg, Max; Bertheau, Yves

    2005-01-01

    A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard. PMID:15859083

  6. Preparation of Phi29 DNA Polymerase Free of Amplifiable DNA Using Ethidium Monoazide, an Ultraviolet-Free Light-Emitting Diode Lamp and Trehalose

    PubMed Central

    Takahashi, Hirokazu; Yamazaki, Hiroyuki; Akanuma, Satoshi; Kanahara, Hiroko; Saito, Toshiyuki; Chimuro, Tomoyuki; Kobayashi, Takayoshi; Ohtani, Toshio; Yamamoto, Kimiko; Sugiyama, Shigeru; Kobori, Toshiro

    2014-01-01

    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. PMID:24505243

  7. Identifying a Core RNA Polymerase Surface Critical for Interactions with a Sigma-Like Specificity Factor

    PubMed Central

    Cliften, Paul F.; Jang, Sei-Heon; Jaehning, Judith A.

    2000-01-01

    Cyclic interactions occurring between a core RNA polymerase (RNAP) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. In this work we have identified regions of the single-subunit yeast mitochondrial RNAP (Rpo41p) important for interaction with its sigma-like specificity factor (Mtf1p). Previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of Mtf1p are required for interaction. In this work we started with an interaction-defective point mutant in Mtf1p (V135A) and used a two-hybrid selection to isolate suppressing mutations in the core polymerase. We identified suppressors in three separate regions of the RNAP which, when modeled on the structure of the closely related phage T7 RNAP, appear to lie on one surface of the protein. Additional point mutations and biochemical assays were used to confirm the importance of each region for Rpo41p-Mtf1p interactions. Remarkably, two of the three suppressors are found in regions required by T7 RNAP for DNA sequence recognition and promoter melting. Although these essential regions of the phage RNAP are poorly conserved with the mitochondrial RNAPs, they are conserved among the mitochondrial enzymes. The organellar RNAPs appear to use this surface in an alternative way for interactions with their separate sigma-like specificity factor, which, like its bacterial counterpart, provides promoter recognition and DNA melting functions to the holoenzyme. PMID:10958696

  8. DNA polymerases engineered by directed evolution to incorporate non-standard nucleotides

    PubMed Central

    Laos, Roberto; Thomson, J. Michael; Benner, Steven A.

    2014-01-01

    DNA polymerases have evolved for billions of years to accept natural nucleoside triphosphate substrates with high fidelity and to exclude closely related structures, such as the analogous ribonucleoside triphosphates. However, polymerases that can accept unnatural nucleoside triphosphates are desired for many applications in biotechnology. The focus of this review is on non-standard nucleotides that expand the genetic “alphabet.” This review focuses on experiments that, by directed evolution, have created variants of DNA polymerases that are better able to accept unnatural nucleotides. In many cases, an analysis of past evolution of these polymerases (as inferred by examining multiple sequence alignments) can help explain some of the mutations delivered by directed evolution. PMID:25400626

  9. UvrD facilitates DNA repair by pulling RNA polymerase backwards.

    PubMed

    Epshtein, Vitaly; Kamarthapu, Venu; McGary, Katelyn; Svetlov, Vladimir; Ueberheide, Beatrix; Proshkin, Sergey; Mironov, Alexander; Nudler, Evgeny

    2014-01-16

    UvrD helicase is required for nucleotide excision repair, although its role in this process is not well defined. Here we show that Escherichia coli UvrD binds RNA polymerase during transcription elongation and, using its helicase/translocase activity, forces RNA polymerase to slide backward along DNA. By inducing backtracking, UvrD exposes DNA lesions shielded by blocked RNA polymerase, allowing nucleotide excision repair enzymes to gain access to sites of damage. Our results establish UvrD as a bona fide transcription elongation factor that contributes to genomic integrity by resolving conflicts between transcription and DNA repair complexes. Furthermore, we show that the elongation factor NusA cooperates with UvrD in coupling transcription to DNA repair by promoting backtracking and recruiting nucleotide excision repair enzymes to exposed lesions. Because backtracking is a shared feature of all cellular RNA polymerases, we propose that this mechanism enables RNA polymerases to function as global DNA damage scanners in bacteria and eukaryotes. PMID:24402227

  10. Molecular docking studies of phytochemicals from Phyllanthus niruri against Hepatitis B DNA Polymerase

    PubMed Central

    Mohan, Mekha; James, Priyanka; Valsalan, Ravisankar; Nazeem, Puthiyaveetil Abdulla

    2015-01-01

    Hepatitis B virus (HBV) infection is the leading cause for liver disorders and can lead to hepatocellular carcinoma, cirrhosis and liver damage which in turn can cause death of patients. HBV DNA Polymerase is essential for HBV replication in the host and hence is used as one of the most potent pharmacological target for the inhibition of HBV. Chronic hepatitis B is currently treated with nucleotide analogues that suppress viral reverse transcriptase activity and most of them are reported to have viral resistance. Therefore, it is of interest to model HBV DNA polymerase to dock known phytochemicals. The present study focuses on homology modeling and molecular docking analysis of phytocompounds from the traditional antidote Phyllanthus niruri and other nucleoside analogues against HBV DNA Polymerase using the software Discovery studio 4.0. 3D structure of HBV DNA Polymerase was predicted based on previously reported alignment. Docking studies revealed that a few phytochemicals from Phyllanthus niruri had good interactions with HBV DNA Polymerase. These compounds had acceptable binding properties for further in vitro validation. Thus the study puts forth experimental validation for traditional antidote and these phytocompounds could be further promoted as potential lead molecule. PMID:26527851

  11. Replication dynamics in fission and budding yeasts through DNA polymerase tracking.

    PubMed

    Vázquez, Enrique; Antequera, Francisco

    2015-10-01

    The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof-read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions. PMID:26293347

  12. A simplified procedure for the analysis of DNA polymerase III levels in Bacillus subtilis strains.

    PubMed Central

    Ciarrocchi, G; Fortunato, A; Attolini, C; Falaschi, A

    1976-01-01

    A simple and reproducible procedure is described which allows the fast and almost quantitative removal of DNA polymerases I and II from DNA polymerase III, in crude extracts of polA+ strains of Bacillus subtilis. The procedure entails streptomycin sulfate and ammonium sulfate fractionations; subsequent analysis of the partially purified preparation by G-200 chromatography, DEAE cellulose chromatography and density gradient sedimentation, shows that the ammonium sulfate fraction contains less than 5% of the total activity as DNA polymerase I and less than 2% as DNA polymerase II. The purification procedure, up to the ammonium sulfate step, was utilized for the analysis of the level of DNA polymerase III in several B. subtilis mutants, with results comparable to those obtained from the corresponding polA- strains following more cumbersome purification procedures. The M.W. of the purified form is of 227.000, somewhat greater than the published values. The early fractions of the purification have revealed the existence of a form with a M.W. of 426.000; the nature of this form, which has been observed in several instances and which is very unstable and short-lived, is under investigation. PMID:826885

  13. Metal A and Metal B Sites of Nuclear RNA Polymerases Pol IV and Pol V Are Required for siRNA-Dependent DNA Methylation and Gene Silencing

    PubMed Central

    Haag, Jeremy R.; Pontes, Olga; Pikaard, Craig S.

    2009-01-01

    Plants are unique among eukaryotes in having five multi-subunit nuclear RNA polymerases: the ubiquitous RNA polymerases I, II and III plus two plant-specific activities, nuclear RNA polymerases IV and V (previously known as Polymerases IVa and IVb). Pol IV and Pol V are not required for viability but play non-redundant roles in small interfering RNA (siRNA)-mediated pathways, including a pathway that silences retrotransposons and endogenous repeats via siRNA-directed DNA methylation. RNA polymerase activity has not been demonstrated for Polymerases IV or V in vitro, making it unclear whether they are catalytically active enzymes. Their largest and second-largest subunit sequences have diverged considerably from Pol I, II and III in the vicinity of the catalytic center, yet retain the invariant Metal A and Metal B amino acid motifs that bind magnesium ions essential for RNA polymerization. By using site-directed mutagenesis in conjunction with in vivo functional assays, we show that the Metal A and Metal B motifs of Polymerases IV and V are essential for siRNA production, siRNA-directed DNA methylation, retrotransposon silencing, and the punctate nuclear localization patterns typical of both polymerases. Collectively, these data show that the minimal core sequences of polymerase active sites, the Metal A and B sites, are essential for Pol IV and Pol V biological functions, implying that both are catalytically active. PMID:19119310

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

    PubMed Central

    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

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

    PubMed

    Su, Yan; Peter Guengerich, F

    2016-01-01

    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. PMID:27248785

  16. Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

    PubMed

    Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

    2015-05-26

    DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. PMID:25881991

  17. Accessory subunit of mitochondrial DNA polymerase from Drosophila embryos. Cloning, molecular analysis, and association in the native enzyme.

    PubMed

    Wang, Y; Farr, C L; Kaguni, L S

    1997-05-23

    A full-length cDNA of the accessory (beta) subunit of mitochondrial DNA polymerase from Drosophila embryos has been obtained, and its nucleotide sequence was determined. The cDNA clone encodes a polypeptide with a deduced amino acid sequence of 361 residues and a predicted molecular mass of 41 kDa. The gene encoding the beta subunit lies within 4 kilobase pairs of that for the catalytic subunit in the Drosophila genome, on the left arm of chromosome 2. The two genes have similar structural features and share several common DNA sequence elements in their upstream regions, suggesting the possibility of coordinate regulation. A human cDNA homolog of the accessory subunit was identified, and its nucleotide sequence was determined. The human sequence encodes a polypeptide with a predicted molecular mass of 43 kDa that shows a high degree of amino acid sequence similarity to the Drosophila beta subunit. Subunit-specific rabbit antisera, directed against the recombinant catalytic and accessory subunit polypeptides overexpressed and purified from Escherichia coli, recognize specifically and immunoprecipitate the native enzyme from Drosophila embryos. Demonstration of the physical association of the two subunits in the Drosophila enzyme and identification of a human accessory subunit homolog provide evidence for a common heterodimeric structure for animal mitochondrial DNA polymerases. PMID:9153213

  18. Differential furanose selection in the active sites of archaeal DNA polymerases probed by fixed-conformation nucleotide analogues.

    PubMed

    Ketkar, Amit; Zafar, Maroof K; Banerjee, Surajit; Marquez, Victor E; Egli, Martin; Eoff, Robert L

    2012-11-13

    DNA polymerases select for the incorporation of deoxyribonucleotide triphosphates (dNTPs) using amino acid side-chains that act as a "steric-gate" to bar improper incorporation of rNTPs. An additional factor in the selection of nucleotide substrates resides in the preferred geometry for the furanose moiety of the incoming nucleotide triphosphate. We have probed the role of sugar geometry during nucleotide selection by model DNA polymerases from Sulfolobus solfataricus using fixed conformation nucleotide analogues. North-methanocarba-dATP (N-MC-dATP) locks the central ring into a RNA-type (C2'-exo, North) conformation near a C3'-endo pucker, and South-methanocarba-dATP (S-MC-dATP) locks the central ring system into a (C3'-exo, South) conformation near a C2'-endo pucker. Dpo4 preferentially inserts N-MC-dATP and in the crystal structure of Dpo4 in complex with N-MC-dAMP, the nucleotide analogue superimposes almost perfectly with Dpo4 bound to unmodified dATP. Biochemical assays indicate that the S. solfataricus B-family DNA polymerase Dpo1 can insert and extend from both N-MC-dATP and S-MC-dATP. In this respect, Dpo1 is unexpectedly more tolerant of substrate conformation than Dpo4. The crystal structure of Dpo4 bound to S-MC-dADP shows that poor incorporation of the Southern pucker by the Y-family polymerase results from a hydrogen bond between the 3'-OH group of the nucleotide analogue and the OH group of the steric gate residue, Tyr12, shifting the S-MC-dADP molecule away from the dNTP binding pocket and distorting the base pair at the primer-template junction. These results provide insights into substrate specificity of DNA polymerases, as well as molecular mechanisms that act as a barrier against insertion of rNTPs. PMID:23050956

  19. DNA Polymerase δ Is Highly Processive with Proliferating Cell Nuclear Antigen and Undergoes Collision Release upon Completing DNA*S⃞

    PubMed Central

    Langston, Lance D.; O'Donnell, Mike

    2008-01-01

    In most cells, 100-1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fast-growing cells, this necessitates rapid recycling of DNA polymerase on the lagging strand. Bacteria produce long Okazaki fragments (1-2 kb) and utilize a highly processive DNA polymerase III (pol III), which is held to DNA by a circular sliding clamp. In contrast, Okazaki fragments in eukaryotes are quite short, 100-250 bp, and thus the eukaryotic lagging strand polymerase does not require a high degree of processivity. The lagging strand polymerase in eukaryotes, polymerase δ (pol δ), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol δ is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol δ is highly processive with PCNA, over at least 5 kb, on Replication Protein A (RPA)-coated primed single strand DNA. The high processivity of pol δ observed in this report contrasts with its role in synthesis of short lagging strand fragments, which require it to rapidly dissociate from DNA at the end of each Okazaki fragment. We find that this dilemma is solved by a “collision release” process in which pol δ ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol δ transfers to a new primed site, provided the new site contains a PCNA clamp. Additional results indicate that the collision release mechanism is intrinsic to the pol3/pol31 subunits of the pol δ heterotrimer. PMID:18635534

  20. DNA polymerase delta is highly processive with proliferating cell nuclear antigen and undergoes collision release upon completing DNA.

    PubMed

    Langston, Lance D; O'Donnell, Mike

    2008-10-24

    In most cells, 100-1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fast-growing cells, this necessitates rapid recycling of DNA polymerase on the lagging strand. Bacteria produce long Okazaki fragments (1-2 kb) and utilize a highly processive DNA polymerase III (pol III), which is held to DNA by a circular sliding clamp. In contrast, Okazaki fragments in eukaryotes are quite short, 100-250 bp, and thus the eukaryotic lagging strand polymerase does not require a high degree of processivity. The lagging strand polymerase in eukaryotes, polymerase delta (pol delta), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol delta is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol delta is highly processive with PCNA, over at least 5 kb, on Replication Protein A (RPA)-coated primed single strand DNA. The high processivity of pol delta observed in this report contrasts with its role in synthesis of short lagging strand fragments, which require it to rapidly dissociate from DNA at the end of each Okazaki fragment. We find that this dilemma is solved by a "collision release" process in which pol delta ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol delta transfers to a new primed site, provided the new site contains a PCNA clamp. Additional results indicate that the collision release mechanism is intrinsic to the pol3/pol31 subunits of the pol delta heterotrimer. PMID:18635534

  1. Comparison of Large Subunits of Type II DNA-dependent RNA Polymerases from Higher Plants.

    PubMed

    Kidd, G H; Link, G; Bogorad, L

    1979-10-01

    Two-dimensional tryptic mapping of (125)I-labeled polypeptides has been employed to compare the large subunits of type II DNA-dependent RNA polymerases from maize, parsley (Petroselinum sativum), and wheat. Maps of the 220 kilodalton (kd) and 140 kd subunits from wheat RNA polymerase II differ from those of the corresponding subunits from parsley enzyme II. The 180 kd subunits from maize and parsley type II enzymes also yield dissimilar tryptic maps. Thus, despite similarities in molecular mass, the large subunits of wheat, parsley, and maize type II RNA polymerases are unique to each individual plant species. PMID:16661032

  2. Temperature and salt effects on the formation of preinitiation complexes between RNA polymerase and phage DNA.

    PubMed

    Escarmis, C; Domingo, E; Warner, R C

    1975-08-21

    The influence of temperature and KCl concentration on the formation of rifampicin-resistant preinitiation complexes by holo RNA polymerase has been compared for T4 DNA and Azotobacter phage A21 DNA. The sharp transition with respect to temperature between an inactive complex of polymerase and DNA and a preinitiation complex reflects an equilibrium between the two complexes, the position of which depends on the temperature and the salt concentration. The transition is shifted to higher temperatures by increasing the KCl concentration. The position of this transition is characteristically different for T4 and A21 DNA. The midpoint for A21 DNA is about 15 degrees C above that for T4 at 0.006 M KCl. At 0.15 M KCl the transition for A21 DNA cannot be observed below 37 degrees C. This difference is responsible for the apparent inhibition of a21 dna transcription by KCl and for the low template activity of A21 DNA under the conditions of the standard assay. Both holo and core RNA polymerases are able to form complexes with A21 DNA that are resistant to attack by rifampicin. The second-order rate constant for the inactivation of the complex with the core enxyme is three times greater than that for the complex with the holoenzyme. PMID:1100115

  3. Deuterium incorporation into Escherichia coli proteins. A neutron-scattering study of DNA-dependent RNA polymerase.

    PubMed

    Lederer, H; May, R P; Kjems, J K; Schaefer, W; Crespi, H L; Heumann, H

    1986-05-01

    Neutron small-angle scattering studies of single protein subunits in a protein-DNA complex require the adjustment of the neutron scattering-length densities of protein and DNA, which is attainable by specific deuteration of the protein. The neutron scattering densities of unlabelled DNA and DNA-dependent RNA polymerase of Escherichia coli match when RNA polymerase is isolated from cells grown in a medium containing 46% D2O and unlabelled glucose as carbon source. Their contrasts vanish simultaneously in a dialysis buffer containing 65% D2O. An expression was evaluated which allows the calculation of the degree of deuteration and match point of any E. coli protein from the D2O content of the growth medium, taking the 2H incorporation into RNA polymerase amino acids to be representative for all amino acids in E. coli proteins. The small-angle scattering results, on which the calculation of the degree of deuteration is based, were confirmed by mass spectrometric measurements. PMID:3516697

  4. Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

    PubMed Central

    Wawrzycka, Aleksandra; Gross, Marta; Wasaznik, Anna; Konieczny, Igor

    2015-01-01

    Although the molecular basis for replisome activity has been extensively investigated, it is not clear what the exact mechanism for de novo assembly of the replication complex at the replication origin is, or how the directionality of replication is determined. Here, using the plasmid RK2 replicon, we analyze the protein interactions required for Escherichia coli polymerase III (Pol III) holoenzyme association at the replication origin. Our investigations revealed that in E. coli, replisome formation at the plasmid origin involves interactions of the RK2 plasmid replication initiation protein (TrfA) with both the polymerase β- and α-subunits. In the presence of other replication proteins, including DnaA, helicase, primase and the clamp loader, TrfA interaction with the β-clamp contributes to the formation of the β-clamp nucleoprotein complex on origin DNA. By reconstituting in vitro the replication reaction on ssDNA templates, we demonstrate that TrfA interaction with the β-clamp and sequence-specific TrfA interaction with one strand of the plasmid origin DNA unwinding element (DUE) contribute to strand-specific replisome assembly. Wild-type TrfA, but not the TrfA QLSLF mutant (which does not interact with the β-clamp), in the presence of primase, helicase, Pol III core, clamp loader, and β-clamp initiates DNA synthesis on ssDNA template containing 13-mers of the bottom strand, but not the top strand, of DUE. Results presented in this work uncovered requirements for anchoring polymerase at the plasmid replication origin and bring insights of how the directionality of DNA replication is determined. PMID:26195759

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

    PubMed Central

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

    2014-01-01

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

  6. Synthesis, DNA Polymerase Incorporation, and Enzymatic Phosphate Hydrolysis of Formamidopyrimidine Nucleoside Triphosphates

    PubMed Central

    Imoto, Shuhei; Patro, Jennifer N.; Jiang, Yu Lin; Oka, Natsuhisa; Greenberg, Marc M.

    2007-01-01

    The nucleoside triphosphates of N6-(2-deoxy-α,β-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy·dGTP) and its C-nucleoside analogue (β-C-Fapy·dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy·dGTP and β-C-Fapy·dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3′-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy·dG observed in cells if 1% of the dGTP pool is converted to Fapy·dGTP. Similarly, polymerase-mediated introduction of β-C-Fapy·dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy·dGTP and β-C-Fapy·dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates. PMID:17090045

  7. Repressor activity of the RpoS/σS-dependent RNA polymerase requires DNA binding.

    PubMed

    Lévi-Meyrueis, Corinne; Monteil, Véronique; Sismeiro, Odile; Dillies, Marie-Agnès; Kolb, Annie; Monot, Marc; Dupuy, Bruno; Duarte, Sara Serradas; Jagla, Bernd; Coppée, Jean-Yves; Beraud, Mélanie; Norel, Françoise

    2015-02-18

    The RpoS/σ(S) sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σ(S)-dependent control, that of a repressor. Negative regulation by σ(S) has been proposed to result largely from competition between σ(S) and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σ(S) binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σ(S) protein proficient for Eσ(S) complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σ(S) requires its binding to DNA. Although the mechanisms of repression by σ(S) are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by Eσ(S). PMID:25578965

  8. Repressor activity of the RpoS/σS-dependent RNA polymerase requires DNA binding

    PubMed Central

    Lévi-Meyrueis, Corinne; Monteil, Véronique; Sismeiro, Odile; Dillies, Marie-Agnès; Kolb, Annie; Monot, Marc; Dupuy, Bruno; Duarte, Sara Serradas; Jagla, Bernd; Coppée, Jean-Yves; Beraud, Mélanie; Norel, Françoise

    2015-01-01

    The RpoS/σS sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σS-dependent control, that of a repressor. Negative regulation by σS has been proposed to result largely from competition between σS and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σS binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σS protein proficient for EσS complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σS requires its binding to DNA. Although the mechanisms of repression by σS are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by EσS. PMID:25578965

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

    PubMed

    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. PMID:25197027

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

    SciTech Connect

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

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

    PubMed Central

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

    2016-01-01

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker–induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress. PMID:27407148

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

    PubMed

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

    2016-07-26

    DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress. PMID:27407148

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

    PubMed

    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. PMID:27072134

  14. Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase

    PubMed Central

    Morin, José A.; Cao, Francisco J.; Lázaro, José M.; Arias-Gonzalez, J. Ricardo; Valpuesta, José M.; Carrascosa, José L.; Salas, Margarita; Ibarra, Borja

    2015-01-01

    During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN). PMID:25800740

  15. Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

    2016-03-01

    We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

  16. Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation

    PubMed Central

    Taladriz, Soraya; Hanke, Tobias; Ramiro, María J.; García-Díaz, Miguel; Lacoba, Mario García de; Blanco, Luis; Larraga, Vicente

    2001-01-01

    We have identified a novel polymerase beta (Pol β)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol β, shows a nuclear localization that contrasts with the mitochondrial localization of Pol β from Crithidia fasciculata, a closely related parasite, the only polymerase β described so far in Trypanosomatidae. Li Pol β, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol β-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol β, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol β mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol β at the infective phase of the parasite. The data suggest a role of Li Pol β in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells. PMID:11557814

  17. Optimization of asymmetric polymerase chain reaction for rapid fluorescent DNA sequencing.

    PubMed

    Wilson, R K; Chen, C; Hood, L

    1990-02-01

    A high-throughput method for the preparation of single-stranded template DNA, which is suitable for sequence analysis using fluorescent labeling chemistry, is described here. In this procedure, the asymmetric polymerase chain reaction is employed to amplify recombinant plasmid or bacteriophage DNA directly from colonies or plaques. The use of amplification primers located at least 200 base pairs 5' to the site of sequencing primer annealing removes the need for extensive purification of the asymmetric polymerase chain reaction product. Instead, the single-stranded product DNA is purified by a simple isopropanol precipitation step and then directly sequenced using fluorescent dye-labeled oligonucleotides. This method significantly reduces the time and labor required for template preparation and improves fluorescent DNA sequencing strategies by providing a much more uniform yield of single-stranded DNA. PMID:2317375

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

    PubMed

    Pannunzio, Nicholas R; Lieber, Michael R

    2016-05-01

    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. PMID:27153532

  19. Genetic evidence that both dNTP-stabilized and strand slippage mechanisms may dictate DNA polymerase errors within mononucleotide microsatellites.

    PubMed

    Baptiste, Beverly A; Jacob, Kimberly D; Eckert, Kristin A

    2015-05-01

    Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, β, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites. We utilized the ratio of insertion to deletion errors as a genetic indicator of mechanism. Strikingly, we observed a statistically significant bias toward deletion errors within mononucleotide repeats for the majority of the 28 DNA template and polymerase combinations examined, with notable exceptions based on sequence and polymerase identity. Using mutator forms of Pol β did not substantially alter the error specificity, suggesting that mispairing-misalignment mechanism is not a primary mechanism. Based on our results for mammalian DNA polymerases representing three structurally distinct families, we suggest that dNTP-stabilized mutagenesis may be an alternative mechanism for mononucleotide microsatellite indel mutation. The change from a predominantly dNTP-stabilized mechanism to a strand-slippage mechanism with increasing microsatellite length may account for the differential rates of tandem repeat mutation that are observed genome-wide. PMID:25758780

  20. Genetic Evidence That Both dNTP-Stabilized and Strand Slippage Mechanisms May Dictate DNA Polymerase Errors Within Mononucleotide Microsatellites

    PubMed Central

    Baptiste, Beverly A.; Jacob, Kimberly D.; Eckert, Kristin A.

    2015-01-01

    Mononucleotide microsatellites are tandem repeats of a single base pair, abundant within coding exons and frequent sites of mutation in the human genome. Because the repeated unit is one base pair, multiple mechanisms of insertion/deletion (indel) mutagenesis are possible, including strand-slippage, dNTP-stabilized, and misincorportion-misalignment. Here, we examine the effects of polymerase identity (mammalian Pols α, β, κ, and η), template sequence, dNTP pool size, and reaction temperature on indel errors during in vitro synthesis of mononucleotide microsatellites. We utilized the ratio of insertion to deletion errors as a genetic indicator of mechanism. Strikingly, we observed a statistically significant bias towards deletion errors within mononucleotide repeats for the majority of the 28 DNA template and polymerase combinations examined, with notable exceptions based on sequence and polymerase identity. Using mutator forms of Pol β did not substantially alter the error specificity, suggesting that mispairing-misalignment mechanism is not a primary mechanism. Based on our results for mammalian DNA polymerases representing three structurally distinct families, we suggest that dNTP-stabilized mutagenesis may be an alternative mechanism for mononucleotide microsatellite indel mutation. The change from a predominantly dNTP-stabilized mechanism to a strand-slippage mechanism with increasing microsatellite length may account for the differential rates of tandem repeat mutation that are observed genome-wide. PMID:25758780

  1. USP7 modulates UV-induced PCNA monoubiquitination by regulating DNA polymerase eta stability

    PubMed Central

    Qian, Jiang; Pentz, Kyle; Zhu, Qianzheng; Wang, Qien; He, Jinshan; Srivastava, Amit K.; Wani, Altaf A.

    2014-01-01

    DNA polymerase eta (Polη) plays unique and pivotal functions in several DNA damage-tolerance pathways. Steady-state level of this short-lived protein is tightly controlled by multiple mechanisms including proteolysis. Here, we have identified the deubiquitinating enzyme, ubiquitin-specific protease 7 (USP7), as a novel regulator of Polη stability. USP7 regulates Polη stability through both indirect and direct mechanisms. Knockout of USP7 increased the steady-state level of Polη and slowed down the turnover of both Polη and p53 proteins through destabilizing their E3 ligase Mdm2. Also, USP7 physically binds Polη in vitro and in vivo. Overexpression of wild-type USP7 but not its catalytically-defective mutants deubiquitinates Polη and increases its cellular steady-state level. Thus, USP7 directly serves as a specific deubiquitinating enzyme for Polη. Furthermore, ectopic expression of USP7 promoted the UV-induced PCNA monoubiquitination in Polη-proficient but not Polη-deficient XPV cells, suggesting that USP7 facilitates UV-induced PCNA monoubiquitination by stabilizing Polη. Taken together, our findings reveal a modulatory role of USP7 in PCNA ubiquitination-mediated stress-tolerance pathways by fine-tuning Polη turnover. PMID:25435364

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

    SciTech Connect

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

    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.

  3. Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology.

    PubMed

    Daher, Rana K; Stewart, Gale; Boissinot, Maurice; Boudreau, Dominique K; Bergeron, Michel G

    2015-04-01

    Recombinase polymerase amplification (RPA) technology relies on three major proteins, recombinase proteins, single-strand binding proteins, and polymerases, to specifically amplify nucleic acid sequences in an isothermal format. The performance of RPA with respect to sequence mismatches of closely-related non-target molecules is not well documented and the influence of the number and distribution of mismatches in DNA sequences on RPA amplification reaction is not well understood. We investigated the specificity of RPA by testing closely-related species bearing naturally occurring mismatches for the tuf gene sequence of Pseudomonas aeruginosa and/or Mycobacterium tuberculosis and for the cfb gene sequence of Streptococcus agalactiae. In addition, the impact of the number and distribution of mismatches on RPA efficiency was assessed by synthetically generating 14 types of mismatched forward primers for detecting five bacterial species of high diagnostic relevance such as Clostridium difficile, Staphylococcus aureus, S. agalactiae, P. aeruginosa, and M. tuberculosis as well as Bacillus atropheus subsp. globigii for which we use the spores as internal control in diagnostic assays. A total of 87 mismatched primers were tested in this study. We observed that target specific RPA primers with mismatches (n > 1) at their 3'extrimity hampered RPA reaction. In addition, 3 mismatches covering both extremities and the center of the primer sequence negatively affected RPA yield. We demonstrated that the specificity of RPA was multifactorial. Therefore its application in clinical settings must be selected and validated a priori. We recommend that the selection of a target gene must consider the presence of closely-related non-target genes. It is advisable to choose target regions with a high number of mismatches (≥36%, relative to the size of amplicon) with respect to closely-related species and the best case scenario would be by choosing a unique target gene. PMID:25481659

  4. Synthesis of site-specific DNA-protein conjugates and their effects on DNA replication.

    PubMed

    Yeo, Jung Eun; Wickramaratne, Susith; Khatwani, Santoshkumar; Wang, Yen-Chih; Vervacke, Jeffrey; Distefano, Mark D; Tretyakova, Natalia Y

    2014-08-15

    DNA-protein cross-links (DPCs) are bulky, helix-distorting DNA lesions that form in the genome upon exposure to common antitumor drugs, environmental/occupational toxins, ionizing radiation, and endogenous free-radical-generating systems. As a result of their considerable size and their pronounced effects on DNA-protein interactions, DPCs can interfere with DNA replication, transcription, and repair, potentially leading to mutagenesis, genotoxicity, and cytotoxicity. However, the biological consequences of these ubiquitous lesions are not fully understood due to the difficulty of generating DNA substrates containing structurally defined, site-specific DPCs. In the present study, site-specific cross-links between the two biomolecules were generated by copper-catalyzed [3 + 2] Huisgen cycloaddition (click reaction) between an alkyne group from 5-(octa-1,7-diynyl)-uracil in DNA and an azide group within engineered proteins/polypeptides. The resulting DPC substrates were subjected to in vitro primer extension in the presence of human lesion bypass DNA polymerases η, κ, ν, and ι. We found that DPC lesions to the green fluorescent protein and a 23-mer peptide completely blocked DNA replication, while the cross-link to a 10-mer peptide was bypassed. These results indicate that the polymerases cannot read through the larger DPC lesions and further suggest that proteolytic degradation may be required to remove the replication block imposed by bulky DPC adducts. PMID:24918113

  5. Identification of 5-Methoxyflavone as a Novel DNA Polymerase-Beta Inhibitor and Neuroprotective Agent against Beta-Amyloid Toxicity.

    PubMed

    Merlo, Sara; Basile, Livia; Giuffrida, Maria Laura; Sortino, Maria Angela; Guccione, Salvatore; Copani, Agata

    2015-11-25

    Cell-cycle reactivation is a core feature of degenerating neurons in Alzheimer's disease (AD) and Parkinson's disease (PD). A variety of stressors, including β-amyloid (Aβ) in the case of AD, can force neurons to leave quiescence and to initiate an ectopic DNA replication process, leading to neuronal death rather than division. As the primary polymerase (pol) involved in neuronal DNA replication, DNA pol-β contributes to neuronal death, and DNA pol-β inhibitors may prove to be effective neuroprotective agents. Currently, specific and highly active DNA pol-β inhibitors are lacking. Nine putative DNA pol-β inhibitors were identified in silico by querying the ZINC database, containing more than 35 million purchasable compounds. Following pharmacological evaluation, only 5-methoxyflavone (1) was validated as an inhibitor of DNA pol-β activity. Cultured primary neurons are a useful model to investigate the neuroprotective effects of potential DNA pol-β inhibitors, since these neurons undergo DNA replication and death when treated with Aβ. Consistent with the inhibition of DNA pol-β, 5-methoxyflavone (1) reduced the number of S-phase neurons and the ensuing apoptotic death triggered by Aβ. 5-Methoxyflavone (1) is the first flavonoid compound able to halt neurodegeneration via a definite molecular mechanism rather than through general antioxidant and anti-inflammatory properties. PMID:26517378

  6. Synthesis of DNA oligonucleotides containing C5-ethynylbenzenesulfonamide-modified nucleotides (EBNA) by polymerases towards the construction of base functionalized nucleic acids.

    PubMed

    Goubet, Astrid; Chardon, Antoine; Kumar, Pawan; Sharma, Pawan K; Veedu, Rakesh N

    2013-02-01

    C5-Ethynylbenzenesulfonamide-modified nucleotide (EBNA) was investigated as substrate of various DNA polymerases. The experiments revealed that KOD, Phusion and Klenow DNA polymerases successfully accepted EBNA-T nucleotide as a substrate and yielded the fully extended DNA. KOD DNA polymerase was found to be the most efficient enzyme to furnish EBNA-T containing DNA in good yields. Phusion DNA polymerase efficiently amplified the template containing EBNA-T nucleotides by PCR. PMID:23265899

  7. In vitro transcription of the Bacillus subtilis phage phi 29 DNA by Bacillus subtilis and Escherichia coli RNA polymerases.

    PubMed Central

    Sogo, J M; Lozano, M; Salas, M

    1984-01-01

    The Escherichia coli RNA polymerase bound to phage phi 29 DNA has been visualized by electron microscopy. Thirteen specific binding sites have been observed at 1.7,2.6,5.5,10.4,13.7,25.2,25.7,26.3,33.5,59.5,69.2,91.7 and 99.6 DNA length units and they have been named A1,A1I,A1II,A1III,A1IV,A2,A2I, A3, A4,B1,B1I,C1 and C2, respectively. The binding sites A1,A2,A3,B1,C1 and C2 coincide with those found with Bacillus subtilis RNA polymerase. The transcription of phage phi 29 DNA with B. subtilis or E. coli RNA polymerases has been studied. With the B. subtilis RNA polymerase eight transcripts were found, starting at positions corresponding to the binding sites A1, A1III, A2,A3,B1I,B2,C1 and C2, respectively. With the E. coli RNA polymerase the same transcripts were found and a new one starting at position corresponding to the A4 binding site. The RNAs starting at binding sites A1,A1III,A2,B1I, B2,C1 and C2 are transcribed from right to left, as expected for early RNA. The RNAs which initiate at positions A3 and A4 are transcribed from left to right and probably correspond to late RNAs. Images PMID:6322128

  8. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.

    PubMed

    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. PMID:25313033

  9. DNA polymerase kappa deficiency does not affect somatic hypermutation in mice.

    PubMed

    Schenten, Dominik; Gerlach, Valerie L; Guo, Caixia; Velasco-Miguel, Susana; Hladik, Christa L; White, Charles L; Friedberg, Errol C; Rajewsky, Klaus; Esposito, Gloria

    2002-11-01

    Somatic hypermutation (SH) in B cells undergoing T cell-dependent immune responses generates high-affinity antibodies that provide protective immunity. Most current models of SH postulate the introduction of a nick into the DNA and subsequent replication-independent, error-prone short-patch synthesis by one or more DNA polymerases. The Pol kappa (DinB1) gene encodes a specialized mammalian DNA polymerase called DNA polymerase kappa (pol kappa), a member of the recently discovered Y family of DNA polymerases. The mouse PolK gene is expressed at high levels in the seminiferous tubules of the testis and in the adrenal cortex, and at lower levels in most other cells of the body including B lymphocytes. In vitro studies showed that pol kappa can act as an error-prone polymerase, although they failed to ascribe a clear function to this enzyme. The ability of pol kappa to generate mutations when extending primers on undamaged DNA templates identifies this enzyme as a potential candidate for the introduction of nucleotide changes in the immunoglobulin (Ig) genes during the process of SH. Here we show that pol kappa-deficient mice are viable, fertile and able to mount a normal immune response to the antigen (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma-globulin (NP-GC). They also mutate their Ig genes normally. However, pol kappa-deficient embryonic fibroblasts are abnormally sensitive to killing following exposure to ultraviolet (UV) radiation, suggesting a role of pol kappa in translesion DNA synthesis. PMID:12555660

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

    SciTech Connect

    Fischer, H.; Erdmann, S.; Holler, E. )

    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.

  11. Polymerase chain reaction for the specific detection of Escherichia coli/Shigella.

    PubMed

    Spierings, G; Ockhuijsen, C; Hofstra, H; Tommassen, J

    1993-09-01

    The outer membrane protein PhoE of members of the family Enterobacteriaceae consists of conserved membrane-spanning segments and hypervariable surface-exposed regions. Two oligonucleotides based on DNA sequences encoding two different cell-surface-exposed regions of the Escherichia coli K12 PhoE protein were tested for their specificity in polymerase chain reactions. They reacted with all strains of the species E. coli/Shigella tested, except for strain S. boydii serovar 13, which is known to represent a different DNA-relatedness group. The probes did not react with any other Enterobacteriaceae tested, including strains of Escherichia blattae, Escherichia hermanii, Escherichia vulneris and Escherichia adecarboxylata, except for an Escherichia fergusonnii strain, which is most closely related to E. coli. Therefore, the primer couple showed a high degree of species-specificity. In addition, a second primer couple based on two conserved regions of the phoE genes was tested. This primer couple recognized a broad group of closely related enteric bacteria including Salmonella and Shigella. PMID:8310181

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

    PubMed Central

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

    1990-01-01

    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. Images PMID:1696290

  13. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation.

    PubMed

    Zhang, Jixiang; Xie, Shaojun; Cheng, Jinkui; Lai, Jinsheng; Zhu, Jian-Kang; Gong, Zhizhong

    2016-06-01

    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

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

    SciTech Connect

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

    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.

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

    PubMed

    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. PMID:27271319

  16. Pre-steady-state kinetic analysis of the incorporation of anti-HIV nucleotide analogs catalyzed by human X- and Y-family DNA polymerases.

    PubMed

    Brown, Jessica A; Pack, Lindsey R; Fowler, Jason D; Suo, Zucai

    2011-01-01

    Nucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established both in vitro and in vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypass in vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases β, λ, η, ι, κ, and Rev1 incorporating the active, 5'-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5'-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs. PMID:21078938

  17. Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens.

    PubMed Central

    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 now feasible and may form the basis of the next generation of automated microbial identification systems. However, identification of pathogens by this strategy is hampered by the frequent contamination of reagents used for the amplification reaction, in particular Taq polymerase, with exogenous bacterial DNA. Here, we describe detailed investigations on the use of 8-methoxypsoralen and long-wave UV light to eliminate contaminating DNA in polymerase chain reaction reagents. The clinical utility of the developed procedure was demonstrated in a case of paucibacillary osteomyelitis, for which no specific bacterial agent had been cultured. Images PMID:8458958

  18. Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase

    NASA Astrophysics Data System (ADS)

    Harada, Yoshie; Ohara, Osamu; Takatsuki, Akira; Itoh, Hiroyasu; Shimamoto, Nobuo; Kinosita, Kazuhiko

    2001-01-01

    Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5pNnm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

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

    PubMed

    Pastor-Palacios, Guillermo; López-Ramírez, Varinia; Cardona-Felix, Cesar S; Brieba, Luis G

    2012-01-01

    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. PMID:23226232

  20. In Vitro Lesion Bypass Studies of O(4)-Alkylthymidines with Human DNA Polymerase η.

    PubMed

    Williams, Nicole L; Wang, Pengcheng; Wu, Jiabin; Wang, Yinsheng

    2016-04-18

    Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O(4)-alkylthymidine (O(4)-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O(4)-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O(4)-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O(4)-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O(4)-iPrdT, O(4)-iBudT, and O(4)-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O(4)-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η. PMID:27002924

  1. Specific initiation by RNA polymerase I in a whole-cell extract from yeast.

    PubMed Central

    Schultz, M C; Choe, S Y; Reeder, R H

    1991-01-01

    A protocol is described for making a soluble whole-cell extract from yeast (Saccharomyces cerevisiae) that supports active and specific transcription initiation by RNA polymerases I, II, and III. Specific initiation by polymerase I decreases in high-density cultures, paralleling the decrease in abundance of the endogenous 35S rRNA precursor. This extract should be useful for studying the molecular mechanisms that regulate rRNA transcription in yeast. Images PMID:1992452

  2. PolDIP2 interacts with human PrimPol and enhances its DNA polymerase activities.

    PubMed

    Guilliam, Thomas A; Bailey, Laura J; Brissett, Nigel C; Doherty, Aidan J

    2016-04-20

    Translesion synthesis (TLS) employs specialized DNA polymerases to bypass replication fork stalling lesions. PrimPol was recently identified as a TLS primase and polymerase involved in DNA damage tolerance. Here, we identify a novel PrimPol binding partner, PolDIP2, and describe how it regulates PrimPol's enzymatic activities. PolDIP2 stimulates the polymerase activity of PrimPol, enhancing both its capacity to bind DNA and the processivity of the catalytic domain. In addition, PolDIP2 stimulates both the efficiency and error-free bypass of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxoG) lesions by PrimPol. We show that PolDIP2 binds to PrimPol's catalytic domain and identify potential binding sites. Finally, we demonstrate that depletion of PolDIP2 in human cells causes a decrease in replication fork rates, similar to that observed in PrimPol(-/-)cells. However, depletion of PolDIP2 in PrimPol(-/-)cells does not produce a further decrease in replication fork rates. Together, these findings establish that PolDIP2 can regulate the TLS polymerase and primer extension activities of PrimPol, further enhancing our understanding of the roles of PolDIP2 and PrimPol in eukaryotic DNA damage tolerance. PMID:26984527

  3. Plasimids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, Sanford A.; Martinez, Susana; Lopez, Paloma; Espinosa, Manuel

    1991-01-01

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of Streptococcus pneumoniae. Plasmid pSM22, the vector containing the pneumocccal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme.

  4. Plasmids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, S.A.; Martinez, S.; Lopez, P.; Espinosa, M.

    1991-03-26

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of Streptococcus pneumoniae. Plasmid pSM22, the vector containing the pneumocccal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme. 1 figure.

  5. Plasmids containing the gene for DNA polymerase I from Streptococcus pneumoniae

    DOEpatents

    Lacks, S.A.; Martinez, S.; Lopez, P.; Espinosa, M.

    1987-08-28

    A method is disclosed for cloning the gene which encodes a DNA polymerase-exonuclease of /und Streptococcus/ /und pneumoniae/. Plasmid pSM22, the vector containing the pneumococcal polA gene, facilitates the expression of 50-fold greater amounts of the PolI enzyme. 1 fig., 1 tab.

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

    ERIC Educational Resources Information Center

    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…

  7. PolDIP2 interacts with human PrimPol and enhances its DNA polymerase activities

    PubMed Central

    Guilliam, Thomas A.; Bailey, Laura J.; Brissett, Nigel C.; Doherty, Aidan J.

    2016-01-01

    Translesion synthesis (TLS) employs specialized DNA polymerases to bypass replication fork stalling lesions. PrimPol was recently identified as a TLS primase and polymerase involved in DNA damage tolerance. Here, we identify a novel PrimPol binding partner, PolDIP2, and describe how it regulates PrimPol's enzymatic activities. PolDIP2 stimulates the polymerase activity of PrimPol, enhancing both its capacity to bind DNA and the processivity of the catalytic domain. In addition, PolDIP2 stimulates both the efficiency and error-free bypass of 8-oxo-7,8-dihydrodeoxyguanosine (8-oxoG) lesions by PrimPol. We show that PolDIP2 binds to PrimPol's catalytic domain and identify potential binding sites. Finally, we demonstrate that depletion of PolDIP2 in human cells causes a decrease in replication fork rates, similar to that observed in PrimPol−/− cells. However, depletion of PolDIP2 in PrimPol−/− cells does not produce a further decrease in replication fork rates. Together, these findings establish that PolDIP2 can regulate the TLS polymerase and primer extension activities of PrimPol, further enhancing our understanding of the roles of PolDIP2 and PrimPol in eukaryotic DNA damage tolerance. PMID:26984527

  8. Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.

    PubMed

    Boyle, David S; Lehman, Dara A; Lillis, Lorraine; Peterson, Dylan; Singhal, Mitra; Armes, Niall; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie

    2013-01-01

    Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes. PMID:23549916

  9. Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification

    PubMed Central

    Boyle, David S.; Lehman, Dara A.; Lillis, Lorraine; Peterson, Dylan; Singhal, Mitra; Armes, Niall; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie

    2013-01-01

    ABSTRACT Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes. PMID:23549916

  10. Genetic diversity in marine algal virus communities as revealed by sequence analysis of DNA polymerase genes.

    PubMed Central

    Chen, F; Suttle, C A; Short, S M

    1996-01-01

    Algal-virus-specific PCR primers were used to amplify DNA polymerase gene (pol) fragments (683 to 689 bp) from the virus-sized fraction (0.02 to 0.2 microns) concentrated from inshore and offshore water samples collected from the Gulf of Mexico. Algal-virus-like DNA pol genes were detected in five samples collected from the surface and deep chlorophyll maximum. PCR products from an offshore station were cloned, and the genetic diversity of 33 fragments was examined by restriction fragment length polymorphism and sequence analysis. The five different genotypes or operational taxonomic units (OTUs) that were identified on the basis of restriction fragment length polymorphism banding patterns were present in different relative abundances (9 to 34%). One clone from each OTU was sequenced, and phylogenetic analysis showed that all of the OTUs fell within the family Phycodnaviridae. Four of the OTUs fell within a group of viruses (MpV) which infect the photosynthetic picoplankter Micromonas pusilla. The genetic diversity among these genotypes was as large as that previously found for MpV isolates from different oceans. The remaining genotype formed its own clade between viruses which infect M. pusilla and Chrysochromulina brevifilum. These results imply that marine virus communities contain a diverse assemblage of MpV-like viruses, as well as other unknown members of the Phycodnaviridae. PMID:8702280

  11. A role for DNA polymerase alpha in epigenetic control of transcriptional silencing in fission yeast.

    PubMed

    Nakayama Ji; Allshire, R C; Klar, A J; Grewal, S I

    2001-06-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase alpha (pol(alpha)) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that pol(alpha) mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polalpha and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Pol(alpha) in vitro. Moreover, silencing-defective mutant Pol(alpha) displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Pol(alpha), in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  12. An artificial processivity clamp made with streptavidin facilitates oriented attachment of polymerase-DNA complexes to surfaces.

    PubMed

    Williams, John G K; Steffens, David L; Anderson, Jon P; Urlacher, Teresa M; Lamb, Donald T; Grone, Daniel L; Egelhoff, Jolene C

    2008-10-01

    Single molecule analysis of individual enzymes can require oriented immobilization of the subject molecules on a detection surface. As part of a technology development project for single molecule DNA sequencing, we faced the multiple challenges of immobilizing both a DNA polymerase and its DNA template together in an active, stable complex capable of highly processive DNA synthesis on a nonstick surface. Here, we report the genetic modification of the archaeal DNA polymerase 9 degrees N in which two biotinylated peptide 'legs' are inserted at positions flanking the DNA-binding cleft. Streptavidin binding on either side of the cleft both traps the DNA template in the polymerase and orients the complex on a biotinylated surface. We present evidence that purified polymerase-DNA-streptavidin complexes are active both in solution and immobilized on a surface. Processivity is improved from <20 nt in the unmodified polymerase to several thousand nucleotides in the engineered complexes. High-molecular weight DNA synthesized by immobilized complexes is observed moving above the surface even as it remains tethered to the polymerase. Pre-formed polymerase-DNA-streptavidin complexes can be stored frozen and subsequently thawed without dissociation or loss of activity, making them convenient for use in single molecule analysis. PMID:18723573

  13. High expression of functional adenovirus DNA polymerase and precursor terminal protein using recombinant vaccinia virus.

    PubMed Central

    Stunnenberg, H G; Lange, H; Philipson, L; van Miltenburg, R T; van der Vliet, P C

    1988-01-01

    Initiation of Adenovirus (Ad) DNA replication occurs by a protein-priming mechanism in which the viral precursor terminal protein (pTP) and DNA polymerase (pol) as well as two nuclear DNA-binding proteins from uninfected HeLa cells are required. Biochemical studies on the pTP and DNA polymerase proteins separately have been hampered due to their low abundance and their presence as a pTP-pol complex in Ad infected cells. We have constructed a genomic sequence containing the large open reading frame from the Ad5 pol gene to which 9 basepairs from a putative exon were ligated. When inserted behind a modified late promoter of vaccinia virus the resulting recombinant virus produced enzymatically active 140 kDa Ad DNA polymerase. The same strategy was applied to express the 80 kDa pTP gene in a functional form. Both proteins were overexpressed at least 30-fold compared to extracts from Adenovirus infected cells and, when combined, were fully active for initiation in an in vitro Adenovirus DNA replication system. Images PMID:3362670

  14. dNTP-dependent Conformational Transitions in the Fingers Subdomain of Klentaq1 DNA Polymerase

    PubMed Central

    Rothwell, Paul J.; Allen, William J.; Sisamakis, Evangelos; Kalinin, Stanislav; Felekyan, Suren; Widengren, Jerker; Waksman, Gabriel; Seidel, Claus A. M.

    2013-01-01

    DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the “nucleotide-binding” state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state. PMID:23525110

  15. Gastric cancer associated variant of DNA polymerase beta (Leu22Pro) promotes DNA replication associated double strand breaks

    PubMed Central

    Rozacky, Jenna; Nemec, Antoni A.; Sweasy, Joann B.; Kidane, Dawit

    2015-01-01

    DNA polymerase beta (Pol β) is a key enzymefor the protection against oxidative DNA lesions via itsrole in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5′ phosphate group (5′-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5′-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation. PMID:26090616

  16. Gastric cancer associated variant of DNA polymerase beta (Leu22Pro) promotes DNA replication associated double strand breaks.

    PubMed

    Rozacky, Jenna; Nemec, Antoni A; Sweasy, Joann B; Kidane, Dawit

    2015-09-15

    DNA polymerase beta (Pol β) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol β, is responsible for removal of the 5' phosphate group (5'-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol β (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5'-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation. PMID:26090616

  17. Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

    PubMed Central

    Narita, Yohei; Ryo, Akihide; Kawashima, Daisuke; Sugimoto, Atsuko; Kanda, Teru; Kimura, Hiroshi

    2013-01-01

    Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein is known as a regulator which recognizes phosphorylated Ser/Thr-Pro motifs and increases the rate of cis and trans amide isomer interconversion, thereby altering the conformation of its substrates. We found that Pin1 knockdown using short hairpin RNA (shRNA) technology resulted in strong suppression of productive Epstein-Barr virus (EBV) DNA replication. We further identified the EBV DNA polymerase catalytic subunit, BALF5, as a Pin1 substrate in glutathione S-transferase (GST) pulldown and immunoprecipitation assays. Lambda protein phosphatase treatment abolished the binding of BALF5 to Pin1, and mutation analysis of BALF5 revealed that replacement of the Thr178 residue by Ala (BALF5 T178A) disrupted the interaction with Pin1. To further test the effects of Pin1 in the context of virus infection, we constructed a BALF5-deficient recombinant virus. Exogenous supply of wild-type BALF5 in HEK293 cells with knockout recombinant EBV allowed efficient synthesis of viral genome DNA, but BALF5 T178A could not provide support as efficiently as wild-type BALF5. In conclusion, we found that EBV DNA polymerase BALF5 subunit interacts with Pin1 through BALF5 Thr178 in a phosphorylation-dependent manner. Pin1 might modulate EBV DNA polymerase conformation for efficient, productive viral DNA replication. PMID:23221557

  18. Novel enzyme immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue.

    PubMed Central

    Merkelbach, S.; Gehlen, J.; Handt, S.; Füzesi, L.

    1997-01-01

    Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molecules could be achieved by binding the DNA onto a silica column before further purification. Viral DNA sequences could be amplified by PCR in DNA extracted from routinely processed paraffin blocks from cases with clinically or morphologically suspected cytomegalovirus or Epstein-Barr virus infections. The PCR products were specified by a novel liquid hybridization assay called PCR-enzyme-linked immunosorbent assay. Using this assay, the time-consuming Southern hybridization could be replaced and the time requirement for the detection of PCR products could be reduced from 1 day to 4 hours. The assay system described here represents a reliable, sensitive, and specific method for the detection of viral DNA from paraffin-embedded tissue samples. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9137080

  19. RNA polymerase III-specific general transcription factor IIIC contains a heterodimer resembling TFIIF Rap30/Rap74

    PubMed Central

    Taylor, Nicholas M. I.; Baudin, Florence; von Scheven, Gudrun; Müller, Christoph W.

    2013-01-01

    Transcription of tRNA-encoding genes by RNA polymerase (Pol) III requires the six-subunit general transcription factor IIIC that uses subcomplexes τA and τB to recognize two gene-internal promoter elements named A- and B-box. The Schizosaccharomyces pombe τA subcomplex comprises subunits Sfc1, Sfc4 and Sfc7. The crystal structure of the Sfc1/Sfc7 heterodimer reveals similar domains and overall domain architecture to the Pol II-specific general transcription factor TFIIF Rap30/Rap74. The N-terminal Sfc1/Sfc7 dimerization module consists of a triple β-barrel similar to the N-terminal TFIIF Rap30/Rap74 dimerization module, whereas the C-terminal Sfc1 DNA-binding domain contains a winged-helix domain most similar to the TFIIF Rap30 C-terminal winged-helix domain. Sfc1 DNA-binding domain recognizes single and double-stranded DNA by an unknown mechanism. Several features observed for A-box recognition by τA resemble the recognition of promoters by bacterial RNA polymerase, where σ factor unfolds double-stranded DNA and stabilizes the non-coding DNA strand in an open conformation. Such a function has also been proposed for TFIIF, suggesting that the observed structural similarity between Sfc1/Sfc7 and TFIIF Rap30/Rap74 might also reflect similar functions. PMID:23921640

  20. DNA polymerase activity in heat killing and hyperthermic radiosensitization of mammalian cells as observed after fractionated heat treatments.

    PubMed

    Jorritsma, J B; Burgman, P; Kampinga, H H; Konings, A W

    1986-03-01

    Possible relations between hyperthermic inactivation of alpha and beta DNA polymerase activity and hyperthermic cell killing or hyperthermic radiosensitization were investigated. Ehrlich Ascites Tumor (EAT) cells and HeLa S3 cells were treated with fractionated doses of hyperthermia. The heating schedules were chosen such that the initial heat treatment resulted in either thermotolerance or thermosensitization (step-down heating) for the second heat treatment. The results show that for DNA polymerase activity and heat radiosensitization (cell survival) no thermotolerance or thermosensitization is observed. Thus hyperthermic cell killing and DNA polymerase activity are not correlated. The correlation of hyperthermic radiosensitization and DNA polymerase activity was substantially less than observed in previous experiments with normotolerant and thermotolerant HeLa S3 cells. We conclude that alpha and beta DNA polymerase inactivation is not always the critical cellular process responsible for hyperthermic cell killing or hyperthermic radiosensitization. Other possible cellular systems that might determine these processes are discussed. PMID:3754338

  1. The Human Tim-Tipin Complex Interacts Directly with DNA Polymerase ϵ and Stimulates Its Synthetic Activity*

    PubMed Central

    Aria, Valentina; De Felice, Mariarita; Di Perna, Roberta; Uno, Shuji; Masai, Hisao; Syväoja, Juhani E.; van Loon, Barbara; Hübscher, Ulrich; Pisani, Francesca M.

    2013-01-01

    The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ϵ. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ϵ directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ϵ bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork. PMID:23511638

  2. GSH2 promoter methylation in pancreatic cancer analyzed by quantitative methylation-specific polymerase chain reaction

    PubMed Central

    GAO, FEI; HUANG, HAO-JIE; GAO, JUN; LI, ZHAO-SHEN; MA, SHU-REN

    2015-01-01

    Tumor suppressor gene silencing via promoter hypermethylation is an important event in pancreatic cancer pathogenesis. Aberrant DNA hypermethylation events are highly tumor specific, and may provide a diagnostic tool for pancreatic cancer patients. The objective of the current study was to identify novel methylation-related genes that may potentially be used to establish novel therapeutic and diagnostic strategies against pancreatic cancer. The methylation status of the GS homeobox 2 (GSH2) gene was analyzed using the sodium bisulfite sequencing method. The GSH2 methylation ratio was examined in primary carcinomas and corresponding normal tissues derived from 47 patients with pancreatic cancer, using quantitative methylation-specific polymerase chain reaction. Methylation ratios were found to be associated with the patient's clinicopathological features. GSH2 gene methylation was detected in 26 (55.3%) of the 47 pancreatic cancer patients, indicating that it occurs frequently in pancreatic cancer. A significant association with methylation was observed for tumor-node-metastasis stage (P=0.031). GSH2 may be a novel methylation-sensitive tumor suppressor gene in pancreatic cancer and may be a tumor-specific biomarker of the disease. PMID:26171036

  3. Mechanism of Microhomology-Mediated End-Joining Promoted by Human DNA Polymerase Theta

    PubMed Central

    Kent, Tatiana; Chandramouly, Gurushankar; McDevitt, Shane Michael; Ozdemir, Ahmet Y.; Pomerantz, Richard T.

    2014-01-01

    Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break repair pathway that utilizes sequence microhomology to recombine broken DNA. Although MMEJ is implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase θ (Polθ) performs MMEJ of DNA containing 3’ single-strand DNA overhangs with two or more base-pairs of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Polθ in human cells. Our data support a mechanism whereby Polθ facilitates end-joining and microhomology annealing then utilizes the opposing overhang as a template in trans which stabilizes the DNA synapse. Polθ exhibits a preference for DNA containing a 5’-terminal phosphate, similar to polymerases involved in non-homologous end-joining. Lastly, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Polθ which likely promote DNA synapse formation. PMID:25643323

  4. Conformational Dynamics of Bacteriophage T7 DNA Polymerase and its Processivity Factor, Escherichia coli thioredoxin

    SciTech Connect

    Akabayov, B.; Akabayov, S; Lee , S; Tabor, S; Kulczyk , A; Richardson, C

    2010-01-01

    Gene 5 of bacteriophage T7 encodes a DNA polymerase (gp5) responsible for the replication of the phage DNA. Gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. Thioredoxin (trx) of Escherichia coli binds tightly (Kd = 5 nM) to a unique segment in the thumb subdomain of gp5 and increases processivity. We have probed the molecular basis for the increase in processivity. A single-molecule experiment reveals differences in rates of enzymatic activity and processivity between gp5 and gp5/trx. Small angle X-ray scattering studies combined with nuclease footprinting reveal two conformations of gp5, one in the free state and one upon binding to trx. Comparative analysis of the DNA binding clefts of DNA polymerases and DNA binding proteins show that the binding surface contains more hydrophobic residues than other DNA binding proteins. The balanced composition between hydrophobic and charged residues of the binding site allows for efficient sliding of gp5/trx on the DNA. We propose a model for trx-induced conformational changes in gp5 that enhance the processivity by increasing the interaction of gp5 with DNA.

  5. Single-Molecule Investigation of Response to Oxidative DNA Damage by a Y-Family DNA Polymerase.

    PubMed

    Raper, Austin T; Gadkari, Varun V; Maxwell, Brian A; Suo, Zucai

    2016-04-12

    Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo and rescue stalled DNA replication machinery. Dpo4, a well-characterized model Y-family DNA polymerase, is known to catalyze translesion synthesis across a variety of DNA lesions including 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxo-dG). Our previous X-ray crystallographic, stopped-flow Förster resonance energy transfer (FRET), and computational simulation studies have revealed that Dpo4 samples a variety of global conformations as it recognizes and binds DNA. Here we employed single-molecule FRET (smFRET) techniques to investigate the kinetics and conformational dynamics of Dpo4 when it encountered 8-oxo-dG, a major oxidative lesion with high mutagenic potential. Our smFRET data indicated that Dpo4 bound the DNA substrate in multiple conformations, as suggested by three observed FRET states. An incoming correct or incorrect nucleotide affected the distribution and stability of these states with the correct nucleotide completely shifting the equilibrium toward a catalytically competent complex. Furthermore, the presence of the 8-oxo-dG lesion in the DNA stabilized both the binary and ternary complexes of Dpo4. Thus, our smFRET analysis provided a basis for the enhanced efficiency which Dpo4 is known to exhibit when replicating across from 8-oxo-dG. PMID:27002236

  6. Uranyl mediated photofootprinting reveals strong E. coli RNA polymerase--DNA backbone contacts in the +10 region of the DeoP1 promoter open complex.

    PubMed Central

    Jeppesen, C; Nielsen, P E

    1989-01-01

    Employing a newly developed uranyl photofootprinting technique (Nielsen et al. (1988) FEBS Lett. 235, 122), we have analyzed the structure of the E. coli RNA polymerase deoP1 promoter open complex. The results show strong polymerase DNA backbone contacts in the -40, -10, and most notably in the +10 region. These results suggest that unwinding of the -12 to +3 region of the promoter in the open complex is mediated through polymerase DNA backbone contacts on both sides of this region. The pattern of bases that are hyperreactive towards KMnO4 or uranyl within the -12 to +3 region furthermore argues against a model in which this region is simply unwound and/or single stranded. The results indicate specific protein contacts and/or a fixed DNA conformation within the -12 to +3 region. Images PMID:2503811

  7. A polymerase chain reaction method for the amplification of full-length envelope genes of HIV-1 from DNA samples containing single molecules of HIV-1 provirus.

    PubMed

    McClure, P; Curran, R; Boneham, S; Ball, J K

    2000-07-01

    Polymerase chain reaction (PCR) amplification of full-length envelope genes from the human immunodeficiency virus type 1 (HIV-1) directly from uncultured clinical samples is difficult. This paper describes a comparative assessment of the performance of three thermostable polymerases in an HIV-1 full-length envelope gene PCR. The PCR method utilising Expand HiFi polymerase was successful when using DNA samples extracted from a variety of sources including blood, semen and various tissues. This method generated high and specific yields of product from samples containing as little as one copy of HIV-1 proviral DNA. The resulting PCR products were suitable for a variety of downstream analytical methods including DNA sequence analysis. PMID:10921844

  8. Enzymatic synthesis of modified oligonucleotides by PEAR using Phusion and KOD DNA polymerases.

    PubMed

    Wang, Xuxiang; Zhang, Jianye; Li, Yingjia; Chen, Gang; Wang, Xiaolong

    2015-02-01

    Antisense synthetic oligonucleotides have been developed as potential gene-targeted therapeutics. We previously reported polymerase-endonuclease amplification reaction (PEAR) for amplification of natural and 5'-O-(1-thiotriphosphate) (S)-modified oligonucleotides. Here, we extended the PEAR technique for enzymatic preparation of 2'-deoxy-2'-fluoro-(2'-F) and 2'-F/S double-modified oligonucleotides. The result showed that KOD and Phusion DNA polymerase could synthesize oligonucleotides with one or two modified nucleotides, and KOD DNA polymerase is more suitable than Phusion DNA polymerase for PEAR amplification of 2'-F and 2'-F/S double modified oligonucleotides. The composition of PEAR products were analyzed by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection and showed that the sequence of the PEAR products are maintained at an extremely high accuracy (>99.9%), and after digestion the area percent of full-length modified oligonucleotides reaches 89.24%. PEAR is suitable for synthesis of modified oligonucleotides efficiently and with high purity. PMID:25517220

  9. Impact of ribonucleotide incorporation by DNA polymerases β and λ on oxidative base excision repair

    PubMed Central

    Crespan, Emmanuele; Furrer, Antonia; Rösinger, Marcel; Bertoletti, Federica; Mentegari, Elisa; Chiapparini, Giulia; Imhof, Ralph; Ziegler, Nathalie; Sturla, Shana J.; Hübscher, Ulrich; van Loon, Barbara; Maga, Giovanni

    2016-01-01

    Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols β and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol β, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol β- and λ-deficient cell extracts suggest that Pol β levels can greatly affect rNMP incorporation opposite oxidative DNA lesions. PMID:26917111

  10. Two-step polymerase chain reactions and restriction endonuclease analyses detect and differentiate ompA DNA of Chlamydia spp.

    PubMed Central

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1992-01-01

    Specific and sensitive amplification of major outer membrane protein (MOMP) gene (ompA) DNA sequences of Chlamydia species with various MOMP genotypes was achieved by a two-step polymerase chain reaction (PCR). Degenerate, inosine-containing oligonucleotide primers homologous to the 5' and 3' ends of the translated regions of all chlamydial MOMP genes were used in a PCR to amplify a DNA fragment of approximately 1,120 bp. A portion of this DNA fragment was amplified in a second genus-specific reaction that yielded a DNA fragment of approximately 930 bp. A pair of degenerate oligonucleotide primers homologous to internal sequences of the primary DNA fragment was used in this PCR. This method detected three cognate chlamydial genomes in a background of 1 microgram of unrelated DNA. MOMP genes of 13 representative chlamydial MOMP genotypes of the species C. trachomatis, C. pneumoniae, and C. psittaci were amplified. In a secondary PCR, group-specific detection was achieved by the simultaneous use of one genus-specific primer and three primers derived from different fingerprint regions of three major groups of chlamydiae. This multiplex PCR differentiated the groups by the length of the amplified DNA fragments and detected the simultaneous presence of DNA sequences of the Chlamydia spp. with different MOMP genotypes. Further differentiation as ompA restriction fragment length polymorphism types among all chlamydial strains with the various MOMP genotypes analyzed here was achieved by restriction endonuclease analysis of the secondary PCR products. DNA sequences corresponding to the ompA restriction fragment length polymorphism type B577 of C. psittaci were detected in two of seven milk samples from cases of bovine mastitis. Images PMID:1349899

  11. The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains.

    PubMed Central

    Moser, M J; Holley, W R; Chatterjee, A; Mian, I S

    1997-01-01

    Prior sequence analysis studies have suggested that bacterial ribonuclease (RNase) Ds comprise a complete domain that is found also in Homo sapiens polymyositis-scleroderma overlap syndrome 100 kDa autoantigen and Werner syndrome protein. This RNase D 3'-->5' exoribonuclease domain was predicted to have a structure and mechanism of action similar to the 3'-->5' exodeoxyibonuclease (proofreading) domain of DNA polymerases. Here, hidden Markov model (HMM) and phylogenetic studies have been used to identify and characterise other sequences that may possess this exonuclease domain. Results indicate that it is also present in the RNase T family; Borrelia burgdorferi P93 protein, an immunodominant antigen in Lyme disease; bacteriophage T4 dexA and Escherichia coli exonuclease I, processive 3'-->5' exodeoxyribonucleases that degrade single-stranded DNA; Bacillus subtilis dinG, a probable helicase involved in DNA repair and possibly replication, and peptide synthase 1; Saccharomyces cerevisiae Pab1p-dependent poly(A) nuclease PAN2 subunit, required for shortening mRNA poly(A) tails; Caenorhabditis elegans and Mus musculus CAF1, transcription factor CCR4-associated factor 1; Xenopus laevis XPMC2, prevention of mitotic catastrophe in fission yeast; Drosophila melanogaster egalitarian, oocyte specification and axis determination, and exuperantia, establishment of oocyte polarity; H.sapiens HEM45, expressed in tumour cell lines and uterus and regulated by oestrogen; and 31 open reading frames including one in Methanococcus jannaschii . Examination of a multiple sequence alignment and two three-dimensional structures of proofreading domains has allowed definition of the core sequence, structural and functional elements of this exonuclease domain. PMID:9396823

  12. [THE HIGHLY EFFECTIVE DETECTION OF DNA RICKETTSIA USING TECHNIQUE OF POLYMERASE CHAIN REACTION IN REAL-TIME].

    PubMed

    Kartashov, M Yu; Mikryukova, T P; Ternovoi, V A; Moskvitina, N S; Loktev, V B

    2015-12-01

    The article considers development of highly effective technique of detection of genetic material of ricketsia based on polymerase chain reaction in real-time using original primers to the most conservative sites of gene of citrate synthase (gItA). The analytical sensitivity of the developed polymerase chain reaction in real-time test permits to detect from 80 genome equivalents in analyzed sample during three hours. The high specificity of test-system is substantiated by detection of nucleotide sequences of amplificated fragments of gene gltA. The approbation ofthe polymerase chain reaction in real-time test is carried out on collection of 310 ticks of species I. persulcatus, I. pavlovskyi, D. reticulatus. It is demonstrated that the developed alternate ofprimers and probe permits with high degree of sensitivity and specifcity to detect DNA of different species of ricketsia widespread on territory of Russia (R. sibirica, R. raoultii, R. helvetica, R. tarasevichiae). The proposed polymerase chain reaction in real-time test can be appliedfor isolation of fragment of gene gltA with purpose for detecting nucleotide sequence and subsequent genetic typing of ricketsia. The application ofthe proposed technique can facilitate task of monitoring hot spots of ricketsiosis. PMID:27032252

  13. Cloning DPB3, the gene encoding the third subunit of DNA polymerase II of Saccharomyces cerevisiae.

    PubMed Central

    Araki, H; Hamatake, R K; Morrison, A; Johnson, A L; Johnston, L H; Sugino, A

    1991-01-01

    DNA polymerase II purified from Saccharomyces cerevisiae contains polypeptides with apparent molecular masses of greater than 200, 80, 34, 30 and 29 kDa, the two largest of which (subunits A and B) are encoded by the essential genes POL2 and DPB2. By probing a lambda gt11 expression library of yeast DNA with antiserum against DNA polymerase II, we isolated a single gene, DPB3, that encodes both the 34- and 30-kDa polypeptides (subunit C and C'). The nucleotide sequence of DPB3 contained an open reading frame encoding a 23-kDa protein, significantly smaller than the observed molecular masses, 34- or 30-kDa, which might represent post-translationally modified forms of the DPB3 product. The predicted amino acid sequence contained a possible NTP-binding motif and a glutamate-rich region. NTP-binding motif and a glutamate-rich region. A dpb3 deletion mutant (dpb3 delta) was viable and yielded a DNA polymerase II lacking the 34- and 30-kDa polypeptides. dpb3 delta strains exhibited an increased spontaneous mutation rate, suggesting that the DPB3 product is required to maintain fidelity of chromosomal replication. Since a fifth, 29-kDa polypeptide was present in DNA polymerase II preparations from wild-type cell extracts throughout purification, the subunit composition appears to be A, B, C (or C and C') and D. The 5' nontranscribed region of DPB3 contained the MulI-related sequence ACGCGA, while the 0.9-kb DPB3 transcript accumulated periodically during the cell cycle and peaked at the G1/S boundary. The level of DPB3 transcript thus appears to be under the same cell cycle control as those of POL2, DPB2 and other DNA replication genes. DPB3 was mapped to chromosome II, 30 cM distal to his7. Images PMID:1923754

  14. Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III

    PubMed Central

    Proshkina, Galina M.; Shematorova, Elena K.; Proshkin, Sergey A.; Zaros, Cécile; Thuriaux, Pierre; Shpakovski, George V.

    2006-01-01

    RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner. PMID:16877568

  15. Single-Nucleotide Polymorphism Identification Assays Using a Thermostable DNA Polymerase and Delayed Extraction MALDI-TOF Mass Spectrometry

    PubMed Central

    Haff, Lawrence A.; Smirnov, Igor P.

    1997-01-01

    We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to delayed-extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications. PMID:9110177

  16. Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction.

    PubMed

    Zhang, Yinbo; Baranovskiy, Andrey G; Tahirov, Emin T; Tahirov, Tahir H; Pavlov, Youri I

    2016-07-01

    DNA polymerases (pols) are sophisticated protein machines operating in the replication, repair and recombination of genetic material in the complex environment of the cell. DNA pol reactions require at least two divalent metal ions for the phosphodiester bond formation. We explore two understudied roles of metals in pol transactions with emphasis on polα, a crucial enzyme in the initiation of DNA synthesis. We present evidence that the combination of many factors, including the structure of the template/primer, the identity of the metal, the metal turnover in the pol active site, and the influence of the concentration of nucleoside triphosphates, affect DNA pol synthesis. On the poly-dT70 template, the increase of Mg(2+) concentration within the range typically used for pol reactions led to the severe loss of the ability of pol to extend DNA primers and led to a decline in DNA product sizes when extending RNA primers, simulating the effect of "counting" of the number of nucleotides in nascent primers by polα. We suggest that a high Mg(2+) concentration promotes the dynamic formation of unconventional DNA structure(s), thus limiting the apparent processivity of the enzyme. Next, we found that Zn(2+) supported robust polα reactions when the concentration of nucleotides was above the concentration of ions; however, there was only one nucleotide incorporation by the Klenow fragment of DNA pol I. Zn(2+) drastically inhibited polα, but had no effect on Klenow, when Mg(2+) was also present. It is possible that Zn(2+) perturbs metal-mediated transactions in pol active site, for example affecting the step of pyrophosphate removal at the end of each pol cycle necessary for continuation of polymerization. PMID:27235627

  17. Specific and Rapid Detection of Mycobacterium tuberculosis Complex in Clinical Samples by Polymerase Chain Reaction

    PubMed Central

    Singh, Anamika; Kashyap, Vijendra Kumar

    2012-01-01

    Background. Tuberculosis, a global health problem and highly prevalent in India, has always been a serious problem with respect to definitive diagnosis. Polymerase chain reaction (PCR) techniques are now widely used for early detection and species differentiation of mycobacteria, but mostly with their own limitations. We aim to detect and differentiate Mycobacterium tuberculosis (Mtb) infections by choosing appropriate target sequences, ideally present in all mycobacterial species (MTB complex) and absent in others. Methods. Amplification of three target sequences from unrelated genes, namely, hsp 65 (165 bp), dnaJ (365 bp), and insertion element IS 6110 (541 bp) by PCR was carried out in clinical samples from suspected cases of tuberculosis/ mycobacterioses and healthy controls. Results. The sensitivity of this method ranged from 73.33% to 84.61%, and the specificity was 80%. The PCR method was significantly better (P = 0.03 and P = 0.009) than both smear and culture methods. Conclusion. Our trimarker-based PCR method could specifically detect M. tuberculosis and MTB complex infection from that of major pathogenic NTM and nonpathogenic mycobacteria. This method, by well distinguishing between MTB complex and NTM, presented a fast and accurate method to detect and diagnose mycobacterial infections more efficiently and could thereby help in better patient management particularly considering the increase in mycobacterial infections due to emergence of NTM over the past decades. PMID:23093958

  18. Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two specific sites.

    PubMed

    Fels, A; Hu, K; Riesner, D

    2001-11-15

    Pospiviroidae, with their main representative potato spindle tuber viroid (PSTVd), are replicated via a rolling circle mechanism by the host-encoded DNA-dependent RNA polymerase II (pol II). In the first step, the (+)-strand circular viroid is transcribed into a (-)-strand oligomer intermediate. As yet it is not known whether transcription is initiated by promotors at specific start sites or is distributed non-specifically over the whole circle. An in vitro transcription extract was prepared from a non-infected potato cell culture which exhibited transcriptional activity using added circular PSTVd (+)-strand RNA as template. In accordance with pol II activity, transcription could be inhibited by alpha-amanitin. RT-PCR revealed the existence of at least two different start sites and primer extension identified these as nucleotides A(111) and A(325). The sequences of the first 7 nt transcribed are very similar, (105)GGAGCGA(111) and (319)GGGGCGA(325). GC-boxes are located at a distance of 15 and 16 nt upstream, respectively, in the native viroid structure, which may act to facilitate initiation. The GC-boxes may have a similar function to the GC-rich hairpin II in the (-)-strand intermediate, as described previously. The results are compared with the corresponding features of avocado sunblotch viroid, which belongs to a different family of viroids and exhibits different transcription initiation properties. PMID:11713308

  19. Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems.

    PubMed

    Terpe, Kay

    2013-12-01

    During the genomics era, the use of thermostable DNA polymerases increased greatly. Many were identified and described-mainly of the genera Thermus, Thermococcus and Pyrococcus. Each polymerase has different features, resulting from origin and genetic modification. However, the rational choice of the adequate polymerase depends on the application itself. This review gives an overview of the most commonly used DNA polymerases used for PCR application: KOD, Pab (Isis™), Pfu, Pst (Deep Vent™), Pwo, Taq, Tbr, Tca, Tfi, Tfl, Tfu, Tgo, Tli (Vent™), Tma (UITma™), Tne, Tth and others. PMID:24177730

  20. Mismatch extension of DNA polymerases and high-accuracy single nucleotide polymorphism diagnostics by gold nanoparticle-improved isothermal amplification.

    PubMed

    Chen, Feng; Zhao, Yue; Fan, Chunhai; Zhao, Yongxi

    2015-09-01

    Sequence mismatches may induce nonspecific extension reaction, causing false results for SNP diagnostics. Herein, we systematically investigated the impact of various 3'-terminal mismatches on isothermal amplification catalyzed by representative DNA polymerases. Despite their diverse efficiencies depending on types of mismatch and kinds of DNA polymerase, all 12 kinds of single 3'-terminal mismatches induced the extension reaction. Generally, only several mismatches (primer-template, C-C, G-A, A-G, and A-A) present an observable inhibitory effect on the amplification reaction, whereas other mismatches trigger amplified signals as high as those of Watson-Crick pairs. The related mechanism was deeply discussed, and a primer-design guideline for specific SNP analysis was summarized. Furthermore, we found that the addition of appropriate gold nanoparticles (AuNPs) can significantly inhibit mismatch extension and enhance the amplification specificity. Also the high-accuracy SNP analysis of human blood genomic DNA has been demonstrated by AuNPs-improved isothermal amplification, the result of which was verified by sequencing (the gold standard method for SNP assay). Collectively, this work provides mechanistic insight into mismatch behavior and achieves accurate SNP diagnostics, holding great potential for the application in molecular diagnostics and personalized medicine. PMID:26249366

  1. Suppression of rolling circle amplification by nucleotide analogs in circular template for three DNA polymerases.

    PubMed

    Tang, Suming; Wei, Hua; Hu, Tianyu; Jiang, Jiquan; Chang, Jinglin; Guan, Yifu; Zhao, Guojie

    2016-08-01

    Among wide applications of nucleotide analogs, their roles in enzyme catalytic reactions are significant in both fundamental and medical researches. By introducing analogs into circular templates, we succeeded in determining effects of four analogs on RCA efficiency for three different DNA polymerases. Results showed an obvious suppression effect for 2'-OMeRNA modification, which might be due to the size of the C2'-modified moieties. 2'-F RNA, LNA and PS had little interference, suggesting good analog candidates for application in RCA. Different polymerases and nucleobases made a little difference according to analogs we used. These results are useful for understanding polymerase catalytic mechanism and analogs applications in RCA reaction. PMID:27151504

  2. Coumarins as Potential Inhibitors of DNA Polymerases and Reverse Transcriptases. Searching New Antiretroviral and Antitumoral Drugs.

    PubMed

    Garro, Hugo A; Pungitore, Carlos R

    2015-01-01

    Human Immunodeficiency Virus (HIV) is the viral agent of Acquired Immunodeficiency Syndrome (AIDS), and at present, there is no effective vaccine against HIV. Reverse Transcriptase (RT) is an essential enzyme for retroviral replication, such as HIV as well as for other RNA infectious viruses like Human T lymphocyte virus. Polymerases act in DNA metabolism, modulating different processes like mitosis, damage repair, transcription and replication. It has been widely documented that DNA Polymerases and Reverse Transcriptases serve as molecular targets for antiviral and antitumoral chemotherapy. Coumarins are oxygen heterocycles that are widely distributed throughout the plant kingdom. Natural coumarins have attraction due to their bioactive properties such as tumor promotion inhibitory effects, and anti-HIV activity. Coumarins and derivates exhibit potent inhibitory effects on HIV-1 replication in lymphocytes and compounds isolated from Calophyllum inophyllum or DCK derivates showed inhibitory activity against human RT. Furthermore, natural isocoumarins isolated from cultures of fungi or hydroxycoumarins were able to inhibit human DNA polymerase. In view of their importance as drugs and biologically active natural products, and their medicinally useful properties, extensive studies have been carried out on the synthesis of coumarin compounds in recent years. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), a class of antiretroviral chemotherapeutic agents, act by binding to an allosteric pocket showing, generally, low toxicity. This work tries to summarize the investigation about natural and synthetic coumarins with the ability to inhibit key enzymes that play a crucial role in DNA metabolism and their possible application as antiretroviral and antitumoral agents. PMID:26179474

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

    PubMed

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

    2016-07-17

    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 shown to serve as ligands for a single, critical Zn(2+) ion. The cysteines farthest from the C terminal were not required for activity, and a role for these amino acids has yet to be defined. Additionally, it is shown that Afu Pol-D activity is slowed by the template strand hypoxanthine, extending previous results that demonstrated inhibition by uracil. Hypoxanthine was a weaker inhibitor than uracil. Investigations with isolated DP2, which has a measurable polymerase activity, localised the deaminated base binding site to this subunit. Uracil and hypoxanthine slowed Afu Pol-D "in trans", that is, a copied DNA strand could be inhibited by a deaminated base in the alternate strand of a replication fork. The error rate of Afu Pol-D, measured in vitro, was 0.24×10(-5), typical for a polymerase that has been proposed to carry out genome replication in the Archaea. Deleting the 3'-5' proofreading exonuclease activity reduced fidelity twofold. The results presented in this publication considerably increase our knowledge of Pol-D. PMID:27320386

  4. Duality of polynucleotide substrates for Phi29 DNA polymerase: 3′→5′ RNase activity of the enzyme

    PubMed Central

    Lagunavicius, Arunas; Kiveryte, Zivile; Zimbaite-Ruskuliene, Vilma; Radzvilavicius, Tomas; Janulaitis, Arvydas

    2008-01-01

    Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3′→5′ DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3′ to 5′ polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is ∼10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection. PMID:18230765

  5. Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase

    PubMed Central

    Jarchow-Choy, Sarah K.; Krueger, Andrew T.; Liu, Haibo; Gao, Jianmin; Kool, Eric T.

    2011-01-01

    Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3′-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson–Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)n polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)n, fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA. PMID:20947563

  6. Sequence rearrangement and duplication of double stranded fibronectin cDNA probably occurring during cDNA synthesis by AMV reverse transcriptase and Escherichia coli DNA polymerase I.

    PubMed Central

    Fagan, J B; Pastan, I; de Crombrugghe, B

    1980-01-01

    Two cloned cDNAs derived from the mRNA for cell fibronectin have been sequenced, providing evidence that transcription with AMV reverse transcriptase or Escherichia coli DNA polymerase I may not always result in double stranded cDNA that is exactly homologous with its mRNA template. Instead, the sequences of these cloned cDNAs are consistent with the duplication and rearrangement of sequences during synthesis of double stranded cDNA. PMID:6159581

  7. Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

    PubMed Central

    Burak, Matthew J.; Guja, Kip E.; Garcia-Diaz, Miguel

    2015-01-01

    8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases. PMID:26220180

  8. Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

    PubMed Central

    Marczynski, G T; Schultz, P W; Jaehning, J A

    1989-01-01

    We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested. Images PMID:2677667

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

    PubMed

    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. PMID:26920159

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

    PubMed Central

    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 K354, R355, and K367 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 system

  11. DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I.

    PubMed Central

    Zhang, W; Evans, D H

    1995-01-01

    A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli. The protein is DNA polymerase I. Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA. Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates. Both kinds of reactions are unaffected by added ATP. Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules. The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand. Thus strand transfer occurs with the same polarity as nick translation. These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function. Images PMID:8524652

  12. Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

    PubMed

    Rohrman, Brittany; Richards-Kortum, Rebecca

    2015-02-01

    Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads. PMID:25560368

  13. DnaJA1/Hsp40 Is Co-Opted by Influenza A Virus To Enhance Its Viral RNA Polymerase Activity

    PubMed Central

    Cao, Mengmeng; Wei, Candong; Zhao, Lili; Wang, Jingfeng; Jia, Qiannan; Wang, Xue

    2014-01-01

    ABSTRACT The RNA-dependent RNA polymerase (RdRp) of influenza A virus is a heterotrimeric complex composed of the PB1, PB2, and PA subunits. The interplay between host factors and the three subunits of the RdRp is critical to enable viral RNA synthesis to occur in the nuclei of infected cells. In this study, we newly identified host factor DnaJA1, a member of the type I DnaJ/Hsp40 family, acting as a positive regulator for influenza virus replication. We found that DnaJA1 associates with the bPB2 and PA subunits and enhances viral RNA synthesis both in vivo and in vitro. Moreover, DnaJA1 could be translocated from cytoplasm into the nucleus upon influenza virus infection. The translocation of DnaJA1 is specifically accompanied by PB1-PA nuclear import. Interestingly, we observed that the effect of DnaJA1 on viral RNA synthesis is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, while the J domain normally mediates the Hsp70-DnaJ interaction required for regulating Hsp70 ATPase activity. Therefore, we propose that DnaJA1 is co-opted by the influenza A virus to enter the nucleus and to enhance its RNA polymerase activity in an Hsp70 cochaperone-independent manner. IMPORTANCE The interplay between host factors and influenza virus RNA polymerase plays a critical role in determining virus pathogenicity and host adaptation. In this study, we newly identified a host protein, DnaJA1/Hsp40, that is co-opted by influenza A virus RNA polymerase to enhance its viral RNA synthesis in the nuclei of infected cells. We found that DnaJA1 associates with both PB2 and PA subunits and translocates into the nucleus along with the nuclear import of the PB1-PA dimer during influenza virus replication. Interestingly, the effect of DnaJA1 is mainly dependent on its C-terminal substrate-binding domain and not on its typical J domain, which is required for its Hsp70 cochaperone function. To our knowledge, this is the first report on a member of the

  14. Enhancing helicase-dependent amplification by fusing the helicase with the DNA polymerase.

    PubMed

    Motré, Aurélie; Li, Ying; Kong, Huimin

    2008-08-15

    In this study, we have engineered a new bifunctional protein named "helimerase", by physically linking Thermoanaerobacter tengcongensis UvrD helicase (TteUvrD) and Bacillus stearothermophilus DNA polymerase I Large Fragment (Bstpol) using a coiled-coil. TteUvrD is fused with one part of the coiled-coil, WinZip-A2 (WZA2), through a linker L1 and possesses a Maltose Binding Protein (MBP) tag at the N terminal end. Bstpol is fused with the other part of the coiled-coil, WinZip-B1 (WZB1), through the same linker and possesses a His tag at the N terminal end. Therefore, the two fusion proteins MBP-WZA2-L1-TteUvrD and His-WZB1-L1-Bstpol composed the helimerase. We showed that this complex could be formed in vivo as well as in vitro and possessed specific activities of both TteUvrD and Bstpol. Moreover, we demonstrated that significantly longer fragments could be amplified by the helimerase in Helicase-Dependent Amplification (HDA) reactions instead of using TteUvrD and Bstpol proteins that do not form a complex. PMID:18556147

  15. Improvement of polymerase chain reaction-based Bt11 maize detection method by reduction of non-specific amplification.

    PubMed

    Mano, Junichi; Yanaka, Yuka; Akiyama, Hiroshi; Teshima, Reiko; Furui, Satoshi; Kitta, Kazumi

    2010-01-01

    The Bt11 maize-specific qualitative detection method based on polymerase chain reaction (PCR) in the JAS analytical test handbook has been widely used for administrative monitoring of GM crops and quality control of commercially distributed grains. In the present investigation, some apparently false-positive detections were observed in assays using the Bt11 maize-specific method, and these erroneous results were proved to have been caused by non-specific DNA amplification. We improved the detection method to reduce non-specific amplification by decreasing the concentration of magnesium ions in the PCR mixture. The subsequent evaluation of analytical performance demonstrated no marked difference between the currently used and the improved methods, except for the reduced non-specific amplification. We conclude that the currently used standard method should be replaced with the improved method for the reliable detection of Bt11 maize. PMID:20208407

  16. Identification of a Taq DNA polymerase inhibitor from the red seaweed Symphyocladia latiuscula.

    PubMed

    Jin, Hyung Joo; Oh, Mi Young; Jin, Deuk Hee; Hong, Yong Ki

    2008-07-01

    Two inhibitors of Taq DNA polymerase were isolated from the marine red alga Symphyocladia latiuscula. The inhibitors were purified by methanol extraction, molecular fractionation below 3000 MW and reverse-phase HPLC. The purified compound SL-1 containing three bromines was identified as 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (C7H5Br3O3: MW374) by NMR and MS analyses. The purified compound SL-2 was identified as 2,3, 6-tribromo-4,5-dihydroxybenzyl methyl ether(C8H7Br3O3: MW388). In a 25-microl reaction mixture containing 1.5 units of Taq DNA polymerase, the enzyme was completely inhibited by 0.5 microg SL-1 or 5 microg SL-2. PMID:19195384

  17. Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.

    PubMed Central

    Lee, S H; Eki, T; Hurwitz, J

    1989-01-01

    Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase delta that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase delta on a DNA singly primed phi X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase delta, in contrast to DNA polymerase alpha, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase alpha and delta are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I. Images PMID:2571990

  18. Concordance study: methods of quantifying corn and soybean genomic DNA intended for real-time polymerase chain reaction applications.

    PubMed

    Jenkins, G Ronald; Helber, Jennifer T; Freese, Larry D

    2012-08-29

    Quantitative real-time polymerase chain reaction (qPCR) is a technology commonly used for the detection and quantification of genetically engineered (GE) traits in grains and oilseeds. The method involves measuring copy numbers of taxon-specific, endogenous control genes exposed to the same manipulations as trait-specific target genes. Accurate DNA quantification is essential for successful and predictable results with qPCR. A systematic study of seven different DNA quantification methods, incorporating different chemistries and different instrumentation, were evaluated on corn and soy DNA that was extracted using two distinct extraction methods. A time course study showed that corn and soy DNA was stable under typical laboratory storage conditions. CornCTAB and cornQiagen DNA extracts produced statistically similar quantification values when measured by picogreen PG(TD700), PG(Lum20/20), Hoescht(TD700), and Hoescht(Lum20/20) methods, suggesting that these methods can be used interchangeably to quantify DNA in corn samples prior to initiation of qPCR. Soy(Qiagen) provided greater stochastic measurement variability when quantification methods were compared, whereas soyCTAB had statistically significant differences when a PG method was compared to a Hoescht method of DNA quantification. Finally, agarose gel electrophoresis data revealed more pronounced degradation for Qiagen-extracted DNA compared with CTAB extracts in both corn and soy. Consequently, Ct values generated by qPCR suggested that absolute copy numbers of PCR amplifiable targets were not concordant between Qiagen and CTAB DNA extracts. Understanding measurement uncertainty from component steps used in qPCR can contribute toward harmonizing methods for the detection of GE traits in grains and oilseeds. PMID:22866775

  19. Detection of MYCN Amplification in Serum DNA Using Conventional Polymerase Chain Reaction.

    PubMed

    Ma, Youngeun; Lee, Ji Won; Park, Soo Jin; Yi, Eun Sang; Choi, Young Bae; Yoo, Keon Hee; Sung, Ki Woong; Koo, Hong Hoe

    2016-09-01

    Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and is characterized by a wide range of clinical behaviors. Amplification of MYCN is a well-known poor prognostic factor in NB patients. As the MYCN amplification status is usually tested using tumor specimens, lengthy and invasive procedures are unavoidable. To evaluate the possibility of detecting MYCN amplification without invasive procedure, we performed conventional polymerase chain reaction (PCR) analysis to identify MYCN amplification using the preserved serum DNA. PCR of serum DNA was done in 105 NB patients whose MYCN status had been confirmed by fluorescence in situ hybridization. MYCN amplification was evaluated as the ratio of signal intensities between MYCN and NAGK (M/N ratio). When regarding the tissue FISH results as a reference, 10 patients had MYCN-amplified (MNA) NB, and 95 had non-MNA NB. The M/N ratio of the MNA group (median 2.56, range 1.01-3.58) was significantly higher than that of the non-MNA group (median 0.97, range 0.67-5.18) (P < 0.001). In the receiver operating characteristic curve analysis, the area under the curve was 0.957 (95% confidence interval 0.898-1.000; P < 0.001), and it showed 90.9% sensitivity and 97.9% specificity with the selected cut-off value set as 1.6. The detection of MYCN amplification using conventional PCR analysis of serum samples seems to be a simple and promising method to evaluate the MYCN status of NB patients. Further study with a larger set of patients is needed to confirm the accuracy of this result. PMID:27510381

  20. CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

    PubMed Central

    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

  1. Flavonoid glycoside: a new inhibitor of eukaryotic DNA polymerase alpha and a new carrier for inhibitor-affinity chromatography.

    PubMed

    Mizushina, Yoshiyuki; Ishidoh, Tomomi; Kamisuki, Shinji; Nakazawa, Satoshi; Takemura, Masaharu; Sugawara, Fumio; Yoshida, Hiromi; Sakaguchi, Kengo

    2003-02-01

    Two flavonoid glycosides, kaempferol 3-O-(6"-acetyl)-beta-glucopyranoside (KAG) and quercetin 3-O-(6"-acetyl)-beta-glucopyranoside (QAG), were found to be inhibitors of eukaryotic DNA polymerases from a Japanese vegetable, Petasites japonicus. These compounds inhibited the activities of mammalian replicative DNA polymerases (i.e., pol alpha, delta, and epsilon), but not other pol beta, eta, kappa, and lambda activities. KAG was a stronger inhibitor and more selective to pol alpha than QAG. The IC(50) values of KAG for pol alpha, delta, and epsilon were 41, 164, and 127 microM, respectively. The pol alpha inhibition by KAG was non-competitive with respect to both the DNA template-primer and the dNTP substrate. KAG and QAG did not influence the activities of prokaryotic DNA polymerases or other mammalian DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, human telomerase, human DNA topoisomerase I and II, T7 RNA polymerase, and bovine deoxyribonuclease I. Therefore, we concluded that these flavonoid glycosides are moderate replicative DNA polymerase inhibitors leaning more relatively to pol alpha, and could be used as chromatographic carriers to purify the DNA polymerases rather than cytotoxic agents. We then made a KAG-conjugated column such as the epoxy-activated Sepharose 6B. In the column, pol alpha was selectively adsorbed and eluted. PMID:12565887

  2. Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

    PubMed Central

    Oyofo, B A; Thornton, S A; Burr, D H; Trust, T J; Pavlovskis, O R; Guerry, P

    1992-01-01

    Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples. Images PMID:1400961

  3. Involvement of DNA polymerase beta overexpression in the malignant transformation induced by benzo[a]pyrene

    PubMed Central

    Zhao, Wei; Wu, Mei; Lai, Yanhao; Deng, Wenwen; Liu, Yuan; Zhang, Zunzhen

    2014-01-01

    Objective To explore the relationship between DNA polymerase β (pol β) overexpression and benzo[a]pyrene (BaP) carcinogenesis. Methods Firstly, mouse embryonic fibroblasts that express wild-type level of DNA polymerase β (pol β cell) and high level of pol β (pol β oe cell) were treated by various concentrations of BaP to determine genetic instability induced by BaP under differential expression levels of pol β. Secondly, malignant transformation of pol β cells by low concentration of BaP (20 μM) was determined by soft agar colony formation assay and transformation focus assay. Thirdly, the mRNA and protein levels of BaP-transformed pol β cells (named pol β-T cells) was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, and the genetic instability of these cells were examined by HPRT gene mutation assay and random amplified polymorphic DNA (RAPD) assay. Results Pol β cells were successfully transformed into malignant pol β-T cells by an exposure to low concentration of BaP for 6 months. Pol β-T cells exhibited increased levels of pol β gene expression, HPRT gene mutation frequency and polymorphisms of RAPD products that were comparable to those of pol β oe cells. Conclusion Pol β overexpression and its-associated genetic instability may play a key role in BaP carcinogenesis. PMID:23652152

  4. Structural basis for the suppression of skin cancers by DNA polymerase [eta

    SciTech Connect

    Silverstein, Timothy D.; Johnson, Robert E.; Jain, Rinku; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2010-09-13

    DNA polymerase {eta} (Pol{eta}) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of Pol{eta} (also known as POLH) in humans causes the variant form of xeroderma pigmentosum (XPV). We present the crystal structures of Saccharomyces cerevisiae Pol{eta} (also known as RAD30) in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures reveal that the ability of Pol{eta} to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the two Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. We also show by structural, biochemical and genetic analysis that the two Ts are maintained in a stable configuration in the active site via interactions with Gln55, Arg73 and Met74. Together, these features define the basis for Pol{eta}'s action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.

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

    PubMed

    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. PMID:14963331

  6. Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase.

    PubMed

    Manrao, Elizabeth A; Derrington, Ian M; Laszlo, Andrew H; Langford, Kyle W; Hopper, Matthew K; Gillgren, Nathaniel; Pavlenok, Mikhail; Niederweis, Michael; Gundlach, Jens H

    2012-04-01

    Nanopore technologies are being developed for fast and direct sequencing of single DNA molecules through detection of ionic current modulations as DNA passes through a pore's constriction. Here we demonstrate the ability to resolve changes in current that correspond to a known DNA sequence by combining the high sensitivity of a mutated form of the protein pore Mycobacterium smegmatis porin A (MspA) with phi29 DNA polymerase (DNAP), which controls the rate of DNA translocation through the pore. As phi29 DNAP synthesizes DNA and functions like a motor to pull a single-stranded template through MspA, we observe well-resolved and reproducible ionic current levels with median durations of ∼28 ms and ionic current differences of up to 40 pA. Using six different DNA sequences with readable regions 42-53 nucleotides long, we record current traces that map to the known DNA sequences. With single-nucleotide resolution and DNA translocation control, this system integrates solutions to two long-standing hurdles to nanopore sequencing. PMID:22446694

  7. Comparison of DNA polymerases for improved forensic analysis of challenging samples.

    PubMed

    Nilsson, Martina; Grånemo, Joakim; Buś, Magdalena M; Havsjö, Mikael; Allen, Marie

    2016-09-01

    Inhibitors of polymerase chain reaction (PCR) amplification often present a challenge in forensic investigations of e.g., terrorism, missing persons, sexual assaults and other criminal cases. Such inhibitors may be counteracted by dilution of the DNA extract, using different additives, and selecting an inhibitory resistant DNA polymerase. Additionally, DNA in forensic samples is often present in limited amounts and degraded, requiring special analyses of short nuclear targets or mitochondrial DNA. The present study evaluated the enzymes AmpliTaq Gold, HotStarTaq Plus, KAPA3G Plant, and KAPA2G Robust, with regard to their ability to overcome inhibitory effects. Our data showed that diluting the extracts and adding bovine serum albumin may increase the yield of the PCR product. However, the largest impact was observed when alternative enzymes were utilized, instead of the commonly used AmpliTaq Gold. KAPA2G Robust presented the highest amplification efficiency in the presence of the inhibitor ammonium nitrate. Moreover, the KAPA3G Plant enzyme had the highest efficiency in amplifying degraded DNA from old buried bone material. KAPA3G Plant and KAPA2G Robust may thus be useful for counteracting inhibitors and improving the analysis of challenging samples. PMID:27299290

  8. Rapid isolation of DNA from fresh and preserved fish scales for polymerase chain reaction.

    PubMed

    Yue, G H; Orban, L

    2001-05-01

    We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence. PMID:14961356

  9. Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-06-17

    Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems. PMID:24873435

  10. Flexible double-headed cytosine-linked 2'-deoxycytidine nucleotides. Synthesis, polymerase incorporation to DNA and interaction with DNA methyltransferases.

    PubMed

    Kielkowski, Pavel; Cahová, Hana; Pohl, Radek; Hocek, Michal

    2016-03-15

    New types of double-headed 2'-deoxycytidine 5'-O-triphosphates (dC(XC)TPs) bearing another cytosine or 5-fluorocytosine linked through a flexible propargyl, homopropargyl or pent-1-ynyl linker to position 5 were prepared by the aqueous Sonogashira cross-coupling reactions of 5-iodo-dCTP with the corresponding (fluoro)cytosine-alkynes. The modified dC(XC)TPs were good substrates for DNA polymerases and were used for enzymatic synthesis of cytosine-functionalized DNA by primer extension or PCR. The cytosine- or fluorocytosine-linked DNA probes did not significantly inhibit DNA methyltransferases and did not cross-link to these proteins. PMID:26899597

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

    PubMed

    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. PMID:27019227

  12. A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

    USGS Publications Warehouse

    Rocke, Tonie E.; Smith, Susan R.; Miyamoto, Amy; Shadduck, Daniel J.

    2002-01-01

    A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 × 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

  13. A serotype-specific polymerase chain reaction for identification of Pasteurella multocida serotype 1

    USGS Publications Warehouse

    Rocke, T.E.; Smith, S.R.; Miyamoto, A.; Shadduck, D.J.

    2002-01-01

    A serotype-specific polymerase chain reaction (PCR) assay was developed for detection and identification of Pasteurella multocida serotype 1, the causative agent of avian cholera in wild waterfowl. Arbitrarily primed PCR was used to detect DNA fragments that distinguish serotype 1 from the other 15 serotypes of P. multocida (with the exception of serotype 14). Oligonucleotide primers were constructed from these sequences, and a PCR assay was optimized and evaluated. PCR reactions consistently resulted in amplification products with reference strains 1 and 14 and all other serotype 1 strains tested, with cell numbers as low as 2.3 cells/ml. No amplification products were produced with other P. multocida serotypes or any other bacterial species tested. To compare the sensitivity and further test the specificity of this PCR assay with traditional culturing and serotyping techniques, tissue samples from 84 Pekin ducks inoculated with field strains of P. multocida and 54 wild lesser snow geese collected during an avian cholera outbreak were provided by other investigators working on avian cholera. PCR was as sensitive (58/64) as routine isolation (52/64) in detecting and identifying P. multocida serotype 1 from the livers of inoculated Pekins that became sick or died from avian cholera. No product was amplified from tissues of 20 other Pekin ducks that received serotypes other than type 1 (serotype 3, 12 ?? 3, or 10) or 12 control birds. Of the 54 snow geese necropsied and tested for P. multocida, our PCR detected and identified the bacteria from 44 compared with 45 by direct isolation. The serotype-specific PCR we developed was much faster and less labor intensive than traditional culturing and serotyping procedures and could result in diagnosis of serotype 1 pasteurellosis within 24 hr of specimen submission.

  14. DNA polymerase θ (POLQ), double-strand break repair, and cancer.

    PubMed

    Wood, Richard D; Doublié, Sylvie

    2016-08-01

    DNA polymerase theta (pol θ) is encoded in the genomes of many eukaryotes, though not in fungi. Pol θ is encoded by the POLQ gene in mammalian cells. The C-terminal third of the protein is a family A DNA polymerase with additional insertion elements relative to prokaryotic homologs. The N-terminal third is a helicase-like domain with DNA-dependent ATPase activity. Pol θ is important in the repair of genomic double-strand breaks (DSBs) from many sources. These include breaks formed by ionizing radiation and topoisomerase inhibitors, breaks arising at stalled DNA replication forks, breaks introduced during diversification steps of the mammalian immune system, and DSB induced by CRISPR-Cas9. Pol θ participates in a route of DSB repair termed "alternative end-joining" (altEJ). AltEJ is independent of the DNA binding Ku protein complex and requires DNA end resection. Pol θ is able to mediate joining of two resected 3' ends harboring DNA sequence microhomology. "Signatures" of Pol θ action during altEJ are the frequent utilization of longer microhomologies, and the insertion of additional sequences at joining sites. The mechanism of end-joining employs the ability of Pol θ to tightly grasp a 3' terminus through unique contacts in the active site, allowing extension from minimally paired primers. Pol θ is involved in controlling the frequency of chromosome translocations and preserves genome integrity by limiting large deletions. It may also play a backup role in DNA base excision repair. POLQ is a member of a cluster of similarly upregulated genes that are strongly correlated with poor clinical outcome for breast cancer, ovarian cancer and other cancer types. Inhibition of pol θ is a compelling approach for combination therapy of radiosensitization. PMID:27264557

  15. Regulation of Mutagenic DNA Polymerase V Activation in Space and Time

    PubMed Central

    Robinson, Andrew; McDonald, John P.; Caldas, Victor E. A.; Patel, Meghna; Wood, Elizabeth A.; Punter, Christiaan M.; Ghodke, Harshad; Cox, Michael M.; Woodgate, Roger; Goodman, Myron F.; van Oijen, Antoine M.

    2015-01-01

    Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template. PMID:26317348

  16. Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

    PubMed Central

    Perera, Lalith; Freudenthal, Bret D.; Beard, William A.; Shock, David D.; Pedersen, Lee G.; Wilson, Samuel H.

    2015-01-01

    DNA polymerases facilitate faithful insertion of nucleotides, a central reaction occurring during DNA replication and repair. DNA synthesis (forward reaction) is “balanced,” as dictated by the chemical equilibrium by the reverse reaction of pyrophosphorolysis. Two closely spaced divalent metal ions (catalytic and nucleotide-binding metals) provide the scaffold for these reactions. The catalytic metal lowers the pKa of O3′ of the growing primer terminus, and the nucleotide-binding metal facilitates substrate binding. Recent time-lapse crystallographic studies of DNA polymerases have identified an additional metal ion (product metal) associated with pyrophosphate formation, leading to the suggestion of its possible involvement in the reverse reaction. Here, we establish a rationale for a role of the product metal using quantum mechanical/molecular mechanical calculations of the reverse reaction in the confines of the DNA polymerase β active site. Additionally, site-directed mutagenesis identifies essential residues and metal-binding sites necessary for pyrophosphorolysis. The results indicate that the catalytic metal site must be occupied by a magnesium ion for pyrophosphorolysis to occur. Critically, the product metal site is occupied by a magnesium ion early in the pyrophosphorolysis reaction path but must be removed later. The proposed dynamic nature of the active site metal ions is consistent with crystallographic structures. The transition barrier for pyrophosphorolysis was estimated to be significantly higher than that for the forward reaction, consistent with kinetic activity measurements of the respective reactions. These observations provide a framework to understand how ions and active site changes could modulate the internal chemical equilibrium of a reaction that is central to genome stability. PMID:26351676

  17. Regulation of Mutagenic DNA Polymerase V Activation in Space and Time.

    PubMed

    Robinson, Andrew; McDonald, John P; Caldas, Victor E A; Patel, Meghna; Wood, Elizabeth A; Punter, Christiaan M; Ghodke, Harshad; Cox, Michael M; Woodgate, Roger; Goodman, Myron F; van Oijen, Antoine M

    2015-08-01

    Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD'2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD'. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD'2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template. PMID:26317348

  18. Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ

    PubMed Central

    Yoon, Jung-Hoon; Park, Jeseong; Conde, Juan; Wakamiya, Maki; Prakash, Louise; Prakash, Satya

    2015-01-01

    Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans. PMID:26680302

  19. Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ.

    PubMed

    Yoon, Jung-Hoon; Park, Jeseong; Conde, Juan; Wakamiya, Maki; Prakash, Louise; Prakash, Satya

    2015-12-15

    Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions; however, little is known about the protein factors that affect their function in human cells. In yeast, Rev1 plays a noncatalytic role as an indispensable component of Polζ, and Polζ together with Rev1 mediates a highly mutagenic mode of TLS. However, how Rev1 functions in TLS and mutagenesis in human cells has remained unclear. Here we determined the role of Rev1 in TLS opposite UV lesions in human and mouse fibroblasts and showed that Rev1 is indispensable for TLS mediated by Polη, Polι, and Polκ but is not required for TLS by Polζ. In contrast to its role in mutagenic TLS in yeast, Rev1 promotes predominantly error-free TLS opposite UV lesions in humans. The identification of Rev1 as an indispensable scaffolding component for Polη, Polι, and Polκ, which function in TLS in highly specialized ways opposite a diverse array of DNA lesions and act in a predominantly error-free manner, implicates a crucial role for Rev1 in the maintenance of genome stability in humans. PMID:26680302

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

    PubMed Central

    Kim, Sung-Kun; Castro, Aaron; Kim, Edward S.; Dinkel, Austin P.; Liu, Xiaoyun; Castro, Miguel

    2016-01-01

    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. PMID:26820310

  1. Structure of Human DNL Polymerase k Inserting dATP Opposite an 8-OxoG DNA Lesion

    SciTech Connect

    Vasquez-Del Carpio, R.; Silverstein, T; Lone, S; Swan, M; Choudhury, J; Johnson, R; Pratkash, S; Aggarwal, A

    2009-01-01

    The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Pol? is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Pol? is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases.

  2. Rescue of Newcastle disease virus from cloned cDNA using an RNA polymerase II promoter.

    PubMed

    Li, Bao-Yu; Li, Xue-Rui; Lan, Xi; Yin, Xiang-Pin; Li, Zhi-Yong; Yang, Bin; Liu, Ji-Xing

    2011-06-01

    A new system was developed to improve the efficiency and simplify the procedure of recovery of Newcastle disease virus (NDV) from cloned cDNA. A full-length cDNA clone of mesogenic NDV vaccine strain Mukteswar was assembled from five subgenomic cDNA fragments and cloned into a plasmid allowing transcription driven by cellular RNA polymerase II. The full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequences, resulted in the synthesis of antigenomic RNA with exact termini. Without supplying T7 RNA polymerase, infectious NDV could be generated efficiently in some eukaryotic cell lines by simultaneous transcription of antigenomic RNA from the full-length plasmid and expression of NP, P and L proteins from helper plasmids introduced by cotransfection. The efficiency of recovery with the conventional T7 promoter system based on BRS-T7 cells and the cytomegalovirus (CMV) promoter system was compared, and the results demonstrate that the new system facilitates the generation of recombinant NDV and more efficient than the T7 rescue system using BRS-T7. PMID:21327786

  3. A Specific Qualitative Detection Method for Peanut (Arachis Hypogagea) in Foods Using Polymerase Chain Reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A qualitative method for detection of peanuts in foods using polymerase chain reaction was developed. A universal primer pair CP 03-5 /CP 03-3 was designed to confirm the validity of the DNAs for PCR. The plant-specific amplified fragments were detected from 13 kinds of plants using the universal pr...

  4. A Specific Qualitative Detection Method for Peanut (Arachis Hypogaea) in Foods Using Polymerase Chain Reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a qualitative detection method for peanuts in foods using polymerase chain reaction (PCR). We designed a universal primer pair CP 03-5’/ CP 03-3’ to confirm the validity of the DNAs for PCR. The plant specific amplified fragments were detected from 13 kinds of plants using the universal...

  5. Electrochemical Branched-DNA Assay for Polymerase Chain Reaction-Free Detection and Quantification of Oncogenes in Messenger RNA

    SciTech Connect

    Lee, Ai Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

    2008-12-01

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcript in the population of messenger RNA (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify targets 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-napthyl-phosphate. The specificity and sensitivity of assay enabled direct detection of target transcript in as little as 4.6 ng mRNA 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 transcript in total mRNA population. The approach thus provides a simple, sensitive, accurate and quantitative tool alternate to the RQ-PCR for early disease diagnosis.

  6. Evidence for the kinetic partitioning of polymerase activity on G-quadruplex DNA

    PubMed Central

    Eddy, Sarah; Maddukuri, Leena; Ketkar, Amit; Zafar, Maroof K.; Henninger, Erin E.; Pursell, Zachary F.; Eoff, Robert L.

    2015-01-01

    We have investigated the action of the human DNA polymerase epsilon (hpol ε) and eta (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA relative to non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with near equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain greater than 25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~100-fold more accurate than hpol ε when comparing the mis-insertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model where a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication. PMID:25903680

  7. Deregulated expression of DNA polymerase β is involved in the progression of genomic instability

    PubMed Central

    Luo, Qingying; Lai, Yanhao; Liu, Shukun; Wu, Mei; Liu, Yuan; Zhang, Zunzhen

    2013-01-01

    Deregulated expression of DNA polymerase beta (pol β) has been implicated in genomic instability that leads to tumorigenesis, yet the mechanisms underlying the pol β-mediated genetic instability remain elusive. In this study, we investigated the roles of deregulated expression of pol β in spontaneous and xenobiotic-induced genetic instability using mouse embryonic fibroblasts (MEFs) that express distinct pol β levels (wild-type, null and over-expression) as a model system. Three genetic instability endpoints, DNA strand breaks, chromosome breakage and gene mutation, were examined under various expression levels of pol β by comet assay, micronuclei test and hprt mutation assay. Our results demonstrate that neither pol β deficiency nor pol β over-expression is sufficient for accumulation of spontaneous DNA damage that promotes a hyper-proliferation phenotype. However, pol β null cells exhibit increased sensitivity to exogenous DNA damaging agents with increased genomic instability compared with pol β wild-type and over-expression cells. This finding suggests that a pol β deficiency may underlie genomic instability induced by exogenous DNA damaging agents. Interestingly, pol β over-expression cells exhibit less chromosomal or DNA damage, but display a higher hprt mutation frequency upon methyl methanesulfonate exposure compared with the other two cell types. Our results therefore indicate that an excessive amount of pol β may promote genomic instability, presumably through an error-prone repair response, although it enhances overall BER capacity for induced DNA damage. PMID:22576475

  8. Cytosolic DNA triggers mitochondrial apoptosis via DNA damage signaling proteins independently of AIM2 and RNA polymerase III.

    PubMed

    Wenzel, Michael; Wunderlich, Michael; Besch, Robert; Poeck, Hendrik; Willms, Simone; Schwantes, Astrid; Kremer, Melanie; Sutter, Gerd; Endres, Stefan; Schmidt, Andreas; Rothenfusser, Simon

    2012-01-01

    A key host response to limit microbial spread is the induction of cell death when foreign nucleic acids are sensed within infected cells. In mouse macrophages, transfected DNA or infection with modified vaccinia virus Ankara (MVA) can trigger cell death via the absent in melanoma 2 (AIM2) inflammasome. In this article, we show that nonmyeloid human cell types lacking a functional AIM2 inflammasome still die in response to cytosolic delivery of different DNAs or infection with MVA. This cell death induced by foreign DNA is independent of caspase-8 and carries features of mitochondrial apoptosis: dependence on BAX, APAF-1, and caspase-9. Although it does not require the IFN pathway known to be triggered by infection with MVA or transfected DNA via polymerase III and retinoid acid-induced gene I-like helicases, it shows a strong dependence on components of the DNA damage signaling pathway: cytosolic delivery of DNA or infection with MVA leads to phosphorylation of p53 (serines 15 and 46) and autophosphorylation of ataxia telangiectasia mutated (ATM); depleting p53 or ATM with small interfering RNA or inhibiting the ATM/ATM-related kinase family by caffeine strongly reduces apoptosis. Taken together, our findings suggest that a pathway activating DNA damage signaling plays an important independent role in detecting intracellular foreign DNA, thereby complementing the induction of IFN and activation of the AIM2 inflammasome. PMID:22140256

  9. Pausing of RNA polymerase II Disrupts DNA-specified Nucleosome Organization to Enable Precise Gene Regulation

    PubMed Central

    Gilchrist, Daniel A.; Santos, Gilberto Dos; Fargo, David C.; Xie, Bin; Gao, Yuan; Li, Leping; Adelman, Karen

    2010-01-01

    Metazoan transcription is controlled either through coordinated recruitment of transcription machinery to the gene promoter, or through regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a striking difference between genes that use these distinct regulatory strategies lies in the “default” chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly-regulated genes whose sequences inherently disfavor nucleosome formation within the gene, but favor occlusion of the promoter by nucleosomes. In contrast, housekeeping genes that lack pronounced Pol II pausing show higher nucleosome occupancy downstream, but their promoters are deprived of nucleosomes regardless of polymerase binding. Our results indicate that a key role of paused Pol II is to compete with nucleosomes for occupancy of highly-regulated promoters, thereby preventing the formation of repressive chromatin architecture to facilitate further or future gene activation. PMID:21074046

  10. Oxidation State of the XRCC1 N-terminal Domain Regulates DNA Polymerase Beta Binding Affinity

    SciTech Connect

    Cuneo, M.; London, R

    2010-01-01

    Formation of a complex between the XRCC1 N-terminal domain (NTD) and DNA polymerase {beta} (Pol {beta}) is central to base excision repair of damaged DNA. Two crystal forms of XRCC1-NTD complexed with Pol {beta} have been solved, revealing that the XRCC1-NTD is able to adopt a redox-dependent alternate fold, characterized by a disulfide bond, and substantial variations of secondary structure, folding topology, and electrostatic surface. Although most of these structural changes occur distal to the interface, the oxidized XRCC1-NTD forms additional interactions with Pol {beta}, enhancing affinity by an order of magnitude. Transient disulfide bond formation is increasingly recognized as an important molecular regulatory mechanism. The results presented here suggest a paradigm in DNA repair in which the redox state of a scaffolding protein plays an active role in organizing the repair complex.

  11. DNA polymerase β deficiency leads to neurodegeneration and exacerbates Alzheimer disease phenotypes.

    PubMed

    Sykora, Peter; Misiak, Magdalena; Wang, Yue; Ghosh, Somnath; Leandro, Giovana S; Liu, Dong; Tian, Jane; Baptiste, Beverly A; Cong, Wei-Na; Brenerman, Boris M; Fang, Evandro; Becker, Kevin G; Hamilton, Royce J; Chigurupati, Soumya; Zhang, Yongqing; Egan, Josephine M; Croteau, Deborah L; Wilson, David M; Mattson, Mark P; Bohr, Vilhelm A

    2015-01-01

    We explore the role of DNA damage processing in the progression of cognitive decline by creating a new mouse model. The new model is a cross of a common Alzheimer's disease (AD) mouse (3xTgAD), with a mouse that is heterozygous for the critical DNA base excision repair enzyme, DNA polymerase β. A reduction of this enzyme causes neurodegeneration and aggravates the AD features of the 3xTgAD mouse, inducing neuronal dysfunction, cell death and impairing memory and synaptic plasticity. Transcriptional profiling revealed remarkable similarities in gene expression alterations in brain tissue of human AD patients and 3xTg/Polβ(+/-) mice including abnormalities suggestive of impaired cellular bioenergetics. Our findings demonstrate that a modest decrement in base excision repair capacity can render the brain more vulnerable to AD-related molecular and cellular alterations. PMID:25552414

  12. DNA polymerase β deficiency leads to neurodegeneration and exacerbates Alzheimer disease phenotypes

    PubMed Central

    Sykora, Peter; Misiak, Magdalena; Wang, Yue; Ghosh, Somnath; Leandro, Giovana S.; Liu, Dong; Tian, Jane; Baptiste, Beverly A.; Cong, Wei-Na; Brenerman, Boris M.; Fang, Evandro; Becker, Kevin G.; Hamilton, Royce J.; Chigurupati, Soumya; Zhang, Yongqing; Egan, Josephine M.; Croteau, Deborah L.; Wilson, David M.; Mattson, Mark P.; Bohr, Vilhelm A.

    2015-01-01

    We explore the role of DNA damage processing in the progression of cognitive decline by creating a new mouse model. The new model is a cross of a common Alzheimer's disease (AD) mouse (3xTgAD), with a mouse that is heterozygous for the critical DNA base excision repair enzyme, DNA polymerase β. A reduction of this enzyme causes neurodegeneration and aggravates the AD features of the 3xTgAD mouse, inducing neuronal dysfunction, cell death and impairing memory and synaptic plasticity. Transcriptional profiling revealed remarkable similarities in gene expression alterations in brain tissue of human AD patients and 3xTg/Polβ+/− mice including abnormalities suggestive of impaired cellular bioenergetics. Our findings demonstrate that a modest decrement in base excision repair capacity can render the brain more vulnerable to AD-related molecular and cellular alterations. PMID:25552414

  13. Arabidopsis DNA polymerase lambda mutant is mildly sensitive to DNA double strand breaks but defective in integration of a transgene

    PubMed Central

    Furukawa, Tomoyuki; Angelis, Karel J.; Britt, Anne B.

    2015-01-01

    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. PMID:26074930

  14. Structure of the DNA-binding and RNA polymerase-binding region of transcription antitermination factor λQ

    PubMed Central

    Vorobiev, Sergey M.; Gensler, Yocheved; Vahedian-Movahed, Hanif; Seetharaman, Jayaraman; Su, Min; Huang, Janet Y.; Xiao, Rong; Kornhaber, Gregory; Montelione, Gaetano T.; Tong, Liang; Ebright, Richard H.; Nickels, Bryce E.

    2014-01-01

    SUMMARY The bacteriophage λ Q protein is a transcription antitermination factor that controls expression of the phage late genes as a stable component of the transcription elongation complex. To join the elongation complex, λQ binds a specific DNA sequence element and interacts with RNA polymerase that is paused during early elongation. λQ’s interaction with the paused early elongation complex involves interactions between λQ and two regions of RNA polymerase: region 4 of the σ70 subunit and the flap domain of the β subunit. We present the 2.1 Å resolution crystal structure of a portion of λQ containing determinants for interaction with DNA, interaction with region 4 of σ70, and interaction with the β flap. The structure provides a framework for interpreting prior genetic and biochemical analysis and sets the stage for future structural studies to elucidate the mechanism by which λQ alters the functional properties of the transcription elongation complex. PMID:24440517

  15. Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase

    SciTech Connect

    Kirouac, Kevin N.; Ling, Hong

    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.

  16. Inhibition of non-templated nucleotide addition by DNA polymerases in primer extension using twisted intercalating nucleic acid modified templates.

    PubMed

    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. PMID:26707394

  17. Biochemical properties of the stimulatory activity of DNA polymerase alpha by the hyper-phosphorylated retinoblastoma protein.

    PubMed

    Takemura, Masaharu

    2002-06-01

    Previously, my colleagues and I have reported that the immunopurified hyper-phosphorylated retinoblastoma protein (ppRb) stimulates the activity of DNA polymerase alpha. I describe here the biochemical characteristics of this stimulatory activity. DNA polymerase alpha-stimulatory activity of ppRb was most remarkable when using activated DNA as a template-primer, rather than using poly(dT)-(rA)(10), poly(dA)-(dT)(12-18), and so on. Kinetic analysis showed that there was no significant difference in K(m) value for deoxyribonucleotides of DNA polymerase alpha in the presence of ppRb. Adding ppRb resulted in the overcoming pause site on the template, but did not affect the rate of misincorporation of incorrect deoxyribonucleotides. By adding ppRb, the optimal concentration of template-primer was shifted to a higher region, but not using M13 singly primed DNA. The ppRb seemed to assist the process that DNA polymerase alpha changed its conformation resulting in appropriate enzyme activity. These results suggest that ppRb affects both template-primer and DNA polymerase alpha and makes appropriate circumstances for the enzyme reaction. PMID:12049795

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

    SciTech Connect

    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. Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

    DOE PAGESBeta

    Walmacq, Celine; Wang, Lanfeng; Chong, Jenny; Scibelli, Kathleen; Lubkowska, Lucyna; Gnatt, Averell; Brooks, Philip J.; Wang, Dong; Kashlev, Mikhail

    2015-01-20

    In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5´-templating base, indicating that it derives from nontemplated synthesismore » according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. The translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, trans-lesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.« less

  20. Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions

    SciTech Connect

    Walmacq, Celine; Wang, Lanfeng; Chong, Jenny; Scibelli, Kathleen; Lubkowska, Lucyna; Gnatt, Averell; Brooks, Philip J.; Wang, Dong; Kashlev, Mikhail

    2015-01-20

    In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5´-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. The translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, trans-lesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

  1. Human DNA Polymerase ϵ Is Able to Efficiently Extend from Multiple Consecutive Ribonucleotides*

    PubMed Central

    Göksenin, A. Yasemin; Zahurancik, Walter; LeCompte, Kimberly G.; Taggart, David J.; Suo, Zucai; Pursell, Zachary F.

    2012-01-01

    Replicative DNA polymerases (Pols) help to maintain the high fidelity of replication in large part through their strong selectivity against mispaired deoxyribonucleotides. It has recently been demonstrated that several replicative Pols from yeast have surprisingly low selectivity for deoxyribonucleotides over their analogous ribonucleotides. In human cells, ribonucleotides are found in great abundance over deoxyribonucleotides, raising the possibility that ribonucleotides are incorporated in the human genome at significant levels during normal cellular functions. To address this possibility, the ability of human DNA polymerase ϵ to incorporate ribonucleotides was tested. At physiological concentrations of nucleotides, human Pol ϵ readily inserts and extends from incorporated ribonucleotides. Almost half of inserted ribonucleotides escape proofreading by 3′ → 5′ exonuclease-proficient Pol ϵ, indicating that ribonucleotide incorporation by Pol ϵ is likely a significant event in human cells. Human Pol ϵ is also efficient at extending from primers terminating in up to five consecutive ribonucleotides. This efficient extension appears to result from reduced exonuclease activity on primers containing consecutive 3′-terminal ribonucleotides. These biochemical properties suggest that Pol ϵ is a likely source of ribonucleotides in human genomic DNA. PMID:23093410

  2. Beta-sitosterol-3-O-beta-D-glucopyranoside: a eukaryotic DNA polymerase lambda inhibitor.

    PubMed

    Mizushina, Yoshiyuki; Nakanishi, Rumi; Kuriyama, Isoko; Kamiya, Kohei; Satake, Toshiko; Shimazaki, Noriko; Koiwai, Osamu; Uchiyama, Yukinobu; Yonezawa, Yuko; Takemura, Masaharu; Sakaguchi, Kengo; Yoshida, Hiromi

    2006-05-01

    Beta-sitosterol-3-O-beta-D-glucopyranoside (compound 1), a steroidal glycoside isolated from onion (Allium cepa L.) selectively inhibited the activity of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta and epsilon, but also showed no effect even on the activity of pol beta which is thought to have a very similar three-dimensional structure to the pol beta-like region of pol lambda. Since parts of compound 1 such as beta-sitosterol (compound 2) and D-glucose (compound 3) did not influence the activities of any enzymes tested, the converted structure of compounds 2 and 3 might be important for pol lambda inhibition. The inhibitory effect of compound 1 on both intact pol lambda (i.e. residues 1-575) and a truncated pol lambda lacking the N-terminal BRCA1 C-terminus (BRCT) domain (133-575, del-1 pol lambda) was dose-dependent, and 50% inhibition was observed at a concentration of 9.1 and 5.4 microM, respectively. The compound 1-induced inhibition of del-1 pol lambda activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. On the basis of these results, the pol lambda inhibitory mechanism of compound 1 is discussed. PMID:16621516

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

    DOE PAGESBeta

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

    2010-01-01

    Humore » man DNA polymerase η (HsPol η ) 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 ApPol η from the thermophilic worm Alvinella pompejana , which inhabits deep-sea hydrothermal vent chimneys. ApPol η shares sequence homology with HsPol η 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 ApPol η is more thermostable than HsPol η , 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, ApPol η provides a robust, human-like Pol η that is more active after exposure to high temperatures and organic solvents.« less