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Joann B. Sweasy - One of the best experts on this subject based on the ideXlab platform.
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DNA Polymerase Beta participates in DNA end joining
Nucleic Acids Research, 2018Co-Authors: Sreerupa Ray, Gregory A Breuer, Michelle Deveaux, Daniel Zelterman, Ranjit S Bindra, Joann B. SweasyAbstract:DNA double strand breaks (DSBs) are one of the most deleterious lesions and if left unrepaired, they lead to cell death, genomic instability and carcinogenesis. Cells combat DSBs by two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred to as alternative NHEJ (aNHEJ), which joins ends but results in deletions and insertions. NHEJ requires processing enzymes including nucleases and Polymerases, although the roles of these enzymes are poorly understood. Emerging evidence indicates that X family DNA Polymerases lambda (Pol λ) and mu (Pol μ) promote DNA end-joining. Here, we show that DNA Polymerase Beta (Pol β), another member of the X family of DNA Polymerases, plays a role in aNHEJ. In the absence of DNA Pol β, fewer small deletions are observed. In addition, depletion of Pol β results in cellular sensitivity to bleomycin and DNA protein kinase catalytic subunit inhibitors due to defective repair of DSBs. In summary, our results indicate that Pol β in functions in aNHEJ and provide mechanistic insight into its role in this process.
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abstract 2492 DNA Polymerase Beta participates in DNA end joining
Cancer Research, 2017Co-Authors: Sreerupa Ray, Gregory A Breuer, Michelle Deveaux, Daniel Zelterman, Ranjit S Bindra, Joann B. SweasyAbstract:DNA double strand breaks (DSBs) are one of the most deleterious lesions. If left unrepaired, DSBs lead to genomic instability and carcinogenesis. The cells combat DSBs by two classical pathways that include homologous recombination (HR) which requires the sister chromatid for sequence homology, and non-homologous end-joining (NHEJ), wherein the two DNA ends are re-joined. Recently a back-up NHEJ pathway has been reported and is referred non-canonical NHEJ which has been given many names, such as alternative NHEJ or microhomology-mediated end joining (MMEJ). The enzymatic mechanisms of non-canonical NHEJ are not well defined. NHEJ requires processing enzymes including nucleases and Polymerases, although the roles of these enzymes in the pathway are poorly understood. Recent studies have implicated a role for DNA Polymerase theta (Pol θ), an A-family Polymerase is essential for non-canonical NHEJ pathway. Emerging evidence indicates X-family Polymerases, mammalian DNA Polymerases lambda (λ) and mu (μ) and yeast Pol 4 promote DNA end-joining. Our laboratory has recently provided evidence for a role for DNA Polymerase Beta (Pol β), another X-family Polymerase, in V(D)J recombination, a process that requires end-joining. Here, using a recently developed fluorescence based assay that monitors non-canonical NHEJ and HR, we provide evidence that Pol β plays a role in the non-canonical NHEJ process. DNA sequencing at the break point junctions revealed Pol β-depleted cells have fewer small deletions than control cells, but significantly greater numbers of insertions and large deletions. We further demonstrate that Pol β-depleted cells have increased sensitivity to DNA damaging agents that induce double-strand breaks and that there is persistent accumulation of DSBs in these cells. In combination, our results suggest that Pol β is critical for double-strand break repair. Citation Format: Sreerupa Ray, Michelle DeVeaux, Gregory Breuer, Ranjit Bindra, Daniel Zelterman, Joann Sweasy. DNA Polymerase Beta participates in DNA end-joining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2492. doi:10.1158/1538-7445.AM2017-2492
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DNA Polymerase Beta germline variant confers cellular response to cisplatin therapy
Molecular Cancer Research, 2017Co-Authors: Antonia A Nemec, Michelle Deveaux, Daniel Zelterman, Laura Abriola, Jane S Merkel, Elisa De Stanchina, Peter M Glazer, Joann B. SweasyAbstract:Resistance to cancer chemotherapies leads to deadly consequences, yet current research focuses only on the roles of somatically acquired mutations in this resistance. The mutational status of the germline is also likely to play a role in the way cells respond to chemotherapy. The carrier status for the POLB rs3136797 germline mutation encoding P242R DNA Polymerase Beta (Pol β) is associated with poor prognosis for lung cancer, specifically in response to treatment with cisplatin. Here, it is revealed that the P242R mutation is sufficient to promote resistance to cisplatin in human cells and in mouse xenografts. Mechanistically, P242R Pol β acts as a translesion Polymerase and prefers to insert the correct nucleotide opposite cisplatin intrastrand cross-links, leading to the activation of the nucleotide excision repair (NER) pathway, removal of crosslinks, and resistance to cisplatin. In contrast, wild-type (WT) Pol β preferentially inserts the incorrect nucleotide initiating mismatch repair and cell death. Importantly, in a mouse xenograft model, tumors derived from lung cancer cells expressing WT Pol β displayed a slower rate of growth when treated with cisplatin, whereas tumors expressing P242R Pol β had no response to cisplatin. Pol β is critical for mediating crosstalk in response to cisplatin. The current data strongly suggest that the status of Pol β influences cellular responses to crosslinking agents and that Pol β is a promising biomarker to predict responses to specific chemotherapies. Finally, these results highlight that the genetic status of the germline is a critical factor in the response to cancer treatment. Implications: Pol β has prognostic biomarker potential in the treatment of cancer with cisplatin and perhaps other intrastrand crosslinking agents. Mol Cancer Res; 15(3); 269–80. ©2017 AACR.
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abstract 3026 gastric cancer associated variant of DNA Polymerase Beta leu22pro induce genomic instability and cellular transformation
Cancer Research, 2015Co-Authors: Dawit Kidane, Jenna Rozacky, Joann B. SweasyAbstract:Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA DNA Polymerase Beta (Pol β) is a key enzyme for the protection of 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 and the Polymerase domain at subsequent steps fills the gaps. Previously, we demonstrated that gastric cancer-associated variant of pol β (Leu22Pro (L22P)) lacks dRP lyase function in-vitro. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which the normal function of dRP lyase of DNA Polymerase Beta is critical to maintain global replication fork rates in normal gastric epithelial cells. We report that replication fork rates in L22P expressing cells are on average half of those observed with wild-type cells. In addition, we show that L22P variant promotes replication dependent double strand breaks that may likely responsible for chromosomal aberrations. These data may implicate normal function of dRP lyase is critical to stabilize replication fork stall and prevent replication fork collapse to DNA double strand breaks. Citation Format: Dawit Kidane, Jenna Rozacky, Joann B. Sweasy. Gastric cancer associated variant of DNA Polymerase Beta (Leu22Pro) induce genomic instability and cellular transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3026. doi:10.1158/1538-7445.AM2015-3026
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gastric cancer associated variant of DNA Polymerase Beta leu22pro promotes DNA replication associated double strand breaks
Oncotarget, 2015Co-Authors: Jenna Rozacky, Joann B. Sweasy, Antonia A Nemec, Dawit KidaneAbstract:// Jenna Rozacky 1 , Antoni A. Nemec 2 , Joann B. Sweasy 3 and Dawit Kidane 1 1 Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Dell Pediatric Research Institute, Austin, TX, USA 2 Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL, USA 3 Departments of Therapeutic Radiology and Genetics, The Yale Comprehensive Cancer Center, New Haven CT, USA Correspondence to: Dawit Kidane, email: // Keywords : DNA Polymerase Beta, gastric cancer Received : April 24, 2015 Accepted : May 31, 2015 Published : June 10, 2015 Abstract 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.
Samuel H. Wilson - One of the best experts on this subject based on the ideXlab platform.
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DNA Polymerase Beta and other gap filling enzymes in mammalian base excision repair
The Enzymes, 2019Co-Authors: William A Beard, Samuel H. WilsonAbstract:DNA Polymerase β plays a central role in the base excision DNA repair pathway that cleanses the genome of apurinic/apyrimidinic (AP) sites. AP sites arise in DNA from spontaneous base loss and DNA damage-specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and damaged base. AP sites are deleterious lesions because they can be mutagenic and/or cytotoxic. DNA Polymerase β contributes two enzymatic activities, DNA synthesis and lyase, during the repair of AP sites; these activities reside on carboxyl- and amino-terminal domains, respectively. Accordingly, its cellular, structural, and kinetic attributes have been extensively characterized and it serves as model enzyme for the nucleotidyl transferase reaction utilized by other replicative, repair, and trans-lesion DNA Polymerases.
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DNA Polymerase Beta substrate specificity side chain modulation of the a rule
Journal of Biological Chemistry, 2009Co-Authors: William A Beard, Lars C Pedersen, Vinod K Batra, David D Shock, Samuel H. WilsonAbstract:Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA Polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA Polymerase (pol) β, a family X Polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol β insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol β bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed Polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.
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Gastrointestinal hyperplasia with altered expression of DNA Polymerase Beta.
Public Library of Science (PLoS), 2009Co-Authors: Katsuhiko Yoshizawa, Elena Jelezcova, Samuel H. Wilson, Ashley R Brown, Julie F Foley, Abraham Nyska, Xiangli Cui, Lorne J Hofseth, Robert M Maronpot, Antonia R SepulvedaAbstract:BACKGROUND:Altered expression of DNA Polymerase Beta (Pol Beta) has been documented in a large percentage of human tumors. However, tumor prevalence or predisposition resulting from Pol Beta over-expression has not yet been evaluated in a mouse model. METHODOLOGY/PRINCIPAL FINDINGS:We have recently developed a novel transgenic mouse model that over-expresses Pol Beta. These mice present with an elevated incidence of spontaneous histologic lesions, including cataracts, hyperplasia of Brunner's gland and mucosal hyperplasia in the duodenum. In addition, osteogenic tumors in mice tails, such as osteoma and osteosarcoma were detected. This is the first report of elevated tumor incidence in a mouse model of Pol Beta over-expression. These findings prompted an evaluation of human gastrointestinal tumors with regard to Pol Beta expression. We observed elevated expression of Pol Beta in stomach adenomas and thyroid follicular carcinomas, but reduced Pol Beta expression in esophageal adenocarcinomas and squamous carcinomas. CONCLUSIONS/SIGNIFICANCE:These data support the hypothesis that balanced and proficient base excision repair protein expression and base excision repair capacity is required for genome stability and protection from hyperplasia and tumor formation
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mutagenesis is elevated in male germ cells obtained from DNA Polymerase Beta heterozygous mice
Biology of Reproduction, 2008Co-Authors: Diwi Allen, Robert W. Sobol, Samuel H. Wilson, Damon C Herbert, Alex C Mcmahan, Vladimir Rotrekl, Christi A WalterAbstract:Gametes carry the DNA that will direct the development of the next generation. By compromising genetic integrity, DNA damage and mutagenesis threaten the ability of gametes to fulfill their biological function. DNA repair pathways function in germ cells and serve to ameliorate much DNA damage and prevent mutagenesis. High base excision repair (BER) activity is documented for spermatogenic cells. DNA Polymerase-Beta (POLB) is required for the short-patch BER pathway. Because mice homozygous null for the Polb gene die soon after birth, mice heterozygous for Polb were used to examine the extent to which POLB contributes to maintaining spermatogenic genomic integrity in vivo. POLB protein levels were reduced only in mixed spermatogenic cells. In vitro short-patch BER activity assays revealed that spermatogenic cell nuclear extracts obtained from Polb heterozygous mice had one third the BER activity of age-matched control mice. Polb heterozygosity had no effect on the BER activities of somatic tissues tested. The Polb heterozygous mouse line was crossed with the lacI transgenic Big Blue mouse line to assess mutant frequency. The spontaneous mutant frequency for mixed spermatogenic cells prepared from Polb heterozygous mice was 2-fold greater than that of wild-type controls, but no significant effect was found among the somatic tissues tested. These results demonstrate that normal POLB abundance is necessary for normal BER activity, which is critical in maintaining a low germline mutant frequency. Notably, spermatogenic cells respond differently than somatic cells to Polb haploinsufficiency..
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xrcc1 and DNA Polymerase Beta in cellular protection against cytotoxic DNA single strand breaks
Cell Research, 2008Co-Authors: Julie K Horton, Mary A Watson, Donna F Stefanick, Daniel T Shaughnessy, Jack A Taylor, Samuel H. WilsonAbstract:Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5'-phosphate and 3'-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1(-/-) mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA Polymerase Beta (pol Beta) is specific to this pathway, whereas pol Beta is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1(-/-), and to a lesser extent in pol Beta(-/-) cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol Beta(-/-) cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1(-/-) cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.
Rajendra Prasad - One of the best experts on this subject based on the ideXlab platform.
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structural insight into the DNA Polymerase Beta deoxyribose phosphate lyase mechanism
DNA Repair, 2005Co-Authors: Rajendra Prasad, Vinod K Batra, Xiaoping Yang, Joseph M Krahn, Lars C Pedersen, William A Beard, Samuel H. WilsonAbstract:A large number of biochemical and genetic studies have demonstrated the involvement of DNA Polymerase Beta (Pol Beta) in mammalian base excision repair (BER). Pol Beta participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5'-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol Beta complexed with 5'-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1' of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120 degrees about the dRP 3'-phosphate is required to position the epsilon-amino Lys72 close to the dRP C1'. This model also suggests that several other side chains are in position to facilitate the Beta-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity.
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8 oxodgtp incorporation by DNA Polymerase Beta is modified by active site residue asn279
Biochemistry, 2000Co-Authors: Holly Miller, Rajendra Prasad, Samuel H. Wilson, Francis Johnson, Arthur P GrollmanAbstract:To understand how the active site of a DNA Polymerase might modulate the coding of 8-oxo-7,8-dihydrodeoxyguanine (8-oxodG), we performed steady-state kinetic analyses using wild-type DNA Polymerase Beta (pol Beta) and two active-site mutants. We compared the coding of these Polymerases by calculating the ratio of efficiencies for incorporation of dATP and dCTP opposite 8-oxodG and for incorporation of 8-oxodGTP opposite dA and dC. For wild-type pol Beta, there is a 2:1 preference for incorporation of dCTP over dATP opposite 8-oxodG using a 5'-phosphorylated 4-base gap substrate. Mutation of either Asn279 or Arg283 to alanine has almost no effect on the ratio. 8-OxodGTP is preferentially incorporated opposite a template dA (24:1) by wild-type pol Beta; mutation of Asn279 to alanine results dramatic change whereby there is preferential incorporation of 8-oxodGTP opposite dC (14:1). This suggests that interactions of 8-oxodGTP with Asn279 in the Polymerase active site may alter the conformation of 8-oxodGTP and therefore alter its misincorporation.
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human DNA Polymerase Beta deoxyribose phosphate lyase substrate specificity and catalytic mechanism
Journal of Biological Chemistry, 1998Co-Authors: Rajendra Prasad, William A Beard, Phyllis R Strauss, Samuel H. WilsonAbstract:DNA Polymerase Beta (Beta-pol) cleaves the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5'-incised by AP endonuclease, resulting in a 5' 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of Beta-pol and with the entire 39-kDa Polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of Beta-pol are similar. However, kcat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5' dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of Beta-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5'-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.
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requirement of mammalian DNA Polymerase Beta in base excision repair
Nature, 1996Co-Authors: Robert W. Sobol, Rajendra Prasad, Julie K Horton, Ralf Kuhn, Hua Gu, Rakesh K Singhal, Klaus Rajewsky, Samuel H. WilsonAbstract:Synthesis of DNA by DNA Polymerase-Beta is distributive on single-stranded DNA templates, but short DNA gaps with a 5' PO4 in the gap are filled processively to completion. In vitro studies have suggested a role of Beta-Polymerase in different types of DNA repair. However, the significance of these studies to the in vivo role of Beta-Polymerase has remained unclear. Because genetic studies are essential for determining the physiological role of a gene, we established embryonic fibroblast cell lines homozygous for a deletion mutation in the gene encoding DNA Polymerase-Beta. Extracts from these cell lines were found to be defective in uracil-initiated base-excision repair. The Beta-Polymerase-deleted cells are normal in viability and growth characteristics, although they exhibit increased sensitivity to monofunctional DNA-alkylating agents, but not to other DNA-damaging agents. Both the deficiency in base-excision repair and hypersensitivity to DNA-alkylating agents are rescued following stable transfection with a wild-type Beta-Polymerase minitransgene. These studies demonstrate that Beta-Polymerase functions specifically in base-excision repair in vivo.
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studies of gapped DNA substrate binding by mammalian DNA Polymerase Beta dependence on 5 phosphate group
Journal of Biological Chemistry, 1994Co-Authors: Rajendra Prasad, William A Beard, Samuel H. WilsonAbstract:Abstract Purified mammalian DNA Polymerase Beta (Beta-pol) fills short gaps of up to 6 nucleotides by a processive mechanism, and this gap-filling activity requires a PO4 group on the 5'-side of the gap (Singhal, R. K., and Wilson, S. H. (1993) J. Biol. Chem. 268, 15906-15911). To assess details of bimolecular binding between Beta-pol and a 5-nucleotide (nt) gapped radiolabeled heteropolymeric DNA substrate, Beta-pol.DNA complexes were formed, photochemically cross-linked using UV light, and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A 39-nt template was annealed with two 17-mer oligonucleotides, generating a 5-nt gap. The results indicate that Beta-pol binds to both the template and primer strands, and binding is strongly enhanced by a 5'-PO4 on the downstream oligonucleotide, even though little cross-linking is observed to this oligonucleotide. The results suggest that Beta-pol recognizes the 5'-side of a long single-stranded gap in DNA, provided it contains a 5'-PO4. Additional Beta-pol.DNA binding measurements were performed using a competition assay to assess the ability of heteropolymeric DNA to inhibit synthesis on a homopolymeric template-primer system. The results indicate that in addition to the 5'-PO4, the length of the single-stranded template nucleic acid adjacent to the 5'-PO4 is also important for tight binding. Proteolysis of the cross-linked Beta-pol.DNA complex with trypsin resulted in a single radiolabeled tryptic product corresponding to nucleic acid cross-linked to the 8-kDa domain. The results demonstrate that the role of the 8-kDa domain is to direct Beta-pol binding to the phosphorylated 5'-position in gapped DNA substrates.
William A Beard - One of the best experts on this subject based on the ideXlab platform.
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DNA Polymerase Beta and other gap filling enzymes in mammalian base excision repair
The Enzymes, 2019Co-Authors: William A Beard, Samuel H. WilsonAbstract:DNA Polymerase β plays a central role in the base excision DNA repair pathway that cleanses the genome of apurinic/apyrimidinic (AP) sites. AP sites arise in DNA from spontaneous base loss and DNA damage-specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and damaged base. AP sites are deleterious lesions because they can be mutagenic and/or cytotoxic. DNA Polymerase β contributes two enzymatic activities, DNA synthesis and lyase, during the repair of AP sites; these activities reside on carboxyl- and amino-terminal domains, respectively. Accordingly, its cellular, structural, and kinetic attributes have been extensively characterized and it serves as model enzyme for the nucleotidyl transferase reaction utilized by other replicative, repair, and trans-lesion DNA Polymerases.
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DNA Polymerase Beta substrate specificity side chain modulation of the a rule
Journal of Biological Chemistry, 2009Co-Authors: William A Beard, Lars C Pedersen, Vinod K Batra, David D Shock, Samuel H. WilsonAbstract:Apurinic/apyrimidinic (AP) sites are continuously generated in genomic DNA. Left unrepaired, AP sites represent noninstructional premutagenic lesions that are impediments to DNA synthesis. When DNA Polymerases encounter an AP site, they generally insert dAMP. This preferential insertion is referred to as the A-rule. Crystallographic structures of DNA Polymerase (pol) β, a family X Polymerase, with active site mismatched nascent base pairs indicate that the templating (i.e. coding) base is repositioned outside of the template binding pocket thereby diminishing interactions with the incorrect incoming nucleotide. This effectively produces an abasic site because the template pocket is devoid of an instructional base. However, the template pocket is not empty; an arginine residue (Arg-283) occupies the space vacated by the templating nucleotide. In this study, we analyze the kinetics of pol β insertion opposite an AP site and show that the preferential incorporation of dAMP is lost with the R283A mutant. The crystallographic structures of pol β bound to gapped DNA with an AP site analog (tertrahydrofuran) in the gap (binary complex) and with an incoming nonhydrolyzable dATP analog (ternary complex) were solved. These structures reveal that binding of the dATP analog induces a closed Polymerase conformation, an unstable primer terminus, and an upstream shift of the templating residue even in the absence of a template base. Thus, dATP insertion opposite an abasic site and dATP misinsertions have common features.
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r Beta gamma fluoromethylene dgtp DNA ternary complex with DNA Polymerase Beta
Journal of the American Chemical Society, 2007Co-Authors: Charles E Mckenna, Vinod K Batra, Lars C Pedersen, William A Beard, Boris A Kashemirov, Thomas G Upton, Myron F Goodman, Samuel H. WilsonAbstract:β,γ-Fluoromethylene analogues of nucleotides are generally considered to be useful mimics of the natural substrates for DNA Polymerases, but direct structural evidence defining their active site interactions has not been available. In addition, the effect of introducing a new chiral center (the CHF carbon) has been unexplored. We report here structural studies of the diastereomeric β,γ-CHF analogues (R, 3; S, 4) of dGTP interacting with the active site of DNA pol β, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. The conjugation of dGMP 5‘-morpholidate with a tetrabutylammonium salt of (fluoromethylene)bisphosphonic acid (6b, prepared like its difluoro analogue 7b via fluorination of tetraisopropyl methylenebisphosphonate carbanion with Selectfluor) gives a 1:1 mixture of 3 and 4 (by 19F NMR, pH 10). The β,γ-CF2 (2) and β,γ-CH2 (1) dGTP analogues were also synthesized. Crystallization from a solution containing 3 + 4 together with a preformed DNA pol β complex o...
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structural insight into the DNA Polymerase Beta deoxyribose phosphate lyase mechanism
DNA Repair, 2005Co-Authors: Rajendra Prasad, Vinod K Batra, Xiaoping Yang, Joseph M Krahn, Lars C Pedersen, William A Beard, Samuel H. WilsonAbstract:A large number of biochemical and genetic studies have demonstrated the involvement of DNA Polymerase Beta (Pol Beta) in mammalian base excision repair (BER). Pol Beta participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5'-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol Beta complexed with 5'-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1' of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120 degrees about the dRP 3'-phosphate is required to position the epsilon-amino Lys72 close to the dRP C1'. This model also suggests that several other side chains are in position to facilitate the Beta-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity.
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minor groove interactions at the DNA Polymerase Beta active site modulate single base deletion error rates
Journal of Biological Chemistry, 2000Co-Authors: Wendy P Osheroff, Samuel H. Wilson, William A Beard, Shang Yin, Thomas A KunkelAbstract:The structures of open and closed conformations of DNA Polymerase Beta (pol Beta) suggests that the rate of single-nucleotide deletions during synthesis may be modulated by interactions in the DNA minor groove that align the templating base with the incoming dNTP. To test this hypothesis, we measured the single-base deletion error rates of wild-type pol Beta and lysine and alanine mutants of Arg(283), whose side chain interacts with the minor groove edge of the templating nucleotide at the active site. The error rates of both mutant enzymes are increased >100-fold relative to wild-type pol Beta. Template engineering experiments performed to distinguish among three possible models for deletion formation suggest that most deletions in repetitive sequences by pol Beta initiate by strand slippage. However, pol Beta also generates deletions by a different mechanism that is strongly enhanced by the substitutions at Arg(283). Analysis of error specificity suggests that this mechanism involves nucleotide misinsertion followed by primer relocation, creating a misaligned intermediate. The structure of pol Beta bound to non-gapped DNA also indicates that the templating nucleotide and its downstream neighbor are out of register in the open conformation and this could facilitate misalignment (dNTP or primer terminus) with the next template base.
Lawrence A Loeb - One of the best experts on this subject based on the ideXlab platform.
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detection and characterization of mammalian DNA Polymerase Beta mutants by functional complementation in escherichia coli
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Joann B. Sweasy, Lawrence A LoebAbstract:Abstract 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.
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mechanisms of mutation by oxidative DNA damage reduced fidelity of mammalian DNA Polymerase Beta
Biochemistry, 1993Co-Authors: Daniel I Feig, Lawrence A LoebAbstract:Reactive oxygen species, produced in cells by a variety of mechanisms, damage DNA and cause mutations. To characterize the types of mutations produced in mammalian cells, we copied DNA damaged by reactive oxygen species with mammalian DNA Polymerase Beta. Double-stranded circular M13mp2 DNA containing a 361-nucleotide single-stranded gap within the lacZ gene was damaged by aerobic incubation with Fe2+ and H2O2. The gap then was filled by purified recombinant rat DNA Polymerase Beta, and the DNA was transfected into Escherichia coli. Mutations within the nonessential lacZ gene for Beta-galactosidase were identified by reduced alpha-complementation. In this system, oxidative damage increased the mutation frequency within the target region by an average of 4.3-fold. At certain sites, the base substitution rate is nearly 300 times greater than would be expected to result from a random distribution of damage. The oxidatively induced mutations fall into two categories: those apparently caused by direct miscoding of modified DNA and those associated with enhanced misincorporation at prexisting Polymerase-specific hot spots. The latter group may be due to a conformational change in the DNA caused by oxidative modification and could be indicative of a novel mutagenic mechanism.
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Mammalian DNA Polymerase Beta can substitute for DNA Polymerase I during DNA replication in Escherichia coli.
Journal of Biological Chemistry, 1992Co-Authors: Joann B. Sweasy, Lawrence A LoebAbstract:Abstract Mammalian DNA Polymerase Beta is the smallest known eukaryotic Polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA Polymerase Beta and E. coli DNA Polymerase I are similar, we wished to determine if DNA Polymerase Beta could substitute for DNA Polymerase I in bacteria. We found that the expression of mammalian DNA Polymerase Beta in E. coli restored growth in a DNA Polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA Polymerase Beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA Polymerase Beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA Polymerase Beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.