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PubMed2002

DNA polymerase beta.

Mammalian DNA polymerase beta(beta-pol) is a single polypeptide chain enzyme of 39kDa. beta-pol has enzymatic activities appropriate for roles in base excision repair and other DNA metabolism events involving gap-filling DNA synthesis. Many crystal structures of beta-pol complexed with dNTP and DNA substrates have been solved, and mouse fibroblast cell lines deleted in the beta-pol gene have been examined. These approaches have enhanced our understanding of structural and functional aspects of beta-pol's role in protecting genomic DNA.

Amino Acid Sequence↗
PubMed1984

Differential substrate specificity of DNA polymerase beta and of a DNA polymerase induced by herpes simplex virus type 2 towards thymidine triphosphate analogues.

Several triphosphates of 5-substituted deoxyuridine (dU), such as 5-ethyl-, 5-n-propyl-, 5-n-hexyl- and 5-isopropyldeoxyuridine triphosphates and 5-trifluorothymidine triphosphate are substrates for HeLa cell DNA polymerase beta (2'-deoxynucleoside-5'-triphosphate:DNA-deoxynucleotidyltransferase, EC 2.7.7.7) and for a DNA polymerase isolated from HeLa cells infected with herpes simplex virus type 2 (HSV-2) strain 75. At the concentration tested (50 microM), all these analogues were incorporated more readily into DNA by the virus-coded enzyme than by DNA polymerase beta from the host cell. The DNA polymerase coded by HSV-2 showed an affinity for deoxythymidine triphosphate (dTTP) and the analogues studied higher than that of DNA polymerase beta. Analogues are preferential substrates for the viral enzyme, since they readily substitute for dTTP during synthesis in vitro. In contrast, arabinosylthymine-5'-triphosphate was readily incorporated into DNA by the host cell DNA polymerase beta, but inhibited the DNA polymerase specified by HSV-2.

DNA Polymerase I↗
PubMed2001

Interactions of the 8-kDa domain of rat DNA polymerase beta with DNA.

Interactions between the isolated 8-kDa domain of the rat DNA polymerase beta and DNA have been studied, using the quantitative fluorescence titration technique. The obtained results show that the number of nucleotide residues occluded in the native 8-kDa domain complex with the ssDNA (the site size) is strongly affected by Mg2+ cations. In the absence of Mg2+, the domain occludes 13 +/- 0.7 nucleotide residues, while in the presence of Mg2+ the site size decreases to 9 +/- 0.6 nucleotides. The high affinity of the magnesium cation binding, as well as the dramatic changes in the monovalent salt effect on the protein-ssDNA interactions in the presence of Mg2+, indicates that the site size decrease results from the Mg2+ binding to the domain. The site size of the isolated domain-ssDNA complex is significantly larger than the 5 +/- 2 site size determined for the (pol beta)5 binding mode formed by an intact polymerase, indicating that the intact enzyme, but not the isolated domain, has the ability to use only part of the domain DNA-binding site in its interactions with the nucleic acid. Salt effect on the intrinsic interactions of the domain with the ssDNA indicates that a net release of m approximately 5 ions accompanies the complex formation. Independence of the number of ions released upon the type of anion in solution strongly suggests that the domain forms as many as seven ionic contacts with the ssDNA. Experiments with different ssDNA oligomers show that the affinity decreases gradually with the decreasing number of nucleotide residues in the oligomer. The data indicate a continuous, energetically homogeneous structure of the DNA-binding site of the domain, with crucial, nonspecific contacts between the protein and the DNA evenly distributed over the entire binding site. The DNA-binding site shows little base specificity. Moreover, the domain has an intrinsic affinity and site size of its complex with the dsDNA conformation, similar to the affinity and site size with the ssDNA. The significance of these results for the mechanistic role of the 8-kDa domain in the functioning of rat pol beta is discussed.

Animals↗
PubMed1995

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

We previously demonstrated that mammalian DNA polymerase beta can substitute for DNA polymerase I of Escherichia coli in DNA replication and in base excision repair. We have now obtained genetic evidence suggesting that DNA polymerase beta can substitute for E. coli DNA polymerase I in the initiation of replication of a plasmid containing a pMB1 origin of DNA replication. Specifically, we demonstrate that a plasmid with a pMB1 origin of replication can be maintained in an E. coli polA mutant in the presence of mammalian DNA polymerase beta. Our results suggest that mammalian DNA polymerase beta can substitute for E. coli DNA polymerase I by initiating DNA replication of this plasmid from the 3' OH terminus of the RNA-DNA hybrid at the origin of replication.

Ampicillin Resistance↗
PubMed2000

Structural homology between DNA binding sites of DNA polymerase beta and DNA topoisomerase II.

Unsaturated long-chain fatty acids selectively bind to the DNA binding sites of DNA polymerase beta and DNA topoisomerase II, and inhibit their activities, although the amino acid sequences of these enzymes are markedly different from each other. Computer modeling analysis revealed that the fatty acid interaction interface in both enzymes has a group of four amino acid residues in common, forming a pocket which binds to the fatty acid molecule. The four amino acid residues were Thr596, His735, Leu741 and Lys983 for yeast DNA topoisomerase II, corresponding to Thr79, His51, Leu11 and Lys35 for rat DNA polymerase beta. Using three-dimensional structure model analysis, we determined the spatial positioning of specific amino acid residues binding to the fatty acids in DNA topoisomerase II, and subsequently obtained supplementary information to build the structural model.

Amino Acid Sequence↗
PubMed2002

Local deformations revealed by dynamics simulations of DNA polymerase Beta with DNA mismatches at the primer terminus.

Nanosecond dynamics simulations for DNA polymerase beta (pol beta)/DNA complexes with three mismatched base-pairs, namely GG, CA, or CC (primer/template) at the DNA polymerase active site, are performed to investigate the mechanism of polymerase opening and how the mispairs may affect the DNA extension step; these trajectories are compared to the behavior of a pol beta/DNA complex with the correct GC base-pair, and assessed with the aid of targeted molecular dynamics (TMD) simulations of all systems from the closed to the open enzyme state. DNA polymerase conformational changes (subdomain closing and opening) have been suggested to play a critical role in DNA synthesis fidelity, since these changes are associated with the formation of the substrate-binding pocket for the nascent base-pair. Here we observe different large C-terminal subdomain (thumb) opening motions in the simulations of pol beta with GC versus GG base-pairs. Whereas the conformation of pol beta in the former approaches the observed open state in the crystal structures, the enzyme in the latter does not. Analyses of the motions of active-site protein/DNA residues help explain these differences. Interestingly, rotation of Arg258 toward Asp192, which coordinates both active-site metal ions in the closed "active" complex, occurs rapidly in the GG simulation. We have previously suggested that this rotation is a key slow step in the closed to open transition. TMD simulations also point to a unique pathway for Arg258 rotation in the GG mispair complex. Simulations of the mismatched systems also reveal distorted geometries in the active site of all the mispair complexes examined. The hierarchy of the distortions (GG>CC>CA) parallels the experimentally deduced inability of pol beta to extend these mispairs. Such local distortions would be expected to cause inefficient DNA extension and polymerase dissociation and thereby might lead to proofreading by an extrinsic exonuclease. Thus, our studies on the dynamics of pol beta opening in mismatch systems provide structural and dynamic insights to explain experimental results regarding inefficient DNA extension following misincorporation; these details shed light on how proofreading may be invoked by the abnormal active-site geometry.

Base Pair Mismatch↗
PubMed2000

Translesion replication by DNA polymerase delta depends on processivity accessory proteins and differs in specificity from DNA polymerase beta.

Mutations caused by DNA damage lead to the development of cancer. The critical step in the formation of these mutations is the replication of unrepaired lesions in DNA by DNA polymerases, a process termed translesion replication. Using a newly developed method for preparation of gapped plasmids, containing a site-specific synthetic abasic site, we analyzed translesion replication with purified mammalian DNA polymerases delta and beta. DNA polymerase delta was found to be unable to replicate through the abasic site. Addition of the sliding DNA clamp PCNA, the clamp loader RFC, and ATP caused a drastic 30-fold increase in translesion replication. Thus, similar to Escherichia coli DNA polymerase III, the processivity accessory proteins enable DNA polymerase delta to bypass blocking lesions. Under comparable conditions, DNA polymerase beta was unable to bypass the abasic site, unless its concentration was greatly increased. Analysis of translesion replication products revealed a marked difference in the specificity of bypass: whereas 90% of bypass events by DNA polymerase delta holoenzyme involved insertion of a dAMP residue opposite the abasic site, DNA polymerase beta tended to skip over the abasic site, producing mainly minus frameshifts (73%). The significance of these results for in vivo translesion replication is discussed.

Adenosine Triphosphate↗
PubMed2001

Impaired repair activity of a truncated DNA polymerase beta protein.

DNA polymerase beta (polbeta) is an essential enzyme for gap filling synthesis in damaged DNA template involved in base excision repair pathway. A truncated polbeta protein is expressed in primary colorectal and breast adenocarcinomas. To determine a possible alteration in the functions of the enzyme, a human cell line named HeLapolbetadelta expressing the truncated form of polbeta has been established. These cells revealed a significantly reduced level of repair activity evaluated by gap filling synthesis and polbeta activity. More importantly, the HeLapolbetadelta cells are hypersensitive to MNNG, a DNA alkylating agent. It appears from the responses that the gap filling synthesis of WT cells, a HeLa cell line overexpressing wild-type polbeta protein, was inhibited by HeLapolbetadelta protein.

Cell Survival↗
PubMed2001

Three-dimensional structural model analysis of the binding site of lithocholic acid, an inhibitor of DNA polymerase beta and DNA topoisomerase II.

The molecular action of lithocholic acid (LCA), a selective inhibitor of mammalian DNA polymerase beta (pol beta), was investigated. We found that LCA could also strongly inhibit the activity of human DNA topoisomerase II (topo II). No other DNA metabolic enzymes tested were affected by LCA. Therefore, LCA should be classified as an inhibitor of both pol beta and topo II. Here, we report the molecular interaction of LCA with pol beta and topo II. By three-dimensional structural model analysis and by comparison with the spatial positioning of specific amino acids binding to LCA on pol beta (Lys60, Leu77, and Thr79), we obtained supplementary information that allowed us to build a structural model of topo II. Modeling analysis revealed that the LCA-interaction interface in both enzymes has a pocket comprised of three amino acids in common, which binds to the LCA molecule. In topo II, the three amino acid residues were Lys720, Leu760, and Thr791. These results suggested that the LCA binding domains of pol beta and topo II are three-dimensionally very similar.

African Swine Fever Virus↗
PubMed2002

Heterogeneity in expression of DNA polymerase beta and DNA repair activity in human tumor cell lines.

The 39-kDa DNA polymerase beta (pol beta) is an essential enzyme in short-patch base excision repair pathway. A wild-type and a truncated forms of pol beta proteins are expressed in primary colorectal and breast adenocarcinomas and in a primary culture of renal cell carcinoma. To test whether pol beta has a contributory role in tumorigenicity of human tumor cell lines, we have undertaken a study to determine expression of pol beta in colon, breast, and prostate tumor cell lines. Unlike primary colon tumor cells, three types of pol beta mRNA have been identified in HCT116, LoVo, and DLD1, colon tumor cell lines. A 111-bp-deleted pol beta transcript was expressed in MCF7, a breast tumor cell line, but not in primary breast tumor cells. An expression of a smaller pol beta transcript has been revealed in DU145, a prostate tumor cell line, whereas, a single base (T) deletion in mRNA at codon 191 was found in prostate cancer tissue. Interestingly, a wild-type pol beta transcript was also expressed in all tumor cell lines similar to primary tumor cells. Furthermore, the cell extract of LoVo exhibited highest gap-filling synthesis function of pol beta when the extract of DU145 showed lowest activity. MNNG, a DNA alkylating agent, enhanced the gap-filling synthesis activity in extracts of LoVo cell line. Furthermore, the cellular viability of LoVo and HCT116 cells is sensitive to MNNG when DU145 cells are resistant. These results demonstrate heterogeneity in pol beta mRNA expression, which may be a risk factor related to tumorigenic activities of tumor cell lines.

Breast Neoplasms↗
PubMed2006

Bioactive isomalabaricane triterpenoids from Rhabdastrella globostellata that stabilize the binding of DNA polymerase beta to DNA.

Four isomalabaricane triterpenoids were isolated from an extract of the sponge Rhabdastrella globostellata that was active in an assay measuring stabilization of the binding of DNA with DNA polymerase beta. The known compounds stelliferin riboside (1) and 3-epi-29-acetoxystelliferin E (2) were shown to induce 29% and 23% binding, respectively, at 28 microg/mL, while the new compound stellettin J (3) induced 5% binding at 28 microg/mL. The new compound stellettin K (4) had no activity in the binding assay. The compounds were characterized by spectroscopic methods. These compounds displayed varying levels of activity toward the A2780 ovarian cancer cell line, revealing structure-based effects on both the level of cytotoxicity and DNA-polymerase beta binding. This is the first report of natural products with the ability to promote stabilization of the DNA-polymerase beta covalent binary complex.

Animals↗
PubMed2001

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA.

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.

Aphidicolin↗
PubMed1995

Phorbol ester abrogates up-regulation of DNA polymerase beta by DNA-alkylating agents in Chinese hamster ovary cells.

Mammalian DNA polymerase beta (beta-pol), a DNA repair polymerase, is known to be constitutively expressed in cultured cells, but treatment of cells with the DNA-alkylating agents MNNG or methyl methanesulfonate has been shown to up-regulate beta-pol mRNA level. To further characterize this response, we prepared a panel of monoclonal antibodies and used one of them to quantify beta-pol in whole cell extracts by immunoblotting. We found that treatment of Chinese hamster ovary cells with either DNA-alkylating agent up-regulated the beta-pol protein level 5-10-fold. This induction appeared to be secondary to DNA alkylation, as induction was not observed with a genetically altered cell line overexpressing the DNA repair enzyme O6-methylguanine-methyltransferase. We also found that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of wild type Chinese hamster ovary cells increased expression of beta-pol protein (approximately 10-fold). Any interrelationship between this TPA response and the DNA-alkylation response was studied by treatment with combinations of MNNG and TPA. The beta-pol up-regulation observed with MNNG treatment was abrogated by TPA, and conversely the up-regulation observed with TPA treatment was abrogated by MNNG.

Alkylating Agents↗
PubMed2005

Reduced DNA gap repair in aging rat neuronal extracts and its restoration by DNA polymerase beta and DNA-ligase.

Synthetic deoxy-oligo duplexes containing short gaps of 1 and 4 nucleotides were used as model substrates to assess the DNA gap repair ability of the neuronal extracts prepared from cerebral cortex of rats of different ages. Our results demonstrate that gap repair activity in neurons decreases markedly with age. The decreased activity could be restored by supplementing the neuronal extracts with pure recombinant rat liver DNA polymerase beta. High levels of DNA polymerase beta supplementation resulted in gap-filling activity that proceeded essentially through addition of nucleotides through a slow distributive strand displacement mode to achieve full template length (32-mer). However, at lower concentrations of DNA polymerase beta, the gap repair takes place quickly through gap filling followed by ligation to downstream primer, in an energy efficient manner. For this to happen, the conditions required are the presence of 5'-PO4 on the downstream primer and supplementation of aging neuronal extracts with DNA-ligase in addition to recombinant DNA polymerase beta. These results demonstrate that aging neurons are unable to affect base excision repair (BER) due to deficiency of DNA polymerase beta and DNA-ligase and fortifying aged neuronal extracts with these two factors can restore the lost BER activity.

Aging↗
PubMed1990

Identification and properties of the catalytic domain of mammalian DNA polymerase beta.

Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.

Animals↗
PubMed1999

The cyanogenic glucoside, prunasin (D-mandelonitrile-beta-D-glucoside), is a novel inhibitor of DNA polymerase beta.

A DNA polymerase beta (pol. beta) inhibitor has been isolated independently from two organisms; a red perilla, Perilla frutescens, and a mugwort, Artemisia vulgaris. These molecules were determined by spectroscopic analyses to be the cyanogenic glucoside, D-mandelonitrile-beta-D-glucoside, prunasin. The compound inhibited the activity of rat pol. beta at 150 microM, but did not influence the activities of calf DNA polymerase alpha and plant DNA polymerases, human immunodefficiency virus type 1 reverse transcriptase, calf terminal deoxynucleotidyl transferase, or any prokaryotic DNA polymerases, or DNA and RNA metabolic enzymes examined. The compound dose-dependently inhibited pol. beta activity, the IC(50) value being 98 microM with poly dA/oligo dT(12-18) and dTTP as the DNA template and substrate, respectively. Inhibition of pol. beta by the compound was competitive with the substrate, dTTP. The inhibition was enhanced in the presence of fatty acid, and the IC(50) value decreased to approximately 40 microM. In the presence of C(10)-decanoic acid, the K(i) value for substrate dTTP decreased by 28-fold, suggesting that the fatty acid allowed easier access of the compound to the substrate-binding site.

Amygdalin↗
PubMedDate not supplied

Modulation by dietary copper of aflatoxin B1-induced activity of DNA repair enzymes poly (ADP-ribose) polymerase, DNA polymerase beta and DNA ligase.

The activity of some nuclear enzymes associated with DNA repair was examined following aflatoxin B1 administration in rats maintained on different levels of dietary copper. Induction of poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was found to be significantly higher in copper-deficient rats. Copper supplementation, even at marginal doses, was able to bring down the induction to the level observed in normal rats. The results emphasize the protective role of copper against the DNA damaging effects of aflatoxin B1.

Aflatoxin B1↗
PubMed2003

Variants of DNA polymerase Beta extend mispaired DNA due to increased affinity for nucleotide substrate.

DNA polymerase beta offers an attractive system to study the biochemical mechanism of polymerase-dependent mutagenesis. Variants of DNA polymerase beta, Y265F and Y265W, were analyzed for misincorporation efficiency and mispair extension ability, relative to wild-type DNA polymerase beta. Our data show that the fidelity of the mutant polymerases is similar to wild-type enzyme on a one-nucleotide gapped DNA substrate. In contrast, with a six-nucleotide gapped DNA, the mutant proteins are slightly more accurate than the wild-type enzyme. The mutagenic potential of Y265F and Y265W is more pronounced when encountering a mispaired DNA substrate. Here, both variants can extend a G:G mispair quite efficiently, and Y265F can also extend a T:G mispair. The kinetic basis of the increased mispair extension efficiency is due to an improved ability to bind to the incoming nucleotide. Y265W extends the G:G mispair even with an incorrect nucleotide substrate. Overall, our results demonstrate that the Y265 hinge residue is important for stabilizing the architecture of the nucleotide binding pocket of DNA polymerase beta, and that alterations of this residue can have significant impacts upon the fidelity of DNA synthesis.

Amino Acid Substitution↗