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Crystal structures of DNA polymerase beta encountering an 8-oxoguanine lesion show how the polymerase selects an otherwise unfavorable conformation of a damaged template base to enable error-free bypass.
Alterations in DNA repair lead to genomic instability and higher risk of cancer. DNA base excision repair (BER) corrects damaged bases, apurinic sites, and single-strand DNA breaks. Here, a regulatory mechanism for DNA polymerase beta (Pol beta) is described. Pol beta was found to form a complex with the protein arginine methyltransferase 6 (PRMT6) and was specifically methylated in vitro and in vivo. Methylation of Pol beta by PRMT6 strongly stimulated DNA polymerase activity by enhancing DNA binding and processivity, while single nucleotide insertion and dRP-lyase activity were not affected. Two residues, R83 and R152, were identified in Pol beta as the sites of methylation by PRMT6. Genetic complementation of Pol beta knockout cells with R83/152K mutant revealed the importance of these residues for the cellular resistance to DNA alkylating agent. Based on our findings, we propose that PRMT6 plays a role as a regulator of BER.
The "A-rule" reflects the preferred incorporation of dAMP opposite abasic lesions in Escherichia coli in vivo. DNA polymerases (pol) from procaryotic and eucaryotic organisms incorporate nucleotides opposite abasic lesions in accordance with the A-rule. However, recent in vivo data demonstrate that A is not preferentially incorporated opposite abasic lesions in eucaryotes. Purified human DNA polymerases beta and alpha are used to measure the specificity of nucleotide incorporation at a site-directed tetrahydrofuran abasic lesion, in 8-sequence contexts, varying upstream and downstream bases adjacent to the lesion. Extension past the lesion is measured in 4 sequence contexts, varying the downstream template base. Pol alpha strongly favors incorporation of dAMP directly opposite the lesion. In marked contrast, pol beta violates the A-rule for incorporation directly opposite the lesion. In addition to incorporation taking place directly opposite the lesion, we also analyze misalignment incorporation directed by a template base downstream from the lesion. Lesion bypass by pol beta occurs predominantly by "skipping over" the lesion, by insertion of a nucleotide complementary to an adjacent downstream template site. Misalignment incorporation for pol beta occurs by a novel "dNTP-stabilized" mechanism resulting in both deletion and base substitution errors. In contrast, pol alpha shows no propensity for this type of synthesis. The misaligned DNA structures generated during dNTP-stabilized lesion bypass do not conform to misaligned structures reported previously.
Residues of DNA polymerase beta (beta-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15N/(13)C/(2)H/(1)H,(13)C-methyl(Leu,Ile,Val)-labeled beta-Pol palm-thumb domain was used for assignments of the 1H, 15N, and 13C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A deltaV303-V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of beta-Pol contacts an edge/face region of the beta sheet of the XRCC1 NTD, while the beta-Pol palm weakly contacts the alpha2 helix.
Interactions between human DNA polymerase beta and the template-primer, as well as gapped DNA substrates, have been studied using quantitative fluorescence titration and analytical ultracentrifugation techniques. In solution, human pol beta binds template-primer DNA substrates with a stoichiometry much higher than predicted on the basis of the crystallographic structure of the polymerase-DNA complex. The obtained stoichiometries can be understood in the context of the polymerase affinity for the dsDNA and the two ssDNA binding modes, the (pol beta)(16) and (pol beta)(5) binding modes, which differ by the number of nucleotide residues occluded by the protein in the complex. The analysis of polymerase binding to different template-primer substrates has been performed using the statistical thermodynamic model which accounts for the existence of different ssDNA binding modes and has allowed us to extract intrinsic spectroscopic and binding parameters. The data reveal that the small 8 kDa domain of the enzyme can engage the dsDNA in interactions, downstream from the primer, in both (pol beta)(16) and (pol beta)(5) binding modes. The affinity, as well as the stoichiometry of human pol beta binding to the gapped DNAs is not affected by the decreasing size of the ssDNA gap, indicating that the enzyme recognizes the ssDNA gaps of different sizes with very similar efficiency. On the basis of the obtained results we propose a plausible model for the gapped DNA recognition by human pol beta. The enzyme binds the ss/dsDNA junction of the gap, using its 31 kDa domain, with slight preference over the dsDNA. Binding only to the junction, but not to the dsDNA, induces an allosteric conformational transition of the enzyme and the entire enzyme-DNA complex which results in binding of the 8 kDa domain with the dsDNA. This, in turn, leads to the significant amplification of the enzyme affinity for the gap over the surrounding dsDNA, independent of the gap size. The presence of the 5'-terminal phosphate, downstream from the primer, has little effect on the affinity, but profoundly affects the ssDNA conformation in the complex. The significance of these results for the mechanistic model of the functioning of human pol beta is discussed.
Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.
When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.
XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase beta (beta-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain-domain interaction in the XRCC1-beta-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment and the linker-BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa domain. The XRCC1-NTD(1-183)(residues 1-183) was found to bind beta-Pol, the beta-Pol 31 kDa domain and the beta-Pol C-terminal palm-thumb (residues 140-335), and the interaction was further localized to XRCC1-NTD(1-157)(residues 1-157). The XRCC1-NTD(1-183)-beta-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36-355 and residues 1-159, were found to interact with beta-Pol, the beta-Pol 31 kDa domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed from the respective beta-Pol constructs. Neither the XRCC1-NTD(1-159), nor the XRCC1(36-355)polypeptide was found to interact with a beta-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75-212) showed no interaction with beta-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD(1-183)was found to bind beta-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K(d) of 0.4-2.4 microM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding interactions involving beta-Pol.
The most frequent DNA lesions in mammalian genomes are removed by the base excision repair (BER) via multiple pathways that involve the replacement of one or more nucleotides at the lesion site. The biological consequences of a BER defect are at present largely unknown. We report here that mouse cells defective in the main BER DNA polymerase beta (Pol beta) display a decreased rate of DNA single-strand breaks (ssb) rejoining after methyl methanesulfonate damage when compared with wild-type cells. In contrast, Pol beta seems to be dispensable for hydrogen peroxide-induced DNA ssb repair, which is equally efficient in normal and defective cells. By using an in vitro repair assay on single abasic site-containing circular duplex molecules, we show that the long-patch BER is the predominant repair route in Pol beta-null cell extract. Our results strongly suggest that the Pol beta-mediated single nucleotide BER is the favorite pathway for repair of N-methylpurines while oxidation-induced ssb, likely arising from oxidized abasic sites, are the substrate for long-patch BER.
Crystallographic characterization of DNA polymerase beta (pol beta) has suggested that multiple-domain and subdomain motions occur during substrate binding and catalysis. NMR studies of [methyl-(13)C]methionine-labeled pol beta were conducted to characterize the structural and dynamic response to ligand binding. The enzyme contains seven methionine residues, one of which is at the amino terminus and is partially removed by the expression system. Three of the methyl resonances were readily assigned using site-directed mutants. Assignment of the resonances of Met155, Met158, and Met191 was more difficult due to the spatial proximity of these residues, so that assignments were based on NOESY-HSQC data and on the response to paramagnetic Co(2+) addition, as well as shift perturbations observed for the site-directed mutants. The response of the methyl resonances to substrate binding was evaluated by the serial addition of a template oligonucleotide, a downstream 5'-phosphorylated oligonucleotide, and a primer oligonucleotide to create a two-nucleotide-gapped DNA substrate. Addition of the single-stranded template DNA resulted in selective broadening of the methyl resonance of Met18 in the 8 kDa lyase domain, and this resonance then shifted and sharpened upon addition of a 5'-phosphate-terminated downstream complementary oligonucleotide. Conversion of the two-nucleotide-gapped DNA substrate to a single-nucleotide-gapped substrate by incorporation of ddCMP produced a small perturbation of the Met236 resonance, which makes contact with the primer strand in the crystal structure. The addition of a second equivalent of ddCTP to form the pol beta-DNA-ddCTP ternary complex resulted in significant shifts for the resonances corresponding to Met155, Met191, Met236, and Met282. The Met155 methyl resonance is severely broadened, while the Met191 and Met282 resonances exhibit significant but less extreme broadening. Since only Met236 makes contact with the substrate, the effects on Met155, Met236, and Met282 result from indirect conformational and dynamic perturbations. Previous crystallographic characterization of this abortive complex indicated that a polymerase subdomain or segment (alpha-helix N) repositions itself to form one face of the binding pocket for the nascent base pair. Met282 serves as a probe for motion in this segment. Addition of Mg(2+)-dATP to pol beta in the absence of DNA produced qualitatively similar but much smaller effects on Met191 and Met155, but did not strongly perturb Met282, leading to the conclusion that Mg(2+)-dATP alone is insufficient to produce the large conformational changes that are observed in the abortive complex involving the gapped DNA with a blocked primer and ddNTP. Thus, the NMR data indicate that the nucleotide-DNA interaction appears to be essential for conformational activation.
During base excision repair, DNA polymerase beta fills 1-6-nucleotide gaps processively, reflecting a contribution of both its 8- and 31-kDa domains to DNA binding. Here we report the fidelity of pol beta during synthesis to fill gaps of 1, 5, 6, or >300 nucleotides. Error rates during distributive synthesis by recombinant rat and human polymerase (pol) beta with a 390-base gap are similar to each other and to previous values with pol beta purified from tissues. The base substitution fidelity of human pol beta when processively filling a 5-nucleotide gap is similar to that with a 361-nucleotide gap, but "closely-spaced" substitutions are produced at a rate at least 60-fold higher than for distributive synthesis. Base substitution fidelity when filling a 1-nucleotide gap is higher than when filling a 5-nucleotide gap, suggesting a contribution of the 8-kDa domain to the dNTP binding pocket and/or a difference in base stacking or DNA structure imposed by pol beta. Nonetheless, 1-nucleotide gap filling is inaccurate, even generating complex substitution-addition errors. Finally, the single-base deletion error rate during processive synthesis to fill a 6-nucleotide gap is indistinguishable from that of distributive synthesis to fill a 390-nucleotide gap. Thus the mechanism of processivity by pol beta does not allow the enzyme to suppress template misalignments.
The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.
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.
DNA polymerase beta (pol beta) is the major DNA polymerase involved in the base excision repair (BER) pathway in mammalian cells and, as a consequence, BER is severely compromised in cells lacking pol beta. Pol beta null (-/-) mouse embryos are not viable and pol beta null cells are hypersensitive to alkylating agents. Using RNA interference (RNAi) technology in mouse cells, we have reduced the pol beta protein and mRNA to undetectable levels. Pol beta knockdown cell lines display a pattern of hypersensitivity to DNA damaging agents similar to that observed in pol beta null cells. Generation of pol beta knock down cells makes it possible to combine the pol beta null phenotype with deficiencies in other DNA repair proteins, thereby helping to elucidate the role of pol beta and its interactions with other proteins in mammalian cells.
Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.
A DNA polymerase with properties similar to mammalian polymerase beta has been isolated to near homogeneity from embryos of Drosophila melanogaster. A combination of exclusion chromatography and sodium dodecyl sulfate-gel electrophoresis indicates that this enzyme is composed of a single polypeptide of molecular weight-110,000. Optimum activity on a nicked template occurs at pH 8.4 in the presence of 15 mM MgCl2 and 250 mM NaCl. Enzyme activity is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmalemide. These properties clearly distinguish this enzyme from polymerase alpha, which has previously been characterized from this tissue. This report represents the first extensive purification of a beta-like polymerase from the Protostomic branch of the animal phylogenetic tree. It furthermore generates the potential for a genetic analysis of the function of polymerase beta in DNA recombination, repair, and synthesis.
DNA polymerase beta, a 38kD protein is essentially considered to be a repair enzyme at least with respect to some forms of DNA damage. Through northern and western blotting, it was found that mRNA and immunologically reactive molecules of DNA-polymerase beta are reduced by 30% and 20% respectively in old rat brain as compared to the young. However, activity gel assay and immunotitration revealed a 50% reduction in the activity in old brain. These data are taken to indicate accumulation of catalytically inactive molecules of DNA-polymerase beta in rat brain with age.
We examined interactions between base excision repair (BER) DNA intermediates and purified human BER enzymes, DNA polymerase beta (pol beta), apurinic/apyrimidinic endonuclease (APE1) and poly(ADP-ribose) polymerase-1 (PARP-1). Studies under steady-state conditions with purified BER enzymes and BER substrates have already demonstrated interplay between these BER enzymes that is sensitive to the respective concentrations of each enzyme. Therefore, in this study, using conditions of enzyme excess over substrate DNA, we further examine the question of interplay between BER enzymes on BER intermediates. The results reveal several important differences compared with data obtained using steady-state assays. Excess PARP-1 antagonizes the action of pol beta, producing a complete block of long patch BER strand-displacement DNA synthesis. Surprisingly, an excess of APE1 stimulates strand-displacement DNA synthesis by pol beta, but this effect is blocked by PARP-1. The APE1 exonuclease function appears to be modulated by the other BER proteins. Excess APE1 over pol beta may allow APE1 to perform both exonuclease function and stimulation of strand-displacement DNA synthesis by pol beta. This enables pol beta to mediate long patch sub-pathway. These results indicate that differences in the stoichiometry of BER enzymes may regulate BER.