The E295K DNA polymerase beta gastric cancer-associated variant interferes with base excision repair and induces cellular transformation

Approximately 30% of human tumors examined for mutations in polymerase beta (pol beta) appear to express pol beta variant proteins (D. Starcevic, S. Dalal, and J. B. Sweasy, Cell Cycle 3:998-1001, 2004). Many of these variants result from a single amino acid substitution. We have previously shown that the K289M and I260M colon and prostate cancer variants, respectively, induce cellular transformation most likely due to sequence-specific mutator activity (S. Dalal et al., Biochemistry 44:15664-15673, 2005; T. Lang et al., Proc. Natl. Acad. Sci. USA 101:6074-6079, 2004; J. B. Sweasy et al., Proc. Natl. Acad. Sci. USA 102:14350-14355, 2005). In the work described here, we show that the E295K gastric carcinoma pol beta variant acts in a dominant-negative manner by interfering with base excision repair. This leads to an increase in sister chromatid exchanges. Expression of the E295K variant also induces cellular transformation. Our data suggest that unfilled gaps are channeled into a homology-directed repair pathway that could lead to genomic instability. The results indicate that base excision repair is critical for maintaining genome stability and could therefore be a tumor suppressor mechanism.     

Hinge residue Ile260 of DNA polymerase beta is important for enzyme activity and fidelity

DNA polymerases ensure efficient insertion of the correct dNTP into the DNA substrate. They have evolved mechanisms for discriminating among very similar dNTP substrates. DNA polymerase beta is a repair polymerase that provides a model system for a direct study of insertion fidelity. In this study, we examined the role of hinge residue Ile260 of the rat Polbeta on enzyme activity and accuracy. We changed residue I260 to every other amino acid residue and used genetic screens to assess the activity and fidelity of the resulting mutants. The I260D, -E, -K, -N, and -R mutants are significantly less active than wild-type Polbeta. Interestingly, I260H and I260Q are active but exhibit mutator activity. This suggests that the nonpolar nature of residue 260 is important for maintaining the activity and fidelity of Polbeta. We employ molecular modeling as an aid in explaining the observed phenotypes and propose a mechanism whereby the positioning of the DNA substrate in the enzyme and within the surface of the hinge may be a key player in forming an optimal active site for phosphodiester bond formation between Watson-Crick base pairs.     

The I260Q variant of DNA polymerase beta extends mispaired primer termini due to its increased affinity for deoxynucleotide triphosphate substrates

DNA polymerase beta plays a key role in base excision repair. We have previously shown that the hydrophobic hinge region of polymerase beta, which is distant from its active site, plays a critical role in the fidelity of DNA synthesis by this enzyme. The I260Q hinge variant of polymerase beta misincorporates nucleotides with a significantly higher catalytic efficiency than the wild-type enzyme. In the study described here, we show that I260Q extends mispaired primer termini. The kinetic basis for extension of mispairs is defective discrimination by I260Q at the level of ground-state binding of the dNTP substrate. Our results suggest that the hydrophobic hinge region influences the geometry of the dNTP binding pocket exclusively. Because the DNA forms part of the binding pocket, our data are also consistent with the interpretation that the mispaired primer terminus affects the geometry of the dNTP binding pocket such that the I260Q variant has a higher affinity for the incoming dNTP than wild-type polymerase beta.     

The hydrophobic hinge region of rat DNA polymerase beta is critical for substrate binding pocket geometry

The hydrophobic hinge of DNA polymerase beta facilitates closing and stabilization of the enzyme once the nucleotide substrate has bound. Alteration of the hydrophobic nature of the hinge by the introduction of a hydrophilic glutamine residue in place of isoleucine 260 results in an inaccurate polymerase. The kinetic basis of infidelity is lack of discrimination during the binding of substrate. The I260Q polymerase beta variant has lower affinity than wild type enzyme for the correct substrate and much higher affinity for the incorrect substrate. Our results demonstrate that the hinge is important for formation of the substrate binding pocket. Our results are also consistent with the interpretation that DNA polymerase beta discriminates the correct from incorrect substrate during the binding step.     

Effect of DNA polymerase beta loop variants on discrimination of O6-methyldeoxyguanosine modification present in the nucleotide versus template substrate

We have examined the mechanism of DNA polymerase beta (pol beta) lesion discrimination using alkylated dNTP versus alkylated DNA template substrates and the pol beta variants R253M and E249K. Both of these amino acid variants are located in the loop region of the palm domain and are known to play a role in pol beta fidelity and discrimination of 3'-azido-3'-deoxythymidine triphosphate substrates. We observed that these variants affect O(6)-methyldeoxyguanosine- (m6G-) modified dNTP discrimination without affecting m6G template translesion synthesis. Under steady-state conditions, the ratio of inherent reactivity values for the m6dGTP substrate relative to the dGTP substrate was greater for both variant polymerases than for wild-type (WT) pol beta. Biochemical assays of translesion synthesis using m6G lesion-containing templates demonstrated no significant differences between the variants and WT. Using N-methyl-N-nitrosourea- (MNU-) modified DNA templates in the HSV-tk in vitro assay, no difference among the enzymes in the frequency of alkylation-induced G to A transition mutations was observed. However, differences among the polymerases in the frequency of alkylation-induced C to A transversions were observed, consistent with a mutator tendency for E249K and an antimutator tendency for R253M. We conclude that a specific interaction at the loop of the palm domain is involved in pol beta discrimination of the m6G lesion when present on the incoming dNTP substrate but not when present in the DNA template. Our data support a role for the flexible loop in pol beta error discrimination.     

Variant forms of DNA polymerase beta in primary lung carcinomas.

DNA polymerase beta (pol beta) provides most of the gap-filling synthesis at apurinic/apyrimidine sites of damaged DNA in the base excision repair pathway. A truncated form of the pol beta protein is expressed in colon and breast cancers. However, the role of the pol beta gene in lung cancer is not known. Thus, we investigated a possible occurrence of pol beta variants in primary lung tumors. The entire cDNA of pol beta obtained by RT-PCR amplification was analyzed for nucleotide sequencing in lung tumor and matched normal lung tissue of the same patient. Three types of variants were detected in squamous, non-small, or large cell carcinomas. The most common variant was a deletion of 87 bp from pol beta cDNA at a site corresponding to exon 11. In addition, a variant exhibiting deletions of 87 and 140 bp together with an insertion of 105 bp was identified in three lung tumors. This is the first report of the occurrence of pol beta variants, possibly splicing variants, in lung cancer. A truncated pol beta protein resulting from variant forms of the gene may impact the function of the enzyme and increase susceptibility to carcinogenesis.     

Is There a Link Between DNA Polymerase Beta and Cancer?

Recent small-scale studies have shown that 30 % of human tumors examined to date express DNA polymerase beta variant proteins. One of the DNA polymerase beta colon cancer-associated mutants, K289M, has been shown to synthesize DNA with a lower fidelity than wild-type Pol beta. Thus, the K289M protein could confer a mutator phenotype to the cell, resulting in genomic instability. Another DNA polymerase beta variant identified in colon carcinoma interferes with base excision repair in cells. This may result in unfilled gaps which can serve as substrates for recombination and result in genomic instability. DNA polymerase beta has also been shown to be overexpressed in a variety of tumors. In some cases, overexpression of polymerase beta in cells confers a transformed phenotype to the cells. In other cases, overexpression results in telomere fusions. Thus, mutant forms or aberrant quantities of polymerase beta confer a mutator phenotype to cells. Combined with the small-scale tumor studies, these mechanistic studies implicate variant forms of DNA polymerase beta in the etiology of human cancer.     

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

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

DNA polymerase I of Mycobacterium tuberculosis: functional role of a conserved aspartate in the hinge joining the M and N helices.

The highly conserved GXD sequence present in the Mycobacterium tuberculosis DNA polymerase I corresponds to a hinge region in the finger subdomain connecting M and N helices of Escherichia coli pol I. An examination of the crystal structures of pol I family polymerases reveals that the invariant aspartate of the hinge forms a salt bridge with the conserved arginine of the O-helix and an H-bond with Gln-708. To clarify the role of this region, we generated and characterized conserved and nonconserved mutant derivatives of this aspartate, the preceding glutamate and the Gln in TB pol I. For comparison, D732A mutein of pol I was also included. The muteins representing conserved aspartate (Asp-707 of TB pol I or Asp-732 of pol I) showed a strong K(m)((dNTP)) effect and minor alteration in K(d)((DNA)), with about 10-20-fold decrease in overall catalytic efficiency. The TB muteins, E706A and Q683A, have less pronounced deviations from the wild-type enzyme. Further examination of D707A of TB pol I showed no alteration in the processivity or the dideoxynucleotide sensitivity patterns. However, both TB pol D707A and homologous E. coli D732A failed to form a stable E.DNA.dNTP ternary complex. These results suggest that the aspartate in the hinge region is catalytically important and is required for dNTP binding and in the formation of a prepolymerase ternary complex.     

The Asp285 variant of DNA polymerase beta extends mispaired primer termini via increased nucleotide binding

Endogenous DNA damage occurs at a rate of at least 20,000 lesions per cell per day. Base excision repair (BER) is a key pathway for maintaining genome stability. Several pol beta variants were identified as conferring resistance to 3'-azido-3'-deoxythymidine (AZT) in Escherichia coli (Kosa et al. (1999) J. Biol. Chem. 274, 3851-3858). Detailed biochemical studies on one of these AZT-resistant variants, His285 to Asp, have shown that the H285D variant of pol beta possesses pre-steady-state kinetics that are similar to the wild-type polymerase. In gap filling assays with 5-bp gapped DNA, H285D showed a slight mutator phenotype. In depth single turnover kinetic analysis revealed that H285D is much more efficient than wild-type pol beta at extending mispaired primer termini. This mispair extension property of H285D is attributed to a greatly increased binding to the next correct nucleotide in the presence of a mispair. This change in K d(dNTP),app is not accompanied by a change in k pol; values for k pol are the same for both H285D and wild-type. Close examination of available structural data, as well as molecular modeling, has shown that residue 285 is able to make several stabilizing contacts in the fingers domain of the polymerase, and the introduction of a negatively charged side chain could have important effects on the enzyme. It is postulated that the loss of the contact between His285, Lys289, and Ile323 is responsible for the ability of H285D to extend mispairs through disruption of contacts near the C-terminal end of pol beta and propagation into the nucleotide binding pocket.     

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

Approximately 30% of human tumors characterized to date express DNA polymerase beta (pol β) 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.     

A DNA polymerase beta mutator mutant with reduced nucleotide discrimination and increased protein stability

DNA polymerase beta (pol beta) offers a simple system to examine the role of polymerase structure in the fidelity of DNA synthesis. In this study, the M282L variant of pol beta (M282Lbeta) was identified using an in vivo genetic screen. Met282, which does not contact the DNA template or the incoming deoxynucleoside triphosphate (dNTP) substrate, is located on alpha-helix N of pol beta. This mutant enzyme demonstrates increased mutagenesis in both in vivo and in vitro assays. M282Lbeta has a 7.5-fold higher mutation frequency than wild-type pol beta; M282Lbeta commits a variety of base substitution and frameshift errors. Transient-state kinetic methods were used to investigate the mechanism of intrinsic mutator activity of M282Lbeta. Results show an 11-fold decrease in dNTP substrate discrimination at the level of ground-state binding. However, during the protein conformational change and/or phosphodiester bond formation, the nucleotide discrimination is improved. X-ray crystallography was utilized to gain insights into the structural basis of the decreased DNA synthesis fidelity. Most of the structural changes are localized to site 282 and the surrounding region in the C-terminal part of the 31-kDa domain. Repositioning of mostly hydrophobic amino acid residues in the core of the C-terminal portion generates a protein with enhanced stability. The combination of structural and equilibrium unfolding data suggests that the mechanism of nucleotide discrimination is possibly affected by the compacting of the hydrophobic core around residue Leu282. Subsequent movement of an adjacent surface residue, Arg283, produces a slight increase in volume of the pocket that may accommodate the incoming correct base pair. The structural changes of M282Lbeta ultimately lead to an overall reduction in polymerase fidelity.     

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.     

The E288K Colon Tumor Variant of DNA Polymerase β Is a Sequence Specific Mutator

DNA polymerase β (pol β) is the main polymerase involved in base excision repair (BER), which is a pathway responsible for the repair of tens of thousands of DNA lesions per cell per day. Our recent efforts in sequencing colon tumors showed that 40% of the tumors sequenced possessed a variant in the coding region of the POLB gene; one of these variants is E288K. Expression of the E288K variant in cells leads to an increase in the frequency of mutations at AT base pairs. In vitro, the E288K variant is as active as and binds one-base-gapped DNA with the same affinity as wild-type pol β. Single-turnover kinetic data for the E288K variant show that its mutator phenotype is specific for misincorporating opposite template A up to 6-fold more than the wild-type enzyme and that this is due to a decrease in the degree of discrimination in nucleotide binding. Molecular modeling suggests that the substitution of Lys at position 288 causes the polymerase to adopt a more open conformation, which may be disrupting the nucleotide binding pocket. This may explain the reduced degree of discrimination at the level of nucleotide binding. The enhanced mutagenesis of the E288K variant could lead to genomic instability and ultimately a malignant tumor phenotype.     

Enhanced polymerase activity permits efficient synthesis by cancer-associated DNA polymerase ϵ variants at low dNTP levels

Amino acid substitutions in the exonuclease domain of DNA polymerase ϵ (Polϵ) cause ultramutated tumors. Studies in model organisms suggested pathogenic mechanisms distinct from a simple loss of exonuclease. These mechanisms remain unclear for most recurrent Polϵ mutations. Particularly, the highly prevalent V411L variant remained a long-standing puzzle with no detectable mutator effect in yeast despite the unequivocal association with ultramutation in cancers. Using purified four-subunit yeast Polϵ, we assessed the consequences of substitutions mimicking human V411L, S459F, F367S, L424V and D275V. While the effects on exonuclease activity vary widely, all common cancer-associated variants have increased DNA polymerase activity. Notably, the analog of Polϵ-V411L is among the strongest polymerases, and structural analysis suggests defective polymerase-to-exonuclease site switching. We further show that the V411L analog produces a robust mutator phenotype in strains that lack mismatch repair, indicating a high rate of replication errors. Lastly, unlike wild-type and exonuclease-dead Polϵ, hyperactive variants efficiently synthesize DNA at low dNTP concentrations. We propose that this characteristic could promote cancer cell survival and preferential participation of mutator polymerases in replication during metabolic stress. Our results support the notion that polymerase fitness, rather than low fidelity alone, is an important determinant of variant pathogenicity.     

The dinB gene encodes a novel E. coli DNA polymerase, DNA pol IV, involved in mutagenesis.

In Escherichia coli, the dinB gene is required for the SOS-induced lambda untargeted mutagenesis pathway and confers a mutator phenotype to the cell when the gene product is overexpressed. Here, we report that the purified DinB protein is a DNA polymerase. This novel E. coli DNA polymerase (pol IV) is shown to be strictly distributive, devoid of proofreading activity, and prone to elongate bulged (misaligned) primer/template structures. Site-directed mutagenesis experiments of dinB also demonstrate that the polymerase activity of DinB is required for its in vivo mutagenicity. Along with the sequence homologies previously found within the UmuC-like protein family, these results indicate that the uncovered DNA polymerase activity may be a common feature of all these homologous proteins.     

The E295K Cancer Variant of Human Polymerase β Favors the Mismatch Conformational Pathway during Nucleotide Selection

DNA polymerase β (pol β) is responsible for gap filling synthesis during repair of damaged DNA as part of the base excision repair pathway. Human pol β mutations were recently identified in a high percentage (∼30%) of tumors. Characterization of specific cancer variants is particularly useful to further the understanding of the general mechanism of pol β while providing context to disease contribution. We showed that expression of the carcinoma variant E295K induces cellular transformation. The poor polymerase activity exhibited by the variant was hypothesized to be caused by the destabilization of proper active site assembly by the glutamate to lysine mutation. Here, we show that this variant exhibits an unusual preference for binding dCTP opposite a templating adenine over the cognate dTTP. Biochemical studies indicate that the noncognate competes with the cognate nucleotide for binding to the polymerase active site with the noncognate incorporation a function of higher affinity and not increased activity. In the crystal structure of the variant bound to dA:dCTP, the fingers domain closes around the mismatched base pair. Nucleotide incorporation is hindered because key residues in the polymerase active site are not properly positioned for nucleotidyl transfer. In contrast to the noncognate dCTP, neither the cognate dTTP nor its nonhydrolyzable analog induced fingers closure, as isomorphous difference Fourier maps show that the cognate nucleotides are bound to the open state of the polymerase. Comparison with published structures provides insight into the structural rearrangements within pol β that occur during the process of nucleotide discrimination.     

Increased rates of genomic deletions generated by mutations in the yeast gene encoding DNA polymerase delta or by decreases in the cellular levels of DNA polymerase delta

In Saccharomyces cerevisiae, POL3 encodes the catalytic subunit of DNA polymerase delta. While yeast POL3 mutant strains that lack the proofreading exonuclease activity of the polymerase have a strong mutator phenotype, little is known regarding the role of other Pol3p domains in mutation avoidance. We identified a number of pol3 mutations in regions outside of the exonuclease domain that have a mutator phenotype, substantially elevating the frequency of deletions. These deletions appear to reflect an increased frequency of DNA polymerase slippage. In addition, we demonstrate that reduction in the level of wild-type DNA polymerase results in a similar mutator phenotype. Lowered levels of DNA polymerase also result in increased sensitivity to the DNA-damaging agent methyl methane sulfonate. We conclude that both the quantity and the quality of DNA polymerase delta is important in ensuring genome stability.