The DNA polymerase activity of Saccharomyces cerevisiae Rev1 is biologically significant

A cell's ability to tolerate DNA damage is directly connected to the human development of diseases and cancer. To better understand the processes underlying mutagenesis, we studied the cell's reliance on the potentially error-prone translesion synthesis (TLS), and an error-free, template-switching pathway in Saccharomyces cerevisiae. The primary proteins mediating S. cerevisiae TLS are three DNA polymerases (Pols): Rev1, Pol ζ (Rev3/7), and Pol η (Rad30), all with human homologs. Rev1's noncatalytic role in recruiting other DNA polymerases is known to be important for TLS. However, the biological significance of Rev1's unusual conserved DNA polymerase activity, which inserts dC, is much less well understood. Here, we demonstrate that inactivating Rev1's DNA polymerase function sensitizes cells to both chronic and acute exposure to 4-nitroquinoline-1-oxide (4-NQO) but not to UV or cisplatin. Full Rev1-dependent resistance to 4-NQO, however, also requires the additional Rev1 functions. When error-free tolerance is disrupted through deletion of MMS2, Rev1's catalytic activity is more vital for 4-NQO resistance, possibly explaining why the biological significance of Rev1's catalytic activity has been elusive. In the presence or absence of Mms2-dependent error-free tolerance, the catalytic dead strain of Rev1 exhibits a lower 4-NQO-induced mutation frequency than wild type. Furthermore, Pol ζ, but not Pol η, also contributes to 4-NQO resistance. These results show that Rev1's catalytic activity is important in vivo when the cell has to cope with specific DNA lesions, such as N(2)-dG.     

Antimutator variants of DNA polymerases

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

Alternative splicing at exon 2 results in the loss of the catalytic activity of mouse DNA polymerase iota in vitro

Y-family DNA polymerase iota (Pol ι) possesses both DNA polymerase and dRP lyase activities and was suggested to be involved in DNA translesion synthesis and base excision repair in mammals. The 129 strain of mice and its derivatives have a natural nonsense codon mutation in the second exon of the Pol ι gene resulting in truncation of the Pol ι protein. These mice were widely used as a Pol ι-null model for in vivo studies of the Pol ι function. However whether 129-derived strains of mice are fully deficient in the Pol ι functions was a subject of discussion since Pol ι mRNA undergoes alternative splicing at exon 2. Here we report purification of mouse Pol ι lacking the region encoded by exon 2, which includes several conserved residues involved in catalysis. We show that the deletion abrogates both the DNA polymerase and dRP lyase activities of Pol ι in the presence of either Mg²⁺ or Mn²⁺ ions. Thus, 129-derived strains of mice express catalytically inactive alternatively spliced Pol ι variant, whose cellular functions, if any exist, remain to be established.     

Antimutator variants of DNA polymerases

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

The DNA polymerase domain of pol(epsilon) is required for rapid,efficient, and highly accurate chromosomal DNA replication,telomere length maintenance,and normal cell senescence in Saccharomyces cerevisiae

Saccharomyces cerevisiae POL2 encodes the catalytic subunit of DNA polymerase epsilon. This study investigates the cellular functions performed by the polymerase domain of Pol2p and its role in DNA metabolism. The pol2-16 mutation has a deletion in the catalytic domain of DNA polymerase epsilon that eliminates its polymerase and exonuclease activities. It is a viable mutant, which displays temperature sensitivity for growth and a defect in elongation step of chromosomal DNA replication even at permissive temperatures. This mutation is synthetic lethal in combination with temperature-sensitive mutants or the 3'- to 5'-exonuclease-deficient mutant of DNA polymerase delta in a haploid cell. These results suggest that the catalytic activity of DNA polymerase epsilon participates in the same pathway as DNA polymerase delta, and this is consistent with the observation that DNA polymerases delta and epsilon colocalize in some punctate foci on yeast chromatids during S phase. The pol2-16 mutant senesces more rapidly than wild type strain and also has shorter telomeres. These results indicate that the DNA polymerase domain of Pol2p is required for rapid, efficient, and highly accurate chromosomal DNA replication in yeast.     

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

The function of the African swine fever virus (ASFV) reparative DNA polymerase, Pol X, was investigated in the context of virus infection. Pol X is a late structural protein that localizes at cytoplasmic viral factories during DNA replication. Using an ASFV deletion mutant lacking the Pol X gene, we have shown that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the growth of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The replication of the mutant virus in Vero cells presents sensitivity to oxidative damage, and mutational analysis of viral DNA shows that deletion of Pol X results in an increase in the mutation frequency in macrophages. Therefore, our data reveal a biological role for ASFV Pol X in the context of the infected cell in the preservation of viral genetic information.     

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.     

UV-B radiation induces epithelial tumors in mice lacking DNA polymerase eta and mesenchymal tumors in mice deficient for DNA polymerase iota

DNA polymerase eta (Pol eta) is the product of the Polh gene, which is responsible for the group variant of xeroderma pigmentosum, a rare inherited recessive disease which is characterized by susceptibility to sunlight-induced skin cancer. We recently reported in a study of Polh mutant mice that Pol eta is involved in the somatic hypermutation of immunoglobulin genes, but the cancer predisposition of Polh-/- mice has not been examined until very recently. Another translesion synthesis polymerase, Pol iota, a Pol eta paralog encoded by the Poli gene, is naturally deficient in the 129 mouse strain, and the function of Pol iota is enigmatic. Here, we generated Polh Poli double-deficient mice and compared the tumor susceptibility of them with Polh- or Poli-deficient animals under the same genetic background. While Pol iota deficiency does not influence the UV sensitivity of mouse fibroblasts irrespective of Polh genotype, Polh Poli double-deficient mice show slightly earlier onset of skin tumor formation. Intriguingly, histological diagnosis after chronic treatment with UV light reveals that Pol iota deficiency leads to the formation of mesenchymal tumors, such as sarcomas, that are not observed in Polh(-/-) mice. These results suggest the involvement of the Pol eta and Pol iota proteins in UV-induced skin carcinogenesis.     

Relationship of Bacillus subtilis DNA polymerase III to bacteriophage PBS2-induced DNA polymerase and to the replication of uracil-containing DNA.

In vivo studies of PBS2 phage replication in a temperature-sensitive Bacillus subtilis DNA polymerase III (Pol III) mutant and a temperature-resistant revertant of this mutant have suggested the possible involvement of Pol III in PBS2 DNA synthesis. Previous results with 6-(p-hydroxyphenylazo)-uracil (HPUra), a specific inhibitor of Pol III and DNA replication in uninfected cells, suggest that Pol III is not involved in phage DNA replication, due to its resistance to this drug. Experiments were designed to examine possible explanations for this apparent contradiction. First, assays of the host Pol III and the phage-induced DNA polymerase activities in extracts indicated that a labile Pol III did not result in a labile phage-induced enzyme, suggesting that this new polymerase is not a modified HPUra-resistant form of Pol III. Indeed the purified phage-induced enzyme was resistant to the active, reduced form of HPUra under all assay conditions tested. Since in vitro Pol III was capable of replicating the uracil-containing DNA found in this phage, the sensitivity of the purified enzyme to reduced HPUra was examined using phage DNA as template-primer and dUTP as substrate; these new substrates did not affect the sensitivity of the host enzyme to the drug.     

Adaptation to DNA damage and stimulation of genetic instability: the double-edged sword mammalian DNA polymerase kappa

A major tolerance mechanism that functions to replicate damaged genomic DNA across lesions that have escaped elimination by repair mechanism is translesion DNA synthesis (TLS). DNA polymerase kappa (Pol kappa), a specialised low-fidelity DNA polymerase which is able to perform DNA synthesis across several damaged bases, is one of the enzymes involved in the process. The mutagenic nature of Pol kappa implies that its expression must be tightly regulated to prevent the formation of excessive genetic disorders along undamaged parts of the genome. Indeed, Pol kappa overexpression, which is notably observed in lung cancer, results not only in increased spontaneous mutagenesis, but also in pleiotropic alterations such as DNA breaks, genetic exchanges and aneuploidy. This review will discuss both aspects of DNA polymerase kappa, which can be considered as a genomic supervisor participating in genome maintenance and when misregulated as a genetic instability enhancer as well.     

Involvement of DNA polymerase beta in DNA replication and mutagenic consequences

Overexpression in mammalian cells of the error-prone DNA polymerase beta (Pol beta) has been found to increase the spontaneous mutagenesis. Here, we investigated a possible mechanism used by Pol beta to be a genetic instability enhancer: its interference in replicative DNA synthesis, which is normally catalysed by the DNA polymerases alpha, delta and epsilon. By taking advantage of the ability to incorporate efficiently into DNA the chain terminator ddCTP as well as the oxidised nucleotide 8-oxo-dGTP, we show here that purified Pol beta can compete with the replicative DNA polymerases during replication in vitro of duplex DNA when added to human cell extracts. We found that involvement of Pol beta lowers replication fidelity and results in a modified error-specificity. Furthermore, we demonstrated that involvement of Pol beta occurred during synthesis of the lagging strand. These in vitro data provide one possible explanation of how overexpression of the enzyme could perturb the genetic instability in mammalian cells. We discuss these findings within the scope of the up-regulation of Pol beta in many cancer cells.     

Role of Escherichia coli DNA polymerase I in conferring viability upon the dnaN159 mutant strain

The Escherichia coli dnaN159 allele encodes a mutant form of the beta-sliding clamp (beta159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain. In addition, we demonstrate that the beta clamp stimulates the processivity of Pol I in vitro and that beta159 is impaired for this activity. The reduced ability of beta159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the beta clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 DeltapolA strain.     

Expansion of CAG triplet repeats by human DNA polymerases λ and β in vitro,is regulated by flap endonuclease 1 and DNA ligase 1

Huntington's disease (HD) is a neurological genetic disorder caused by the expansion of the CAG trinucleotide repeats (TNR) in the N-terminal region of coding sequence of the Huntingtin's (HTT) gene. This results in the addition of a poly-glutamine tract within the Huntingtin protein, resulting in its pathological form. The mechanism by which TRN expansion takes place is not yet fully understood. We have recently shown that DNA polymerase (Pol) β can promote the microhomology-mediated end joining and triplet expansion of a substrate mimicking a double strand break in the TNR region of the HTT gene. Here we show that TNR expansion is dependent on the structure of the DNA substrate, as well as on the two essential Pol β co-factors: flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). We found that Fen1 significantly stimulated TNR expansion by Pol β, but not by the related enzyme Pol λ, and subsequent ligation of the DNA products by Lig1. Interestingly, the deletion of N-terminal domains of Pol λ, resulted in an enzyme which displayed properties more similar to Pol β, suggesting a possible evolutionary mechanism. These results may suggest a novel mechanism for somatic TNR expansion in HD.     

Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae

Most replicases are multi-subunit complexes. DNA polymerase epsilon from Saccharomyces cerevisiae is composed of four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. Pol2p and Dpb2p are essential. To investigate a possible role for the Dpb2p subunit in maintaining the fidelity of DNA replication, we isolated temperature-sensitive mutants in the DPB2 gene. Several of the newly isolated dpb2 alleles are strong mutators, exhibiting mutation rates equivalent to pol2 mutants defective in the 3' --> 5' proofreading exonuclease (pol2-4) or to mutants defective in mismatch repair (msh6). The dpb2 pol2-4 and dpb2 msh6 double mutants show a synergistic increase in mutation rate, indicating that the mutations arising in the dpb2 mutants are due to DNA replication errors normally corrected by mismatch repair. The dpb2 mutations decrease the affinity of Dpb2p for the Pol2p subunit as measured by two-hybrid analysis, providing a possible mechanistic explanation for the loss of high-fidelity synthesis. Our results show that DNA polymerase subunits other than those housing the DNA polymerase and 3' --> 5' exonuclease are essential in controlling the level of spontaneous mutagenesis and genetic stability in yeast cells.     

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

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

DNA polymerase beta can incorporate ribonucleotides during DNA synthesis of undamaged and CPD-damaged DNA

Overexpression of the error-prone DNA polymerase beta (Pol beta) has been found to increase spontaneous mutagenesis by competing with the replicative polymerases during DNA replication. Here, we investigate an additional mechanism potentially used by Pol beta to enhance genetic instability via its ability to incorporate ribonucleotides into DNA. By using an in vitro primer extension assay, we show that purified human and calf thymus Pol beta can synthesize up to 8-mer long RNA. Moreover, Pol beta can efficiently incorporate rCTP opposite G in the absence of dCTP and, to a lesser extent, rATP opposite T in the absence of dATP and rGTP opposite C in the absence of dGTP. Recently, Pol beta was shown to catalyze in vitro translesion replication of a thymine cyclobutane pyrimidine dimer (CPD). Here, we investigate if ribonucleotides could be incorporated opposite the CPD damage and modulate the efficiency of the bypass process. We find that all four rNTPs can be incorporated opposite the CPD lesion, and that this process affects translesion synthesis. We discuss how incorporation of ribonucleotides into DNA may contribute to the high frequency of mutagenesis observed in Pol beta up-regulating cells.     

Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.     

Mutator effects of overproducing DNA polymerase eta (Rad30) and its catalytically inactive variant in yeast

DNA polymerase eta synthesizes DNA in vitro with low fidelity. Based on this, here we report the effects of deletion or increased expression of yeast RAD30 gene, encoding for polymerase eta (Pol eta), on spontaneous mutagenesis in vivo. Deletion of RAD30 did not affect spontaneous mutagenesis. Overproduction of Rad30p was slightly mutagenic in a wild-type yeast strain and moderately mutagenic in strains with inactive 3'-->5'-exonuclease of DNA polymerase epsilon or DNA mismatch repair. These data suggest that excess Rad30p reduces replication fidelity in vivo and that the induced errors may be corrected by exonucleolytic proofreading and DNA mismatch repair. However, the magnitude of mutator effect (only up to 10-fold) suggests that the replication fork is protected from inaccurate synthesis by Pol eta in the absence of DNA damage. Overproduction of catalytically inactive Rad30p was also mutagenic, suggesting that much of the mutator effect results from indirect perturbation of replication rather than from direct misincorporation by Pol eta. Moreover, while excess wild-type Pol eta primarily induced base substitutions in the msh6 and pms1 strains, excess inactive Rad30p induced both base substitutions and frameshifts. This suggests that more than one mutagenic mechanism is operating when RAD30 is overexpressed.