The relative roles in vivo of Saccharomyces cerevisiae Pol eta, Pol zeta, Rev1 protein and Pol32 in the bypass and mutation induction of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer

We have investigated the relative roles in vivo of Saccharomyces cerevisiae DNA polymerase eta, DNA polymerase zeta, Rev1 protein, and the DNA polymerase delta subunit, Pol32, in the bypass of an abasic site, T-T (6-4) photoadduct and T-T cis-syn cyclobutane dimer, by transforming strains deleted for RAD30, REV3, REV1, or POL32 with duplex plasmids carrying one of these DNA lesions located within a 28-nucleotide single-stranded region. DNA polymerase eta was found to be involved only rarely in the bypass of the T-T (6-4) photoadduct or the abasic sites in the sequence context used, although, as expected, it was solely responsible for the bypass of the T-T dimer. We argue that DNA polymerase zeta, rather than DNA polymerase delta as previously suggested, is responsible for insertion in bypass events other than those in which polymerase eta performs this function. However, DNA polymerase delta is involved indirectly in mutagenesis, since the strain lacking its Pol32 subunit, known to be deficient in mutagenesis, shows as little bypass of the T-T (6-4) photoadduct or the abasic sites as those deficient in Pol zeta or Rev1. In contrast, bypass of the T-T dimer in the pol32delta strain occurs at the wild-type frequency.     

Yeast Rev1 is cell cycle regulated, phosphorylated in response to DNA damage and its binding to chromosomes is dependent upon MEC1

Translesion DNA synthesis (TLS) is one of the mechanisms involved in lesion bypass during DNA replication. Three TLS polymerases (Pol) are present in the yeast Saccharomyces cerevisiae: Pol zeta, Pol eta and the product of the REV1 gene. Rev1 is considered a deoxycytidyl transferase because it almost exclusively inserts a C residue in front of the lesion. Even though REV1 is required for most of the UV-induced and spontaneous mutagenesis events, the role of Rev1 is poorly understood since its polymerase activity is often dispensable. Rev1 interacts with several TLS polymerases in mammalian cells and may act as a platform in the switching mechanism required to substitute a replicative polymerase with a TLS polymerase at the sites of DNA lesions. Here we show that yeast Rev1 is a phosphoprotein, and the level of this modification is cell cycle regulated under normal growing conditions. Rev1 is unphosphorylated in G1, starts to be modified while cells are passing S phase and it becomes hyper-phosphorylated in mitosis. Rev1 is also hyper-phosphorylated in response to a variety of DNA damaging agents, including treatment with a radiomimetic drug mostly causing double-strand breaks (DSB). By using the chromosome spreading technique we found the Rev1 is bound to chromosomes throughout the cell cycle, and its binding does not significantly increase in response to genotoxic stress. Therefore, Rev1 phosphorylation does not appear to modulate its binding to chromosomes, suggesting that such modification may influence other aspects of the TLS process. Rev1 binding under damaged and undamaged conditions, is at least partially dependent on MEC1, a gene playing a pivotal role in the DNA damage checkpoint cascade. This genetic dependency may suggest a role for MEC1 in spontaneous mutagenesis events, which require a functional REV1 gene.     

Evidence for a second function for Saccharomyces cerevisiae Rev1p

The function of the Saccharomyces cerevisiae REV1 gene is required for translesion replication and mutagenesis induced by a wide variety of DNA-damaging agents. We showed previously that Rev1p possesses a deoxycytidyl transferase activity, which incorporates dCMP opposite abasic sites in the DNA template, and that dCMP insertion is the major event during bypass of an abasic site in vivo. However, we now find that Rev1p function is needed for the bypass of a T-T (6-4) UV photoproduct, a process in which dCMP incorporation occurs only very rarely, indicating that Rev1p possesses a second function. In addition, we find that Rev1p function is, as expected, required for bypass of an abasic site. However, replication past this lesion was also much reduced in the G-193R rev1-1 mutant, which we find retains substantial levels of deoxycytidyl transferase activity. This mutant is, therefore, presumably deficient principally in the second, at present poorly defined, function. The bypass of an abasic site and T-T (6-4) lesion also depended on REV3 function, but neither it nor REV1 was required for replication past the T-T dimer; bypass of this lesion presumably depends on another enzyme.     

Disruption of the Rev3l-encoded catalytic subunit of polymerase zeta in mice results in early embryonic lethality

Polymerase zeta (Pol zeta) is an error-prone DNA polymerase [1], which in yeast is involved in trans-lesion synthesis (TLS) and is responsible for most of the ultraviolet (UV) radiation-induced and spontaneous mutagenesis [2-4]. Pol zeta consists of three subunits: REV1, a deoxycytidyl-transferase [5]; REV7, of unclear function [6]; and REV3, the catalytic subunit. REV3 alone is sufficient to carry out TLS, but association with REV1 and REV7 enhances its activity [5, 7]. Experiments using human cells treated with UV radiation indicate also that mammalian Pol zeta is involved in TLS [7]. The peculiar mutagenic activity of Pol zeta [4,7,8] suggests a possible role in somatic hypermutation of immunoglobulin (Ig) genes [9]. Here, we report that, unlike in yeast where the REV3 gene is not essential for life [4], disruption of the mouse homologue (Rev3l) resulted in early embryonic lethality. In Rev3l(-/-) embryos, no haematopoietic cells other than erythrocytes could be identified in the yolk sac. Rev3l(-/-) haematopoietic precursors were unable to expand in vitro and no haematopoietic cells could be derived from the intraembryonic haematogenic compartment (splanchnopleura). Fibroblasts could not be derived from the Rev3l(-/-) embryos, and Rev3l(-/-) embryonic stem (ES) cells could not be obtained. This is the first evidence that an enzyme involved in TLS is critical for mammalian development.     

Proliferating cell nuclear antigen promotes translesion synthesis by DNA polymerase zeta

DNA polymerase zeta (Pol zeta), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol zeta, yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol zeta on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol zeta. Efficient PCNA stimulation required that PCNA was prevented from sliding off the damage-containing model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase delta or eta (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damage-induced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol zeta or by DNA polymerase delta.     

Alteration of ultraviolet-induced mutagenesis in yeast through molecular modulation of the REV3 and REV7 gene expression

DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition. Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein. Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.     

DNA polymerase zeta （pol ζ） in higher eukaryotes

Most current knowledge about DNA polymerase zeta （pol ζ） comes from studies of the enzyme in the budding yeast Saccharomyces cerevisiae, where pol ζ consists of a complex of the catalytic subunit Rev3 with Rev7, which associates with Revl. Most spontaneous and induced mutagenesis in yeast is dependent on these gene products, and yeast pol can mediate translesion DNA synthesis past some adducts in DNA templates. Study of the homologous gene products in higher eukaryotes is in a relatively early stage, but additional functions for the eukaryotic proteins are already apparent. Suppression of vertebrate REV3L function not only reduces induced point mutagenesis but also causes larger-scale genome instability by raising the frequency of spontaneous chromosome translocations. Disruption of Rev3L function is tolerated in Drosophila, Arabidopsis, and in vertebrate cell lines under some conditions, but is incompatible with mouse embryonic development. Functions for REV3L and REV7（MAD2B） in higher eukaryotes have been suggested not only in translesion DNA synthesis but also in some forms of homologous recombination, repair of interstrand DNA crosslinks, somatic hypermutation of immunoglobulin genes and cell-cycle control. This review discusses recent developments in these areas.     

The CysB motif of Rev3p involved in the formation of the four‐subunit DNA polymerase ζ is required for defective‐replisome‐induced mutagenesis

Eukaryotic DNA replication is performed by high‐fidelity multi‐subunit replicative B‐family DNA polymerases (Pols) α, δ and ?. Those complexes are composed of catalytic and accessory subunits and organized in multicomplex machinery: the replisome. The fourth B‐family member, DNA polymerase zeta (Pol ζ), is responsible for a large portion of mutagenesis in eukaryotic cells. Two forms of Pol ζ have been identified, a hetero‐dimeric (Pol ζ₂) and a hetero‐tetrameric (Pol ζ₄) ones and recent data have demonstrated that Pol ζ₄ is responsible for damage‐induced mutagenesis. Here, using yeast Pol ζ mutant defective in the assembly of the Pol ζ four‐subunit form, we show in vivo that [4Fe‐4S] cluster in Pol ζ catalytic subunit (Rev3p) is also required for spontaneous (wild‐type cells) and defective‐replisome‐induced mutagenesis – DRIM (pol3‐Y708A, pol2‐1 or psf1‐100 cells), when cells are not treated with any external damaging agents.     

An essential role for REV3 in mammalian cell survival: absence of REV3 induces p53-independent embryonic death

The REV3 gene of budding yeast encodes the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. While REV3-null yeast mutants are viable and exhibit normal growth, Rev3-deficient mice die around midgestation of embryogenesis, which is accompanied by massive apoptosis of cells within the embryo proper. We have investigated whether REV3 is required for the survival of mouse cells and whether the embryonic lethality caused by REV3 deficiency can be rescued by introduction of a Rev3 transgene or by inactivation of p53, the cellular gatekeeper that regulates DNA damage-induced apoptosis. We show that Rev3(-/-) blastocysts were unable to survive and grow in culture but expression of a Rev3 transgene restored their outgrowth. Moreover, Rev3 transgene expression suppressed the apoptosis in E7.5 Rev3(-/-) embryos. The Rev3(-/-) embryonic lethality, however, was not rescued by either Rev3 transgene expression or p53 deficiency. These results reveal an essential role for REV3 in the survival and growth of mammalian cells and suggest that Rev3(-/-) embryonic death occurs in a p53-independent pathway.     

Polymerase zeta dependency of increased adaptive mutation frequencies in nucleotide excision repair-deficient yeast strains

Reversions of an auxotrophy-causing frameshift allele during prolonged starvation of yeast cells were used as a means to elucidate the mechanisms concerned with the generation of spontaneous adaptive mutations in cell cycle-arrested cells. Whereas about 50% of these reversions were previously shown to depend on the non-homologous end joining pathway of DNA double-strand break repair, the origin of the residual 50% remains unknown. In search for a mechanism for generation of the latter fraction of reversions we examined the role of the translesion synthesis (TLS) polymerases zeta, eta and Rev1p in cells with wild-type or impaired nucleotide excision repair (NER) capacity. The basal level of adaptive mutations in the repair-proficient wild type was not influenced by disruptions of the genes coding for these three TLS polymerases. Intriguingly, a deficiency in NER by disruption of RAD14, RAD16 or RAD26 resulted in a significantly higher frequency of adaptive mutation, yet this increase was strictly dependent on an intact REV3 gene, coding for the catalytic subunit of polymerase zeta. Furthermore, we observed that intact REV3 was also required for the occurrence of increased frequencies of adaptive mutants in the NER-proficient wild type following UV irradiation. While in proliferating cells the translesion synthesis function of polymerase zeta is connected to DNA replication, our data suggest that in cell cycle-arrested cells this enzyme is able to carry out either TLS or error-prone polymerization along an undamaged template in the course of repair processes. Such a hitherto unappreciated activity of polymerase zeta in non-replicating cells may contribute to the incidence of mutations in evolution, aging and cancer.     

ATR homolog Mec1 controls association of DNA polymerase zeta-Rev1 complex with regions near a double-strand break

DNA polymerase zeta (Polzeta) and Rev1 contribute to the bypassing of DNA lesions, termed translesion DNA synthesis (TLS). Polzeta consists of two subunits, one encoded by REV3 (the catalytic subunit) and the other encoded by REV7. Rev1 acts as a deoxycytidyl transferase, inserting dCMP opposite lesions. Polzeta and Rev1 have been shown to operate in the same TLS pathway in the budding yeast Saccharomyces cerevisiae. Here, we show that budding yeast Polzeta and Rev1 form a complex and associate together with double-strand breaks (DSBs). As a component of the Polzeta-Rev1 complex, Rev1 plays a noncatalytic role in the association with DSBs. In budding yeast, the ATR-homolog Mec1 plays a central role in the DNA-damage checkpoint response. We further show that Mec1-dependent phosphorylation promotes the Polzeta-Rev1 association with DSBs. Rev1 association with DSBs requires neither the function of the Rad24 checkpoint-clamp loader nor the Rad6-Rad18-mediated ubiquitination of PCNA. Our results reveal a novel role of Mec1 in the localization of the Polzeta-Rev1 complex to DNA lesions and highlight a linkage of TLS polymerases to the checkpoint response.     

Epistatic participation of REV1 and REV3 in the formation of UV-induced frameshift mutations in cell cycle-arrested yeast cells

Mutations arising in times of cell cycle arrest may provide a selective advantage for unicellular organisms adapting to environmental changes. For multicellular organisms, however, they may pose a serious threat, in that such mutations in somatic cells contribute to carcinogenesis and ageing. The budding yeast Saccharomyces cerevisiae presents a convenient model system for studying the incidence and the mechanisms of stationary-phase mutation in a eukaryotic organism. Having studied the emergence of frameshift mutants after several days of starvation-induced cell cycle arrest, we previously reported that all (potentially error-prone) translesion synthesis (TLS) enzymes identified in S. cerevisiae did not contribute to the basal level of spontaneous stationary-phase mutations. However, we observed that an increased frequency of stationary-phase frameshift mutations, brought about by a defective nucleotide excision repair (NER) pathway or by UV irradiation, was dependent on Rev3p, the catalytic subunit of the TLS polymerase zeta (Pol zeta). Employing the same two conditions, we now examined the effect of deletions of the genes coding for polymerase eta (Pol eta) (RAD30) and Rev1p (REV1). In a NER-deficient strain background, the increased incidence of stationary-phase mutations was only moderately influenced by a lack of Pol eta but completely reduced to wild type level by a knockout of the REV1 gene. UV-induced stationary-phase mutations were abundant in wild type and rad30Delta strains, but substantially reduced in a rev1Delta as well as a rev3Delta strain. The similarity of the rev1Delta and the rev3Delta phenotype and an epistatic relationship evident from experiments with a double-deficient strain suggests a participation of Rev1p and Rev3p in the same mutagenic pathway. Based on these results, we propose that the response of cell cycle-arrested cells to an excess of exo- or endogenously induced DNA damage includes a novel replication-independent cooperative function of Rev1p and Pol zeta, which has the potential to generate mutations.     

DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures

DNA polymerase ζ (Pol ζ) and Rev1 are key players in translesion DNA synthesis. The error-prone Pol ζ can also participate in replication of undamaged DNA when the normal replisome is impaired. Here we define the nature of the replication disturbances that trigger the recruitment of error-prone polymerases in the absence of DNA damage and describe the specific roles of Rev1 and Pol ζ in handling these disturbances. We show that Pol ζ/Rev1-dependent mutations occur at sites of replication stalling at short repeated sequences capable of forming hairpin structures. The Rev1 deoxycytidyl transferase can take over the stalled replicative polymerase and incorporate an additional ‘C’ at the hairpin base. Full hairpin bypass often involves template-switching DNA synthesis, subsequent realignment generating multiply mismatched primer termini and extension of these termini by Pol ζ. The postreplicative pathway dependent on polyubiquitylation of proliferating cell nuclear antigen provides a backup mechanism for accurate bypass of these sequences that is primarily used when the Pol ζ/Rev1-dependent pathway is inactive. The results emphasize the pivotal role of noncanonical DNA structures in mutagenesis and reveal the long-sought-after mechanism of complex mutations that represent a unique signature of Pol ζ.     

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.     

Mutagenic and recombinagenic responses to defective DNA polymerase delta are facilitated by the Rev1 protein in pol3-t mutants of Saccharomyces cerevisiae

Defective DNA replication can result in substantial increases in the level of genome instability. In the yeast Saccharomyces cerevisiae, the pol3-t allele confers a defect in the catalytic subunit of replicative DNA polymerase delta that results in increased rates of mutagenesis, recombination, and chromosome loss, perhaps by increasing the rate of replicative polymerase failure. The translesion polymerases Pol eta, Pol zeta, and Rev1 are part of a suite of factors in yeast that can act at sites of replicative polymerase failure. While mutants defective in the translesion polymerases alone displayed few defects, loss of Rev1 was found to suppress the increased rates of spontaneous mutation, recombination, and chromosome loss observed in pol3-t mutants. These results suggest that Rev1 may be involved in facilitating mutagenic and recombinagenic responses to the failure of Pol delta. Genome stability, therefore, may reflect a dynamic relationship between primary and auxiliary DNA polymerases.     

Overexpression of DNA polymerase zeta reduces the mitochondrial mutability caused by pathological mutations in DNA polymerase gamma in yeast

In yeast, DNA polymerase zeta (Rev3 and Rev7) and Rev1, involved in the error-prone translesion synthesis during replication of nuclear DNA, localize also in mitochondria. We show that overexpression of Rev3 reduced the mtDNA extended mutability caused by a subclass of pathological mutations in Mip1, the yeast mitochondrial DNA polymerase orthologous to human Pol gamma. This beneficial effect was synergistic with the effect achieved by increasing the dNTPs pools. Since overexpression of Rev3 is detrimental for nuclear DNA mutability, we constructed a mutant Rev3 isoform unable to migrate into the nucleus: its overexpression reduced mtDNA mutability without increasing the nuclear one.     

Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis

Pol kappa and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Pol kappa. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Pol zeta) and with two other Y-family polymerases, Pol iota and Pol eta. Mouse Pol kappa, Rev7, Pol iota and Pol eta each bind to the same approximately 100 amino acid C-terminal region of Rev1. Furthermore, Rev7 competes directly with Pol kappa for binding to the Rev1 C-terminus. Notwithstanding the physical interaction between Rev1 and Pol kappa, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer-termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein-protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined.     

Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae

Translesion synthesis (TLS) in Saccharomyces cerevisiae requires at least Rev1p and polymerase zeta (Pol zeta), a complex of the Rev3 polymerase and its accessory factor Rev7p. Although their precise role(s) are poorly characterized, in vitro studies suggest that each protein contributes to TLS in a manner dependent on the particular lesion and surrounding DNA sequence. In the present study, strand segregation analysis is used to attempt to identify the role(s) of the Rev1 and Rev7 proteins during TLS. This assay uses double-stranded plasmids containing a genetic marker opposite to a replication blocking lesion (N-2-acetylaminofluorene; AAF) to measure TLS quantitatively and qualitatively in vivo. The AAF adduct is localized within a repetitive sequence in a manner that allows the formation of misaligned primer-template replication intermediates. Elongation from a misaligned intermediate fixes a frameshift mutation (slipped TLS), while extension of the correctly aligned lesion terminus yields error-free (non-slipped) TLS. The results indicate that there is a strong requirement for Rev7p during Pol zeta-mediated TLS measured in vivo. Furthermore, Rev1p is needed only for non-slipped TLS; slipped TLS remains efficient in its absence, revealing a previously uncharacterized Rev1p activity similar to Escherichia coli UmuDC function. Specifically, this activity is required for elongation from a correctly aligned lesion terminus.     

In vivo consequences of putative active site mutations in yeast DNA polymerases alpha, epsilon, delta, and zeta

Several amino acids in the active site of family A DNA polymerases contribute to accurate DNA synthesis. For two of these residues, family B DNA polymerases have conserved tyrosine residues in regions II and III that are suggested to have similar functions. Here we replaced each tyrosine with alanine in the catalytic subunits of yeast DNA polymerases alpha, delta, epsilon, and zeta and examined the consequences in vivo. Strains with the tyrosine substitution in the conserved SL/MYPS/N motif in region II in Pol delta or Pol epsilon are inviable. Strains with same substitution in Rev3, the catalytic subunit of Pol zeta, are nearly UV immutable, suggesting severe loss of function. A strain with this substitution in Pol alpha (pol1-Y869A) is viable, but it exhibits slow growth, sensitivity to hydroxyurea, and a spontaneous mutator phenotype for frameshifts and base substitutions. The pol1-Y869A/pol1-Y869A diploid exhibits aberrant growth. Thus, this tyrosine is critical for the function of all four eukaryotic family B DNA polymerases. Strains with a tyrosine substitution in the conserved NS/VxYG motif in region III in Pol alpha, -delta, or -epsilon are viable and a strain with the homologous substitution in Rev3 is UV mutable. The Pol alpha mutant has no obvious phenotype. The Pol epsilon (pol2-Y831A) mutant is slightly sensitive to hydroxyurea and is a semidominant mutator for spontaneous base substitutions and frameshifts. The Pol delta mutant (pol3-Y708A) grows slowly, is sensitive to hydroxyurea and methyl methanesulfonate, and is a strong base substitution and frameshift mutator. The pol3-Y708A/pol3-Y708A diploid grows slowly and aberrantly. Mutation rates in the Pol alpha, -delta, and -epsilon mutant strains are increased in a locus-specific manner by inactivation of PMS1-dependent DNA mismatch repair, suggesting that the mutator effects are due to reduced fidelity of chromosomal DNA replication. This could result directly from relaxed base selectivity of the mutant polymerases due to the amino acid changes in the polymerase active site. In addition, the alanine substitutions may impair catalytic function to allow a different polymerase to compete at the replication fork. This is supported by the observation that the pol3-Y708A mutation is recessive and its mutator effect is partially suppressed by disruption of the REV3 gene.     

Rev1 is essential for DNA damage tolerance and non-templated immunoglobulin gene mutation in a vertebrate cell line

The majority of DNA damage-induced mutagenesis in the yeast Saccharomyces cerevisiae arises as a result of translesion replication. This process is critically dependent on the deoxycytidyl transferase Rev1p, which forms a complex with the subunits of DNA polymerase zeta, Rev3p and Rev7p. To examine the role of Rev1 in vertebrate mutagenesis and the DNA damage response, we disrupted the gene in DT40 cells. Rev1-deficient DT40 grow slowly and are sensitive to a wide range of DNA-damaging agents. Homologous recombination repair is likely to be intact as basal and damage induced sister chromatid exchange and immunoglobulin gene conversion are unaffected. How ever, the mutant cells show a markedly reduced level of non-templated immunoglobulin gene mutation, indicating a defect in translesion bypass. Furthermore, ultraviolet exposure results in marked chromosome breakage, suggesting that replication gaps created in the absence of Rev1 cannot be efficiently repaired by recombination. Thus, Rev1-dependent translesion bypass and mutagenesis is likely to be a trade-off for the ability to complete replication of a damaged template and thereby maintain genome integrity.