Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.     

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.     

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.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerases.

The complete amino acid (aa) alignment of the N-terminal domain of 33 DNA-dependent DNA polymerases encompassing the putative segments Exo I, Exo II and Exo III, proposed by Bernad et al. [Cell 59 (1989) 219-228] to form a conserved 3'-5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases, allowed us to identify and/or correct some of the most conserved segments (Exo I, II and III) in certain DNA polymerases. In particular, the aa region of T4 DNA polymerase and other eukaryotic (viral and cellular) DNA polymerases previously proposed as Exo I segment 1, did not align with the Exo I segment of Escherichia coli DNA polymerase I (PolI)-like and protein-primed DNA polymerases; instead, a new conserved region of aa similarity was identified in T4 DNA polymerase and eukaryotic (viral and cellular) DNA polymerases as their corresponding Exo I segment. Therefore, according to our alignment, the recently reported T4 DNA polymerase site-directed mutants, D189A and E191A [Reha-Krantz et al., Proc. Natl. Acad. Sci. USA 88 (1991) 2417-2421], do not correspond to what we now consider the critical Exo I motif of PolI. As discussed in this communication, the functional importance of conserved segments Exo I, Exo II and Exo III is supported by site-directed mutagenesis in PolI, and in phi 29, T7 and delta(Sc) DNA polymerases. Furthermore, genetically selected T4 DNA polymerase mutator mutants form two main clusters, centered in the conserved segment Exo III and in the newly identified Exo I segment.     

Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork

Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question. To begin examining the relative intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon. Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa. We also obtained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain. However, the data suggest that neither this effect nor differential replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion. Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork.     

Structural mechanism for coordination of proofreading and polymerase activities in archaeal DNA polymerases

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.     

The multiple biological roles of the 3'-->5' exonuclease of Saccharomyces cerevisiae DNA polymerase delta require switching between the polymerase and exonuclease domains

Until recently, the only biological function attributed to the 3'-->5' exonuclease activity of DNA polymerases was proofreading of replication errors. Based on genetic and biochemical analysis of the 3'-->5' exonuclease of yeast DNA polymerase delta (Pol delta) we have discerned additional biological roles for this exonuclease in Okazaki fragment maturation and mismatch repair. We asked whether Pol delta exonuclease performs all these biological functions in association with the replicative complex or as an exonuclease separate from the replicating holoenzyme. We have identified yeast Pol delta mutants at Leu523 that are defective in processive DNA synthesis when the rate of misincorporation is high because of a deoxynucleoside triphosphate (dNTP) imbalance. Yet the mutants retain robust 3'-->5' exonuclease activity. Based on biochemical studies, the mutant enzymes appear to be impaired in switching of the nascent 3' end between the polymerase and the exonuclease sites, resulting in severely impaired biological functions. Mutation rates and spectra and synergistic interactions of the pol3-L523X mutations with msh2, exo1, and rad27/fen1 defects were indistinguishable from those observed with previously studied exonuclease-defective mutants of the Pol delta. We conclude that the three biological functions of the 3'-->5' exonuclease addressed in this study are performed intramolecularly within the replicating holoenzyme.     

Characterization of a replicative DNA polymerase mutant with reduced fidelity and increased translesion synthesis capacity

Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase alpha, delta, epsilon or zeta, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure-function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 A crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol.     

DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.     

Kinetics of error generation in homologous B-family DNA polymerases

The kinetics of forming a proper Watson–Crick base pair as well incorporating bases opposite furan, an abasic site analog, have been well characterized for the B Family replicative DNA polymerase from bacteriophage T4. Structural studies of these reactions, however, have only been performed with the homologous enzyme from bacteriophage RB69. In this work, the homologous enzymes from RB69 and T4 were compared in parallel reactions to determine the relative abilities of the two polymerases to incorporate correct nucleotides as well as to form improper pairings. The kinetic rates for three different exonuclease mutants for each enzyme were measured for incorporation of an A opposite T and an A opposite furan as well as for the formation of A:C and T:T mismatches. The T4 exonuclease mutants were all ~2- to 7-fold more efficient than the corresponding RB69 exonuclease mutants depending on whether a T or furan was in the templating position and which exonuclease mutant was used. The rates for mismatch formation by T4 were significantly reduced compared with incorporation opposite furan, much more so than the corresponding RB69 mutant. These results show that there are kinetic differences between the two enzymes but they are not large enough to preclude structural assumptions for T4 DNA polymerase based on the known structure of the RB69 DNA polymerase.     

DNA polymerase alpha and models for proofreading.

Using a modified system to measure fidelity at an amber site in phi X174, we have employed DNA polymerase alpha to test different mechanisms for proofreading. DNA polymerase alpha does not exhibit the characteristics of "kinetic proofreading" seen with procaryotic polymerases. Polymerase alpha shows no evidence for a "next nucleotide" effect, and added deoxynucleoside monophosphates do not alter fidelity. Pyrophosphate, which increases error rates with a procaryotic polymerase, appears to weakly improve polymerase alpha fidelity. DNA polymerase alpha does exhibit a dramatic increase in error rate in the presence of a deoxycytidine thiotriphosphate (dCTP alpha S), but this enhanced mutagenesis also occurs under conditions where kinetic proofreading should be otherwise defeated. This particular effect with dCTP alpha S appears specific for DNA polymerase alpha and is not seen with the other polymerases tested.     

Identification of a mutant DNA polymerase delta in Saccharomyces cerevisiae with an antimutator phenotype for frameshift mutations

We propose that a beta-turn-beta structure, which plays a critical role in exonucleolytic proofreading in the bacteriophage T4 DNA polymerase, is also present in the Saccharomyces cerevisiae DNA pol delta. Site-directed mutagenesis was used to test this proposal by introducing a mutation into the yeast POL3 gene in the region that encodes the putative beta-turn-beta structure. The mutant DNA pol delta has a serine substitution in place of glycine at position 447. DNA replication fidelity of the G447S-DNA pol delta was determined in vivo by using reversion and forward assays. An antimutator phenotype for frameshift mutations in short homopolymeric tracts was observed for the G447S-DNA pol delta in the absence of postreplication mismatch repair, which was produced by inactivation of the MSH2 gene. Because the G447S substitution reduced frameshift but not base substitution mutagenesis, some aspect of DNA polymerase proofreading appears to contribute to production of frameshifts. Possible roles of DNA polymerase proofreading in frameshift mutagenesis are discussed.     

A DNA polymerase epsilon mutant that specifically causes +1 frameshift mutations within homonucleotide runs in yeast

The DNA polymerases delta and epsilon are the major replicative polymerases in the yeast Saccharomyces cerevisiae that possess 3' --> 5' exonuclease proofreading activity. Many errors arising during replication are corrected by these exonuclease activities. We have investigated the contributions of regions of Polepsilon other than the proofreading motifs to replication accuracy. An allele, pol2-C1089Y, was identified in a screen of Polepsilon mutants that in combination with an exonuclease I (exo1) mutation could cause a synergistic increase in mutations within homonucleotide runs. In contrast to other polymerase mutators, this allele specifically results in insertion frameshifts. When pol2-C1089Y was combined with deletions of EXO1 or RAD27 (homologue of human FEN1), mutation rates were increased for +1 frameshifts while there was almost no effect on -1 frameshifts. On the basis of genetic analysis, the pol2-C1089Y mutation did not cause a defect in proofreading. In combination with a deletion of the mismatch repair gene MSH2, the +1 frameshift mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the -1 frameshift rate was unchanged. This suggests that the Pol2-C1089Y protein makes +1 frameshift errors during replication of homonucleotide runs and that these errors can be corrected by either mismatch repair (MMR) or proofreading (in short runs). This is the first report of a +1-specific mutator for homonucleotide runs in vivo. The pol2-C1089Y mutation defines a functionally important residue in Polepsilon.     

Crystal structure of an archaebacterial DNA polymerase.

BACKGROUND: Members of the Pol II family of DNA polymerases are responsible for chromosomal replication in eukaryotes, and carry out highly processive DNA replication when attached to ring-shaped processivity clamps. The sequences of Pol II polymerases are distinct from those of members of the well-studied Pol I family of DNA polymerases. The DNA polymerase from the archaebacterium Desulfurococcus strain Tok (D. Tok Pol) is a member of the Pol II family that retains catalytic activity at elevated temperatures. RESULTS: The crystal structure of D. Tok Pol has been determined at 2.4 A resolution. The architecture of this Pol II type DNA polymerase resembles that of the DNA polymerase from the bacteriophage RB69, with which it shares less than approximately 20% sequence identity. As in RB69, the central catalytic region of the DNA polymerase is located within the 'palm' subdomain and is strikingly similar in structure to the corresponding regions of Pol I type DNA polymerases. The structural scaffold that surrounds the catalytic core in D. Tok Pol is unrelated in structure to that of Pol I type polymerases. The 3'-5' proofreading exonuclease domain of D. Tok Pol resembles the corresponding domains of RB69 Pol and Pol I type DNA polymerases. The exonuclease domain in D. Tok Pol is located in the same position relative to the polymerase domain as seen in RB69, and on the opposite side of the palm subdomain compared to its location in Pol I type polymerases. The N-terminal domain of D. Tok Pol has structural similarity to RNA-binding domains. Sequence alignments suggest that this domain is conserved in the eukaryotic DNA polymerases delta and epsilon. CONCLUSIONS: The structure of D. Tok Pol confirms that the modes of binding of the template and extrusion of newly synthesized duplex DNA are likely to be similar in both Pol II and Pol I type DNA polymerases. However, the mechanism by which the newly synthesized product transits in and out of the proofreading exonuclease domain has to be quite different. The discovery of a domain that seems to be an RNA-binding module raises the possibility that Pol II family members interact with RNA.     

Amino acid changes coded by bacteriophage T4 DNA polymerase mutator mutants. Relating structure to function

Previous studies on the selection of bacteriophage T4 mutator mutants have been extended and a method to regulate the mutator activity of DNA polymerase mutator strains has been developed. The nucleotide changes of 17 bacteriophage T4 DNA polymerase mutations that confer a mutator phenotype and the nucleotide substitutions of several other T4 DNA polymerase mutations have been determined. The most striking observation is that the distribution of DNA polymerase mutator mutations is not random; almost all mutator mutations are located in the N-terminal half of the DNA polymerase. It has been shown that the T4 DNA polymerase shares several regions of homology at the protein sequence level with DNA polymerases of herpes, adeno and pox viruses. From studies of bacteriophage T4 and herpes DNA polymerase mutants, and from analyses of similar protein sequences from several organisms, we conclude that DNA polymerase synthetic activities are located in the C-terminal half of the DNA polymerase and that exonucleolytic activity is located nearer the N terminus.     

Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III.

Saccharomyces cerevisiae cdc2 mutants arrest in the S-phase of the cell cycle when grown at the non-permissive temperature, implicating this gene product as essential for DNA synthesis. The CDC2 gene has been cloned from a yeast genomic library in vector YEp13 by complementation of a cdc2 mutation. An open reading frame coding for a 1093 amino acid long protein with a calculated mol. wt of 124,518 was determined from the sequence. This putative protein shows significant homology with a class of eukaryotic DNA polymerases exemplified by human DNA polymerase alpha and herpes simplex virus DNA polymerase. Fractionation of extracts from cdc2 strains showed that these mutants lacked both the polymerase and proofreading 3'-5' exonuclease activity of DNA polymerase III, the yeast analog of mammalian DNA polymerase delta. These studies indicate that DNA polymerase III is an essential component of the DNA replication machinery.     

Involvement of the essential yeast DNA polymerases in induced gene conversion

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.     

Genetic factors affecting the impact of DNA polymerase delta proofreading activity on mutation avoidance in yeast

Base selectivity, proofreading, and postreplication mismatch repair are important for replication fidelity. Because proofreading plays an important role in error correction, we have investigated factors that influence its impact in the yeast Saccharomyces cerevisiae. We have utilized a sensitive mutation detection system based on homonucleotide runs of 4 to 14 bases to examine the impact of DNA polymerase delta proofreading on mutation avoidance. The contribution of DNA polymerase delta proofreading on error avoidance was found to be similar to that of DNA polymerase epsilon proofreading in short homonucleotide runs (A4 and A5) but much greater than the contribution of DNA polymerase epsilon proofreading in longer runs. We have identified an intraprotein interaction affecting mutation prevention that results from mutations in the replication and the proofreading regions, resulting in an antimutator phenotype relative to a proofreading defect. Finally, a diploid strain with a defect in DNA polymerase delta proofreading exhibits a higher mutation rate than a haploid strain. We suggest that in the diploid population of proofreading defective cells there exists a transiently hypermutable fraction that would be inviable if cells were haploids.     

The 3'-->5' exonucleases of DNA polymerases delta and epsilon and the 5'-->3' exonuclease Exo1 have major roles in postreplication mutation avoidance in Saccharomyces cerevisiae

Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5'-->3' Exo1 and the 3'-->5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces cerevisiae by using various genetic backgrounds and highly sensitive mutation detection systems that are based on long and short homonucleotide runs. Genetic interactions were examined among DNA polymerase epsilon (pol2-4) and delta (pol3-01) mutants defective in 3'-->5' proofreading exonuclease, mutants defective in the 5'-->3' exonuclease Exo1, and mismatch repair mutants (msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance. Surprisingly, the mutation rate in an exo1 pol3-01 mutant was comparable to that in an msh2 pol3-01 mutant, suggesting that they participate directly in postreplication mismatch repair as well as in other DNA metabolic processes.