Mismatch repair (MMR) is a major DNA repair pathway in cells from all branches of life that removes replication errors in a strandspecific manner, such that mismatched nucleotides are preferentially removed from the newly replicated strand of DNA. Here we demonstrate a role for MMR in helping create new phenotypes in nondividing cells. We show that mispairs in yeast that escape MMR during replication can later be subject to MMR activity in a replication strand-independent manner in nondividing cells, resulting in either fully wild-type or mutant DNA sequence. In one case, this activity is responsible for what appears to be adaptive mutation. This replication strand-independent MMR activity could contribute to the formation of tumors arising in nondividing cells and could also contribute to mutagenesis observed during somatic hypermutation of Ig genes.DNA mismatch repair | oligonucleotide transformation | 8-oxoG D NA mismatch repair (MMR) recognizes mismatches created in the process of replication and uses some type of strand discrimination signal to selectively remove mismatched nucleotides present on the newly replicated strand of DNA (1-3). In most eukaryotic cells, there are two mismatch recognition complexes with different, but overlapping, specificities: (i) MutSα, a heterodimer of Msh2 and Msh6, recognizing base/base mismatches and small insertion/deletion loops; and (ii) MutSβ, a heterodimer of Msh2 and Msh3, recognizing both small and large loops (1-3). After recognition of a mispair by MutSα or MutSβ, completion of MMR requires association with proteins related to MutL, usually MutLα, which in yeast is a heterodimer of Mlh1 and Pms1 (1-3). DNA on the newly synthesized strand is then excised and the template strand rereplicated.The method of strand discrimination in eukaryotic MMR is still not solved, although major advances have recently been made. Several components of MMR are known to have association with proliferating cell nuclear antigen (PCNA), a sliding clamp that tracks with the replication fork (1-3). As would be predicted by that model, it has recently been shown that MMR is temporally coupled to replication (4) and that one pathway of MutSα-dependent MMR is through recruitment by a PCNA-Msh6 interaction (5). Whatever signals are used for strand discrimination are presumably lost as replication proceeds.To study various aspects of MMR, we have used single-stranded oligonucleotides (oligos) to introduce specific mispairs into Saccharomyces cerevisiae chromosomal DNA. We have shown previously that oligos can be introduced into cells by electroporation and can correct frame-shift mutations in LYS2 and that this process is inhibited by MMR (6). Our results are most consistent with a mechanism in which the oligo anneals to either the leading or lagging strand of replication at the replication fork, with subsequent extension. Mispairs created by the oligos are recognized by MMR, but those mispairs that escape MMR recognition create mutations in the next round of replication (6). For the experiments ...

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants.

8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides.

We have studied single-strand oligonucleotide (oligo) transformation of yeast by using 40-nt long oligos that create multiple base changes to the yeast genome spread throughout the length of the oligos, making it possible to measure the portions of an oligo that are incorporated during transformation. Although the transformation process is greatly inhibited by DNA mismatch repair (MMR), the pattern of incorporation is essentially the same in the presence or absence of MMR, whether the oligo anneals to the leading or lagging strand of DNA replication, or whether phosphorothioate linkages are used at either end. A central core of approximately 15 nt is incorporated with a frequency of >90%; the ends are incorporated with a lower frequency, and loss of the two ends appears to be by different mechanisms. Bases that are 5–10 nt from the 5′ end are generally lost with a frequency of >95%, likely through a process involving flap excision. On the 3′ end, bases 5–10 nt from the 3′ end are lost about 1/3 of the time. These results indicate that oligos can be used to create multiple simultaneous changes to the yeast genome, even in the presence of MMR.

Oxidative damage to DNA constitutes a major threat to the faithful replication of DNA in all organisms and it is therefore important to understand the various mechanisms that are responsible for repair of such damage and the consequences of unrepaired damage. In these experiments, we make use of a reporter system in Saccharomyces cerevisiae that can measure the specific increase of each type of base pair mutation by measuring reversion to a Trp+ phenotype. We demonstrate that increased oxidative damage due to the absence of the superoxide dismutase gene, SOD1, increases all types of base pair mutations and that mismatch repair (MMR) reduces some, but not all, types of mutations. By analyzing various strains that can revert only via a specific CG → AT transversion in backgrounds deficient in Ogg1 (encoding an 8-oxoG glycosylase), we can study mutagenesis due to a known 8-oxoG base. We show as expected that MMR helps prevent mutagenesis due to this damaged base and that Pol η is important for its accurate replication. In addition we find that its accurate replication is facilitated by template switching, as loss of either RAD5 or MMS2 leads to a significant decrease in accurate replication. We observe that these ogg1 strains accumulate revertants during prolonged incubation on plates, in a process most likely due to retromutagenesis.