Mismatch repair-dependent mutagenesis in nondividing cells

Abstract

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 ...

New insights into the mechanism of DNA mismatch repair

“…This hypothesis is supported by two recent articles that came to a similar conclusion, though having used different approaches. In a first report (Rodriguez et al 2012), the authors used single-strand oligo-nucleotides to introduce mutations and revert a non-functional allele of the TRP5 gene in S. cerevisiae . Analysis of the recovery of Trp + revertants after transforming oligos targeting the transcribed or non-transcribed strand, in MMR-proficient or MMR-deficient strains, revealed that when mispair correction is uncoupled from DNA replication, it uses as template for repair either one of the two DNA strands, most likely due to the absence of a DNA strand discrimination signal and is therefore mutagenic.…”

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

“…Although a low level of MMR has been detected in nondividing cells, it lacks strand discrimination, presumably because the normal link with replication is missing (Rodriguez et al 2012). Such residual MMR, however, may provide a mechanism to introduce a potentially beneficial change into both strands of duplex DNA under stress conditions.…”

Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLα endonuclease activation

“…However, biochemical experiments have also shown that MutLα can be activated on relaxed closed circular (RCC) heteroduplex DNA, which contains a small region of unpaired helix that can serve as a site for clamp loading, but in this case, endonuclease action occurs on either strand (9). This reaction provides a potential mechanism for activation of MMR on nonreplicating DNA and is of interest because recent studies indicate that undirected MMR occurring outside of replication can lead to mutation production (11,12).…”