Novel PMS1 Alleles Preferentially Affect the Repair of Primer Strand Loops during DNA Replication

DNA mismatch repair (MMR) contributes to genomic integrity by repairing mismatches generated during replication, by chemical damage, and as “heteroduplex” intermediates during recombination (7, 28, 31, 35, 44). In addition, the MMR system in higher eukaryotes plays a role in response to DNA damage (3, 6, 7, 62). Inherited MMR defects lead to a mutator phenotype, which in humans and mice is associated with increased cancer susceptibility (5, 7, 13, 16, 38, 50). The MMR system of Escherichia coli has been reconstituted in vitro with purified proteins, including the dedicated proteins MutS, MutL, and MutH (44, 56). The MutS protein, a homodimer, first binds the mispair, followed by recruitment of MutL, the endonuclease MutH, the UvrD helicase, four exonucleases, DNA polymerase, and ligase. Together with transient Dam-mediated hemimethylation, these proteins impose strand specificity that leads to specific repair of the newly replicated strand (10, 25, 26, 43, 44, 74). In the budding yeast Saccharomyces cerevisiae, six MutS homologues (Msh proteins Msh1 to Msh6) and four MutL homologues (Mlh proteins, Mlh1 to Mlh3, and Pms1) function in various MMR transactions (7, 28, 31, 35). Unlike E. coli, the MutS and MutL activities of budding yeast and mammals are each comprised of heterodimers. Mismatches in nuclear DNA replication intermediates are recognized by the Msh2/Msh6 and Msh2/Msh3 heterodimers, which have partial functional overlap (7, 35, 42). Msh2/Msh6 operates in the repair of base-base mispairs and 1-nucleotide “insertion/deletion” loops (28, 32, 41), while Msh2/Msh3 functions in the repair of 1- to 4-nucleotide insertion/deletion loops (28, 32, 41). Similarly for the MutL homologs, Mlh1 forms heterodimers with Pms1, Mlh2, or Mlh3 (19, 28, 30, 49, 72). Genetic studies indicate that the Mlh1/Pms1 heterodimer is the primary MutL activity in MMR-mediated mutation avoidance, whereas the Mlh1/Mlh3 complex plays a minor role in Msh2/Msh3-dependent repair of insertion/deletion loops (19, 24, 30, 49, 53, 54). Current data suggest that initial recognition of the mismatch is by Msh2/Msh6 or Msh2/Msh3, possibly aided by PCNA, which is subsequently joined by Mlh1-Pms1 to form a higher order complex on DNA. This complex is thought to be responsible for directing the downstream and less well characterized MMR events, including strand discrimination, excision, and resynthesis (4, 7, 28, 35, 42). Although the MutS and MutL proteins must interact during MMR-mediated mutation avoidance, the nature of these and other protein-protein interactions is not clear. Whereas MLH1, PMS1, and MHS2 deletion mutations appear to result in a null, or near null, MMR state for mutation avoidance, specific pms1 or mlh1 mutant alleles might produce a novel mutator phenotype, for example, by differentially impacting interactions with Msh2/Msh3 or Msh2/Msh6. In turn, these types of alleles might provide additional insights into the function of the MutL homologs, including interactions with other MMR factors. Therefore, we have initiated genetic screens for mutations of PMS1 or MLH1 that result in mutational spectra different from the corresponding MMR-null strains. Here, we report the isolation of two novel pms1 alleles, which in contrast to a pms1Δ strain preferentially elevate +1-bp frameshifts in mononucleotide runs, with little or no effect on −1-bp frameshifts. These pms1 alleles are in close proximity, affecting residues near the end of the protein in a previously uncharacterized but highly conserved amino acid motif. Further analyses suggest that these pms1 alleles do not differentially impact MMR during leading- versus lagging-strand replication but, rather, fail to efficiently repair single nucleotide loops arising on the primer strand. The results presented here reinforce the current view that MMR is associated with the replication apparatus.