Saccharomyces cerevisiae pol30 (proliferating cell nuclear antigen) mutations impair replication fidelity and mismatch repair

Abstract

To understand the role of POL30 in mutation suppression, 11 Saccharomyces cerevisiae pol30 mutator mutants were characterized. These mutants were grouped based on their mutagenic defects. Many pol30 mutants harbor multiple mutagenic defects and were placed in more than one group. Group A mutations (pol30-52, -104, -108, and -126) caused defects in mismatch repair (MMR). These mutants exhibited mutation rates and spectra reminiscent of MMR-defective mutants and were defective in an in vivo MMR assay. The mutation rates of group A mutants were enhanced by a msh2 or a msh6 mutation, indicating that MMR deficiency is not the only mutagenic defect present. Group B mutants (pol30-45, -103, -105, -126, and -114) exhibited increased accumulation of either deletions alone or a combination of deletions and duplications (4 to 60 bp). All deletion and duplication breakpoints were flanked by 3 to 7 bp of imperfect direct repeats. Genetic analysis of one representative group B mutant, pol30-126, suggested polymerase slippage as the likely mutagenic mechanism. Group C mutants (pol30-100, -103, -105, -108, and -114) accumulated base substitutions and exhibited synergistic increases in mutation rate when combined with msh6 mutations, suggesting increased DNA polymerase misincorporation as a mutagenic defect. The synthetic lethality between a group A mutant, pol30-104, and rad52 was almost completely suppressed by the inactivation of MSH2. Moreover, pol30-104 caused a hyperrecombination phenotype that was partially suppressed by a msh2 mutation. These results suggest that pol30-104 strains accumulate DNA breaks in a MSH2-dependent manner.

FIG. 1

Lys2-Bgl reversion spectra from wild-type, msh2, rad27, and pol30 mutants. The sequence shown represents the reversion window (nucleotides 650 to 798) of the lys2-Bgl assay. The location of the nucleotide alteration resulting in functional restoration of LYS2 is indicated by |. Each independent −1 frameshift is indicated by a ⧫ above the deleted nucleotide. Duplications are indicated by (+) followed by the nucleotides inserted. Deletions of greater than one base pair are marked by (−) followed by the nucleotides deleted. The ratio indicated represents the proportion of each particular class of events. The wild-type and msh2 spectra are from reference ; the rad27 spectra is from reference .

FIG. 2

A msh2 mutation rescues the synthetic lethality between pol30-104 and rad52. (a and b) pol30-104 (RDKY3583), pol30-104 msh2 (RDKY3584), pol30-104 rad52 (RDKY3585) and pol30-104 msh2 rad52 (RDKY3586) strains bearing a URA3 POL30 plasmid were grown at 30°C in liquid SD −Ura to saturation; 10-fold serial dilutions of each culture were spotted onto an SD −Ura plate to determine viability (a) and an SD +5FOA plate to determine whether the URA3 POL30 plasmid was required for viability (b). (c and d) pol30-104 msh2 (RDKY3669) and three independent isolates of pol30-104 msh2 rad52 (RDKY3587), all bearing URA3 POL30 and TRP1 MSH2 plasmids, were grown at 30°C in liquid SD −Ura −Trp media to saturation; 10-fold serial dilutions of each culture were spotted onto an SD −Ura −Trp plate to determine viability (c) and an SD −Trp +5FOA plate to determine whether the URA3 POL30 plasmid was required for viability in the presence of the TRP1 MSH2 plasmid (d).