The Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase ␦ holoenzyme activity was achieved at 37 C but not at 14 C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase ␦ but completely defective for interaction with DNA polymerase . Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.

Uracil-DNA-glycosylase has been proposed to function as the first enzyme in strand-directed mismatch repair in eukaryotic organisms, through removal of uracil from dUMP residues periodically inserted into the DNA during DNA replication (Aprelikova, 0. N., V. M. Golubovskaya, T. A. Kusmin, and N. V. Tomilin, Mutat. Res. 213:135-140, 1989). This hypothesis was investigated with Saccharomyces cerevisiae. Mutation frequencies and spectra were determined for an ungi deletion strain in the target SUP4-o tRNA gene by using a forward selection scheme. Mutation frequencies in the SUP4-o gene increased about 20-fold relative to an isogenic wild-type S. cerevisiae strain, and the mutator effect was completely suppressed in the ungl deletion strain carrying the wild-type UNGI gene on a multicopy plasmid. Sixty-nine independently derived mutations in the SUP4-o gene were sequenced. All but five of these were due to GC->AT transitions. From this analysis, we conclude that the mutator phenotype of the ungi deletion strain is the result of a failure to repair spontaneous cytosine deamination events occurring frequently in S. cerevisiae and that the UNGI gene is not required for strand-specific mismatch repair in S. cerevisiae.Strand-directed mismatch repair depends on a mechanism which allows the organism to distinguish between the parental and the newly replicated DNA strand during DNA replication. This form of repair is best understood for Escherichia coli (11,23). In methyl-directed DNA mismatch repair, the strand to be repaired is identified by its temporary lack of methylation at GATC sites during DNA replication.