A MUTATIONAL ANALYSIS OF THE YEAST PROLIFERATING CELL NUCLEAR ANTIGENINDICATES DISTINCT ROLES IN DNA-REPLICATION AND DNA-REPAIR

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 charg ed amino acids to alanines. Although none of the mutant strains contai ning these double-alanine mutations as the sole source of PCNA were te mperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light, Some of those UV-sensitive mu tants had elevated spontaneous mutation rates. In addition, several mu tants suppressed a cold-sensitive mutation in the CDC44 gene, which en codes the large subunit of replication factor C, A cold-sensitive muta nt, which was isolated by random mutagenesis, showed a terminal phenot ype at the restrictive temperature consistent with a defect in DNA rep lication. Several mutant PCNAs were expressed and purified from Escher ichia coli, and their in vitro properties were determined. The cold-se nsitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Par tial restoration of DNA polymerase delta holoenzyme activity was achie ved at 37 degrees C but not at 14 degrees C by inclusion of the macrom olecular crowding agent polyethylene glycol in the assay. The only oth er mutant (pol30-6, DD41,42AA) that showed a growth defect was partial ly defective for interaction with replication factor C and DNA polymer ase 6 but completely defective for interaction with DNA polymerase E. Two other mutants sensitive to DNA damage showed no defect in vitro. T hese results indicate that the latter mutants are specifically impaire d in one or more DNA repair processes whereas pol30-6 and pol30-52 mut ants 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, wh ich are distinct from those required for DNA replication.