Characterization of the repeat-tract instability and mutator phenotypes conferred by a Tn3 insertion in RFC1, the large subunit of the yeast clamp loader

The RFC1 gene encodes the large subunit of the yeast clamp loader (RFC) tha t is a component of eukaryotic DNA polymerase holoenzymes. We identified a mutant allele of RFC1 (rfc1::Tn3) from a large collection of Saccharomyces cerevisiae mutants that were inviable when present in a rad52 null mutation background. Analysis of rfcl::Tn3 strains indicated that they displayed bo th a mutator and repeat-tract instability phenotype. Strains bearing this a llele were characterized in combination with mismatch repair (msh2 Delta, p ms1 Delta), double-strand break repair (rad52), and DNA replication (pol3-0 1, pol30-52, rth1 Delta/rad27 Delta) mutations in both forward mutation and repeat-tract instability assays. This analysis indicated that the rfc1::Tn 3 allele displays synthetic lethality with pol30, pol3, and rad27 mutations . Measurement of forward mutation frequencies in msh2 Delta rfc1:Tn3 and pm s1 Delta rfc1:Tn3 strains indicated that the rfc1::Tn3 mutant displayed a m utation frequency that appeared nearly multiplicative with the mutation fre quency exhibited by mismatch-repair mutants. In repeat-tract instability as says, however, the rfc1::Tn3 mutant displayed a tract instability phenotype that appeared epistatic to the phenotype displayed by mismatch-repair muta nts. From these data we propose that defects in clamp loader function resul t in DNA replication errors, a subset of which are acted upon by the mismat ch-repair system.