Characterization of genetic interactions with RFA1: the role of RPA in DNAreplication and telomere maintenance

Replication protein A (RPA) is a heterotrimeric single-stranded DNA binding protein whose role in DNA replication, recombination and repair has been m ainly elucidated through in vitro biochemical studies utilizing the mammali an complex. However, the identification of homologs of all three subunits i n Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA. In our laboratory, we have previously isolated a missense alle le of the RFA1 gene, encoding the p70 subunit of the RPA complex. Strains c ontaining this mutant allele, rfa1-D228Y, display increased levels of direc t-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay. In this study, we have characterized furt her the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele. Amon g the replication mutants examined, only one displayed a synthetic lethal p henotype, pol12-100, a conditional allele of the B subunit of pol alpha-pri mase. In addition, a delayed senescence phenotype was observed in rfa1-D228 Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a synergistic reduction in telomer e length observed in the double mutants suggests that the shortening of tel omeres may be the cause of the decreased viability in these strains. Furthe rmore, this result represents the first evidence of a role for RPA in telom ere maintenance. (C) 2000 Societe francaise de biochimie et biologie molecu laire/Editions scientifiques et medicales Elsevier SAS.