Characterization of genetic interactions with RFA1: the role of RPA in DNA replication 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 mainly elucidated through in vitro biochemical studies utilizing the mammalian complex. However, the identification of homologs of all three subunits in Saccharomyces cerevisiae offers the opportunity of examining the in vivo role of RPA. In our laboratory, we have previously isolated a missense allele of the RFA1 gene, encoding the p70 subunit of the RPA complex. Strains containing this mutant allele, rfa1-D228Y, display increased levels of direct-repeat recombination, decreased levels of heteroallelic recombination, UV sensitivity and a S-phase delay. In this study, we have characterized further the role of RPA by screening other replication and repair mutants for a synthetic lethal phenotype in combination with the rfa1-D228Y allele. Among the replication mutants examined, only one displayed a synthetic lethal phenotype, pol12-100, a conditional allele of the B subunit of pol alpha-primase. In addition, a delayed senescence phenotype was observed in raf1-D228Y strains containing a null mutation of HDF1, the S. cerevisiae homolog of the 70 kDa subunit of Ku. Interestingly, a synergistic reduction in telomere length observed in the double mutants suggests that the shortening of telomeres may be the cause of the decreased viability in these strains. Furthermore, this result represents the first evidence of a role for RPA in telomere maintenance.
Smith
J
J
Department of Genetics & Development, Columbia University College of Physicians & Surgeons, New York, NY 10032-2704, USA.
Zou
H
H
Rothstein
R
R
D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005804:Genes, Lethal; D009154:Mutation; D010641:Phenotype; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016615:Telomere / Q000235:genetics*
D004268:DNA-Binding Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; C520769:YKU70 protein, S cerevisiae; C081647:high affinity DNA-binding factor, S cerevisiae; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I