Mutations in the Gene Encoding the 34 kDa Subunit of Yeast Replication Protein A Cause Defective S Phase Progression

Abstract

The in vivofunction of the 34 kDa subunit of yeast replication protein A (RPA), encoded by the RFA2gene, has been studied by analyzing the effect of Rpa34 depletion and by producing and characterizing rfa2temperature- sensitive mutants. We show that unbalanced stoichiometry of the RPA subunits does not affect cell growth and cell cycle progression until the level of Rpa34 becomes rate-limiting, at which point cells arrest with a late S/G2 DNA content. Rpa34 is involved in DNA replication in vivo, since rfa2ts mutants are defective in S phase progression and ARS plasmid stability, and rfa2 pol1double mutants are non-viable. Moreover, when shifted to the restrictive temperature, about 50% of the rfa2mutant cells rapidly die while traversing the S phase and the surviving cells arrest in late S/G2 at the RAD9checkpoint. Finally, rfa2mutant cells have a mutator and hyper-recombination phenotype and are more sensitive to hydroxyurea and methyl-methane-sulfonate than wild-type cells.