MUTATIONS IN THE GENE ENCODING THE 34 KDA SUBUNIT OF YEAST REPLICATION PROTEIN-A CAUSE DEFECTIVE S-PHASE PROGRESSION

The in vivo function of the 34 kDa subunit of yeast replication protei n A (RPA), encoded by the RFA2 gene, has been studied by analyzing the effect of Rpa34 depletion and by producing and characterizing rfa2 te mperature-sensitive mutants. We show that unbalanced stoichiometry of the RPA subunits does not affect cell growth and cell cycle progressio n 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 replica tion in vivo, since rfa2 ts mutants are defective in S phase progressi on and ARS plasmid stability, and rfa2 poll double mutants are non-via ble. Moreover, when shifted to the restrictive temperature, about 50% of the rfa2 mutant cells rapidly die while traversing the S phase and the surviving cells arrest in late S/G2 at the RADS checkpoint. Finall y, rfa2 mutant cells have a mutator and hyper-recombination phenotype and are more sensitive to hydroxyurea and methyl-methane-sulfonate tha n wild-type cells. (C) 1995 Academic Press Limited