GENETIC-ANALYSIS OF YEAST RPA1 REVEALS ITS MULTIPLE FUNCTIONS IN DNA METABOLISM

Replication protein A (RPA) is a single-stranded DNA-binding protein i dentified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharom yces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irr adiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 t emperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Som e of the UV- and MMS-sensitive mutants were killed by an HO-induced do uble-strand break at MAT. Physical analysis of recombination in one UV - and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DN A content indicative of incomplete DNA replication. DNA sequence analy sis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles In vivo and functions in DNA rep lication, repair, and recombination, like the single-stranded DNA-bind ing proteins of bacteria and phages.