Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.
Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-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 DNA content indicative of incomplete DNA replication. DNA sequence analysis 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 replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.
Umezu
K
K
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Sugawara
N
N
Chen
C
C
Haber
J E
JE
Kolodner
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism*; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D029701:Saccharomyces cerevisiae Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009153:Mutagens; C497167:RPA1 protein, human; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D008741:Methyl Methanesulfonate; C530845:HO protein, S cerevisiae; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D004265:DNA Helicases