The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein.
Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11).ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA.ssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein.ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.
Kantake
Noriko
N
Sections of Microbiology and of Molecular and Cellular Biology, Center for Genetics and Development, University of California, Davis, California 95616, USA.
Sugiyama
Tomohiko
T
Kolodner
Richard D
RD
Kowalczykowski
Stephen C
SC
D001667:Binding, Competitive; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D009154:Mutation; D009698:Nucleoproteins / Q000378:metabolism; D051135:Rad51 Recombinase; D011995:Recombination, Genetic / Q000235:genetics*; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D012492:Salts / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009698:Nucleoproteins; C082728:RFA1 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D012492:Salts; D014157:Transcription Factors; D051135:Rad51 Recombinase