The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 protein

Noriko Kantake, Tomohiko Sugiyama, Richard D. Kolodner, Stephen C. Kowalczykowski

Research output: Contribution to journalArticle

71 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)23410-23417
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number26
DOIs
StatePublished - Jul 27 2003

ASJC Scopus subject areas

  • Biochemistry

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