Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins

Michael J. McIlwraith, Eric Van Dyck, Jean Yves Masson, Alicja Z. Stasiak, Andrzej Stasiak, Stephen C. West*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

107 Citations (Scopus)

Abstract

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)151-164
Number of pages14
JournalJournal of Molecular Biology
Volume304
Issue number2
DOIs
Publication statusPublished - 24 Nov 2000
Externally publishedYes

Keywords

  • D-loops
  • DNA repair
  • Genetic recombination
  • Heteroduplex DNA
  • Recombinase

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