Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament

Jachen A. Solinger, Géraldine Lutz, Tomohiko Sugiyama, Stephen C. Kowalczykowski, Wolf Dietrich Heyer

Research output: Contribution to journalArticle

89 Scopus citations

Abstract

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.

Original languageEnglish (US)
Pages (from-to)1207-1221
Number of pages15
JournalJournal of Molecular Biology
Volume307
Issue number5
DOIs
StatePublished - Apr 13 2001
Externally publishedYes

Keywords

  • DNA strand exchange
  • DSB repair
  • Genetic recombination
  • Heteroduplex DNA
  • Rad54

ASJC Scopus subject areas

  • Virology

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