A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein

Tomohiko Sugiyama, Elena M. Zaitseva, Stephen C. Kowalczykowski

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Abstract

Protein-promoted DNA strand exchange requires formation of an active presynaptic complex between the DNA-pairing protein and single-stranded DNA (ssDNA). Formation of such a contiguous filament is stimulated by a ssDNA- binding protein. Here, the effects of replication protein A (RPA) on presynaptic complex formation and DNA strand exchange activities of Rad51 protein were examined. Presynaptic complex formation was assessed by measuring ATP hydrolysis. With φX174 ssDNA, the ATPase activity of Rad51 protein is stimulated ~1.4-fold by RPA, provided that Rad51 protein is in excess of the ssDNA concentration; otherwise, RPA inhibits ATPase activity. In contrast, with ssDNA devoid of secondary structure (poly(dT), poly(dA), poly(dI), and etheno-M13 DNA), RPA does not stimulate the already elevated ATPase activity of Rad51 protein, but inhibits activity at low Rad51 protein concentrations. These results suggest that Rad51 protein and RPA exclude one another from ssDNA by competing for the same binding sites and that RPA exerts its effect on presynaptic complex formation by eliminating secondary structure to which Rad51 protein is bound nonproductively. DNA strand exchange catalyzed by Rad51 protein is also greatly stimulated by RPA. The optimal stoichiometry for stimulation is ~20-30 nucleotides of ssDNA/RPA heterotrimer. The ssDNA-binding protein of Escherichia coli can substitute for RPA, showing that the role of RPA is not specific. We conclude that RPA affects both presynaptic complex formation and DNA strand exchange via changes in DNA structure, employing the same mechanism used by the ssDNA- binding protein to effect change in E. coli RecA protein activity.

Original languageEnglish (US)
Pages (from-to)7940-7945
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number12
DOIs
StatePublished - 1997

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Replication Protein A
Saccharomyces cerevisiae Proteins
DNA-Binding Proteins
Yeast
Single-Stranded DNA
Proteins
DNA
Adenosine Triphosphatases
DNA Replication
Escherichia coli
Rec A Recombinases
Escherichia coli Proteins
Stoichiometry
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

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A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. / Sugiyama, Tomohiko; Zaitseva, Elena M.; Kowalczykowski, Stephen C.

In: Journal of Biological Chemistry, Vol. 272, No. 12, 1997, p. 7940-7945.

Research output: Contribution to journalArticle

Sugiyama, Tomohiko ; Zaitseva, Elena M. ; Kowalczykowski, Stephen C. / A single-stranded DNA-binding protein is needed for efficient presynaptic complex formation by the Saccharomyces cerevisiae Rad51 protein. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 12. pp. 7940-7945.
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