Vital roles of the second DNA-binding site of Rad52 protein in yeast homologous recombination

Naoto Arai, Wataru Kagawa, Kengo Saito, Yoshinori Shingu, Tsutomu Mikawa, Hitoshi Kurumizaka, Takehiko Shibata

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    Abstract

    RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with doublestranded DNA to assist Rad51-mediated homologous pairing.

    Original languageEnglish
    Pages (from-to)17607-17617
    Number of pages11
    JournalJournal of Biological Chemistry
    Volume286
    Issue number20
    DOIs
    Publication statusPublished - 2011 May 20

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    ASJC Scopus subject areas

    • Biochemistry
    • Cell Biology
    • Molecular Biology

    Cite this

    Arai, N., Kagawa, W., Saito, K., Shingu, Y., Mikawa, T., Kurumizaka, H., & Shibata, T. (2011). Vital roles of the second DNA-binding site of Rad52 protein in yeast homologous recombination. Journal of Biological Chemistry, 286(20), 17607-17617. https://doi.org/10.1074/jbc.M110.216739