title: Vital roles of the second DNA-binding site of Rad52 protein in yeast homologous recombination.
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 double-stranded DNA to assist Rad51-mediated homologous pairing.
authors: Department of Applied Biological Science, Nihon University College of Bioresource Sciences, Fujisawa-shi, Kanagawa 252-0880, Japan. narai@brs.nihon-u.ac.jp
authors: Naoto
authors: N
authors: Arai
authors: Wataru
authors: W
authors: Kagawa
authors: Kengo
authors: K
authors: Saito
authors: Yoshinori
authors: Y
authors: Shingu
authors: Tsutomu
authors: T
authors: Mikawa
authors: Hitoshi
authors: H
authors: Kurumizaka
authors: Takehiko
authors: T
authors: Shibata
mesh_terms: D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D004271:DNA, Fungal; D046912:Multiprotein Complexes; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase