Heteroduplex joint formation by a stoichiometric complex of Rad51 and Rad52 of Saccharomyces cerevisiae.
Both Rad51 and Rad52 are required for homologous genetic recombination in Saccharomyces cerevisiae. Rad51 promotes heteroduplex joint formation, a general step in homologous recombination. Rad52 facilitates the binding of Rad51 to replication protein A (RPA)-coated single-stranded DNA. The requirement of RPA can be avoided in vitro, if the single-stranded DNA is short. Using short single-stranded DNA and homologous double-stranded DNA, in the absence of RPA, we found that Rad52 (optimal at three per Rad51) was still required for Rad51-promoted heteroduplex joint formation in vitro, as assayed by the formation of D-loops, suggesting another role for Rad52. Rad51 has to bind to the single-stranded DNA before the addition of double-stranded DNA for efficient D-loop formation. Immunoprecipitation and single-stranded DNA-bead precipitation analyses revealed the presence of the free and DNA-bound complexes of Rad51 and Rad52 at a 1 to 2 stoichiometry. In the presence of single-stranded DNA, in addition to Rad51, Rad52 was required for extensive untwisting that is an intermediate step toward D-loop formation. Thus, these results suggest that the formation of the stoichiometric complex of Rad52 with Rad51 on single-stranded DNA is required for the functional binding of the protein-single-stranded DNA complex to the double-stranded DNA to form D-loops.
Arai
Naoto
N
Department of Applied Biological Science, Nihon University College of Bioresource Sciences, Fujisawa-shi, Kanagawa, Japan. narai@brs.nihon-u.ac.jp
Ito
Daisuke
D
Inoue
Tadashi
T
Shibata
Takehiko
T
Takahashi
Hideo
H
D001671:Biochemistry / Q000379:methods; D002021:Buffers; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000737:chemistry; D047468:Immunoprecipitation; D008540:Meiosis; D008938:Mitosis; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D051135:Rad51 Recombinase; D051721:Rad52 DNA Repair and Recombination Protein; D011938:Rec A Recombinases / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
D002021:Buffers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C495151:RAD51 protein, S cerevisiae; D051135:Rad51 Recombinase; D011938:Rec A Recombinases