title: Identification of a second DNA binding site in the human Rad52 protein.
abstract: Rad52 plays essential roles in homology-dependent double-strand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the Rad52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that Rad52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that Rad52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Kagawa
authors.forename: Ako
authors.initials: A
authors.lastname: Kagawa
authors.forename: Kengo
authors.initials: K
authors.lastname: Saito
authors.forename: Shukuko
authors.initials: S
authors.lastname: Ikawa
authors.forename: Takehiko
authors.initials: T
authors.lastname: Shibata
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurumizaka
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016296:Mutagenesis; D020125:Mutation, Missense; D009690:Nucleic Acid Conformation; D010449:Peptide Mapping / Q000379:methods; D051721:Rad52 DNA Repair and Recombination Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein