title: The Lys313 residue of the human Rad51 protein negatively regulates the strand-exchange activity.
abstract: The Rad51 protein, which catalyzes homologous-pairing and strand-exchange reactions, is an essential enzyme for homologous recombinational repair (HRR) and meiotic homologous recombination in eukaryotes. In humans, the conventional Rad51 (HsRad51) protein has a Lys residue at position 313; however, the HsRad51-Q313 protein, in which the Lys313 residue is replaced by Gln, was reported as an isoform, probably corresponding to a polymorphic variant. In this study, we purified the HsRad51-K313 and HsRad51-Q313 isoforms and analyzed their biochemical activities in vitro. Compared to the conventional HsRad51-K313 protein, the HsRad51-Q313 protein exhibited significantly enhanced strand-exchange activity under conditions with Ca(2+), although the difference was not observed without Ca(2+). A double-stranded DNA (dsDNA) unwinding assay revealed that the HsRad51-Q313 protein clearly showed enhanced DNA unwinding activity, probably due to its enhanced filament-formation ability. Mutational analyses of the HsRad51-Lys313 residue revealed that positively charged residues (Lys and Arg), but not negatively charged, polar and hydrophobic residues (Glu, Gln and Met, respectively), at position 313 reduced the strand-exchange and DNA unwinding abilities of the HsRad51 protein. These results suggest that the electrostatic environment around position 313 is important for the regulation of the HsRad51 recombinase activity.
authors.affiliation: Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Ishida
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Takizawa
authors.forename: Isao
authors.initials: I
authors.lastname: Sakane
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kurumizaka
mesh_terms: D002118:Calcium / Q000737:chemistry; D006801:Humans; D009088:Mucoproteins / Q000737:chemistry*; D009154:Mutation; D009690:Nucleic Acid Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D009088:Mucoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C010393:lysin, gastropoda; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D002118:Calcium