The Lys313 residue of the human Rad51 protein negatively regulates the strand-exchange activity.
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.
Ishida
Takako
T
Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Takizawa
Yoshimasa
Y
Sakane
Isao
I
Kurumizaka
Hitoshi
H
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
D009088:Mucoproteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; C010393:lysin, gastropoda; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D002118:Calcium