title: Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.
abstract: Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Louise H
authors.initials: LH
authors.lastname: Fornander
authors.forename: Karolin
authors.initials: K
authors.lastname: Frykholm
authors.forename: Anna
authors.initials: A
authors.lastname: Reymer
authors.forename: Axelle
authors.initials: A
authors.lastname: Renodon-Cornière
authors.forename: Masayuki
authors.initials: M
authors.lastname: Takahashi
authors.forename: Bengt
authors.initials: B
authors.lastname: Nordén
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D002118:Calcium / Q000737:chemistry*; D002413:Cations, Divalent / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D051135:Rad51 Recombinase / Q000737:chemistry* / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D008274:Magnesium; D002118:Calcium