Nucleotide dependent structural and kinetic changes in Xenopus rad51.1-DNA complex stimulating the strand exchange reaction: destacking of DNA bases and restriction of their local motion.
Rad51 is a eukaryotic homologue of RecA and it catalyzes the DNA strand exchange reaction in homologous recombination. This protein, like RecA, requires ATP as a cofactor for activity. We investigated the mechanism of activation of this protein by the nucleotide cofactor by studying the effect of various nucleotides, particularly ATP, ADP and the non-hydrolyzable analog of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) on the DNA binding of a Xenopus Rad51 protein (XRad51.1). DNA binding was studied in solution by monitoring the fluorescence changes of etheno-modified fluorescent poly(dA) or fluorescein-labeled oligo(dT) and by filter binding assay. Active nucleotides (ATP, dATP) changed the DNA binding mode of XRad51.1. In the active complex, the DNA bases were destacked and their motion was highly restricted. Dissociation of XRad51.1 from DNA was accelerated by ATP and dATP, as was dissociation of RecA from DNA. In contrast to these similarities with RecA, the XRad51.1-DNA complex was dissociated by the non-hydrolyzable analog of ATP (ATPgammaS) and this dissociation was not significantly accelerated by ADP. The effect of ATP hydrolysis on the XRad51.1-DNA complex differs from that on the RecA-DNA complex. ATP hydrolysis may not be essential for the strand exchange reaction whereas the changes in the DNA structure by ATP are important.
Maeshima
K
K
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Maraboeuf
F
F
Morimatsu
K
K
Horii
T
T
Takahashi
M
M
D000818:Animals; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D011061:Poly A / Q000378:metabolism; D011485:Protein Binding; D051135:Rad51 Recombinase; D013050:Spectrometry, Fluorescence; D013936:Thymidine / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D029867:Xenopus Proteins; C040459:poly(d-epsilon A); D011061:Poly A; D004247:DNA; C495152:RAD51 protein, Xenopus; D051135:Rad51 Recombinase; D013936:Thymidine