Different and Unexpected Stabilization Effects of ATP and ADP on Human Rad51?ssDNA Complexes
The molecular mechanism of homologous recombination catalyzed by RecA protein in E. colihas been extensively studied and the role of RecA in promoting the pairing of two homologous DNA molecules, branch migration and strand exchange is largely understood. In eukaryotes, Rad51 protein, a eukaryotic homolog of RecA protein, similarly plays a crucial role in homologous recombination. Electron microscopy and neutron scattering studies (1,2) have shown that Rad51 protein forms, like RecA protein, a filamentous complex with DNA for the reaction, suggesting a similar reaction mechanism. HsRad51 protein promotes ATP-dependent homologous pairing and strand transfer reaction
We have purified human Rad51 (HsRad51) protein to investigate the mechanism of homologous recombination in higher eukaryotes. Fluorescence measurements of a fluorescein-labeled oligonucleotide, oligo(dT*) and a chemically modified fluorescent poly(deA) analogue show that HsRad51 dissociates from ssDNA upon addition of ATP in complete contrast to what is observed for Xenopus XRad51 and RecA proteins (5). The reaction was complete within 20 minutes. Fluorescence anisotropy experiments with oligo(dT*) yielded the same result. This finding was totally unexpected since it is known that ATP is necessary for the strand exchange reaction. To further address this conundrum regarding ATP-dependence, the dissociation of ssDNA from HsRad51 upon addition of ATP and other nucleotides cofactors (ADP, dATP) was investigated in long-time kinetic experiments. We found that dATP behaved like ATP and induced HsRad51 dissociation from the protein-ssDNA complex. Quite unexpectedly, however, not only does ADP not dissociate HsRad51 from ssDNA (once again, an effect opposite to that found for RecA) but leads to a further increase of fluorescence with time. Our findings are discussed in terms of the relation between binding mechanism and strand exchange reaction, and the role of nucleotides cofactors.
1. Ogawa, T., Yu, X., Shinohara, A., and Egelman, E. H. (1993) Science 259, 1896-9.
Hye-Kyung Kim1*, Katsumi Morimatsu1, Eimer Tuite1, Bengt Nordén1, and Masayuki Takahashi2
1Department of Physical Chemistry,