抄録
Many anti-cancer strategies are based on the induction of DNA double strand break (DSB). The effectiveness of these strategies can be counteracted by the cell's ability to repair DSBs through homologous recombination (HR). Therefore, we want to understand the mechanism through which HR operates. We are analyzing the effect of mutations in the ATPase of Rad54, which is essential for HR. Rad54 possess a variety of biochemical activities that can affect Rad51 function in the critical steps of HR. Our in vivo study shows that Rad54's ATPase activity is not only important for HR but also affects its cellular behavior. Specifically, we show that it is not necessary for recruitment of the protein into DNA damaged induced structures at sites of DNA damage, known as foci, but rather that it influences Rad54 dissociation from foci. Furthermore, our data show that Rad54 but not its ATPase activity is required downstream of DNA break resection. In addition, our data reveal Rad54's ATPase activity differentially affects the behavior of the pool of Rad54 in a focus that is bound to DNA versus the pool that is not bound to DNA; the DNA-bound fraction can no longer dissociate, while the unbound fraction that is not actively engaged in DNA repair can still reversible interact with the focus. Finally, we show that the ATPase activity of Rad54 affects the locations of foci, because after preferential movement to the nuclear periphery, they remain there in the mutant cells.
