Department of Cell Biology and Genetics, Cancer Genomics Center, 3000 CA Rotterdam, Netherlands.
J Cell Biol. 2011 Mar 7;192(5):735-50. doi: 10.1083/jcb.201011025. Epub 2011 Feb 28.
Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54's ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54's ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non-DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus.
Rad54 是 DNA 依赖的 ATP 酶家族 SWI/SNF 蛋白家族的成员，通过同源重组修复 DNA 双链断裂 (DSBs)。在这里，我们证明 Rad54 是同源重组蛋白 Rad51 和 Brca2 在 DSBs 处及时积累所必需的。由于复制蛋白 A 和 Nbs1 的积累不受 Rad54 耗竭的影响，因此 Rad54 位于 DSB 切除的下游。Rad54 介导的 Rad51 积累不依赖于 Rad54 的 ATP 酶活性。因此，我们的实验表明 SWI/SNF 蛋白可能具有与其 ATP 酶活性无关的功能。然而，Rad54 焦点形成的定量实时分析表明，Rad54 的 ATP 酶活性对于 Rad54 从 DNA 上的解聚以及 Rad54 在 DSB 处的周转是必需的。尽管非 DNA 结合的 Rad54 分数可逆地与焦点相互作用，而与 Rad54 的 ATP 酶状态无关，但在没有 Rad54 水解 ATP 的情况下，DNA 结合的分数被固定。最后，我们表明 Rad54 的 ATP 水解对于核内 DSB 修复位点的重新分布是必需的。
