Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102-1192, USA.
Nucleic Acids Res. 2011 Mar;39(6):2153-64. doi: 10.1093/nar/gkq1139. Epub 2010 Nov 21.
DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the 'template-strand switch' mechanism. This mechanism involves replication fork regression into a Holliday junction ('chicken foot structure'), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion.
DNA 损伤会导致 DNA 复制叉停滞，这对细胞可能是致命的。同源重组 (HR) 在 DNA 损伤绕过中起着重要作用。人们认为，HR 的关键蛋白 Rad51 通过其 DNA 链入侵活性有助于 DNA 损伤绕过。在这里，我们使用模型停滞的复制叉发现，RAD51 和 RAD54 协同作用可以通过“模板链转换”机制促进体外 DNA 损伤绕过。该机制涉及复制叉退回到 Holliday 连接（“鸡脚结构”），使用新生的滞后 DNA 链作为模板进行 DNA 合成，然后恢复复制叉。我们的结果表明，RAD54 可以通过其分支迁移活性催化模型复制叉的回归和恢复，但强烈偏向于叉恢复。我们发现 RAD51 调节该反应；通过抑制叉恢复并刺激叉回归，它促进了鸡脚结构的积累，我们表明这对于体外 DNA 聚合酶的 DNA 损伤绕过是必不可少的。这些结果表明，RAD51 与 RAD54 合作可能在 DNA 损伤绕过中具有不同于 DNA 链入侵的新作用。
