Department of Cancer Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
Mol Cell. 2010 Jun 11;38(5):649-61. doi: 10.1016/j.molcel.2010.03.020.
DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication, and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage.
为了完成染色体复制并保持细胞活力和基因组稳定性，会绕过阻碍复制的 DNA 损伤。在酵母中，Rad5 是一种 PCNA 多聚泛素连接酶和 DNA 依赖性 ATP 酶，与假定的肿瘤抑制因子同源，并通过未知机制控制无差错的损伤绕过。为了在体内确定该机制，我们研究了 Rad5 的作用，并分析了在复制起点处存在的特定停滞叉处进行损伤绕过期间形成的 DNA 结构。Rad5 介导形成重组依赖性的 X 形 DNA 结构，在姐妹染色单体之间包含 Holliday 连接。缺乏这些损伤诱导的染色单体连接的突变体在解决停滞叉、重新启动复制和完成染色体复制方面存在缺陷。Rad5 多聚泛素连接酶和 ATP 酶结构域都有助于复制叉重组。我们的结果表明，Rad5 的多种活性与同源重组因子协同作用，使复制模板转换事件能够在停滞叉处连接姐妹染色单体，并绕过 DNA 损伤。
