RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress
RecO and RecR Are Necessary for RecA Loading in Response to DNA Damage and Replication Fork Stress
复制标题
DOI：
10.1128/jb.01494-14
复制
发表时间：
2014-08-01
影响因子：
3.2
通讯作者：
Simmons, Lyle A.
中科院分区：
文献类型：
--
作者：
Lenhart, Justin S.;Brandes, Eileen R.;Simmons, Lyle A.
RecA 对于维持细菌细胞基因组完整性至关重要。尽管 RecA 在真细菌中几乎无处不在，但在枯草芽孢杆菌中促进 RecA 加载和修复中心组装的途径仍然知之甚少。在这里，我们表明，在 DNA 损伤或损伤无关的复制叉停滞后，RecA 立即与 DNA 聚合酶复合物（复制体）快速共定位。在大肠杆菌中，RecFOR 和 RecBCD 途径用于加载 RecA，这两种途径之间的选择取决于修复中的损伤类型。我们在枯草芽孢杆菌中发现，RecA 快速定位到修复中心严格依赖于 RecO 和 RecR，以响应所检查的所有类型的损伤，包括位点特异性双链断裂和损伤无关的复制叉停滞。此外，我们提供的证据表明，虽然 RecF 不是体内 RecA 修复中心形成所必需的，但 RecF 确实提高了修复中心组装的效率，表明 RecF 可能影响 RecA 成核或丝延伸的初始阶段。我们进一步确定单链 DNA 结合蛋白 (SSB) 作为 RecA 修复中心组装的重要附加成分。 SSB C 末端的截断会损害枯草芽孢杆菌形成修复中心以响应损伤和损伤无关的叉停滞的能力。根据这些结果，我们得出结论，RecOR 依赖于 SSB 招募到复制体中，对于将 RecA 加载和组织到修复中心以响应 DNA 损伤和复制叉停滞是必要的。
RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.