Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USA.
Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, Michigan, USA.
J Bacteriol. 2014 Aug;196(15):2851-60. doi: 10.1128/JB.01494-14. Epub 2014 Jun 2.
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.
RecA 是维持细菌细胞基因组完整性的核心。尽管 RecA 在真细菌中几乎普遍存在，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知之甚少。在这里，我们发现 RecA 在 DNA 损伤或非依赖性复制叉停滞后立即与 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，而这两种途径的选择取决于正在修复的损伤类型。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，这适用于所有检查过的损伤类型，包括特定的双链断裂和非依赖性复制叉停滞。此外，我们提供的证据表明，尽管 RecF 在体内形成 RecA 修复中心时不是必需的，但 RecF 确实提高了修复中心组装的效率，这表明 RecF 可能会影响 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) 是组装 RecA 修复中心的另一个重要组成部分。SSB C 端的截断会损害枯草芽孢杆菌在应对损伤和非依赖性叉停滞时形成修复中心的能力。有了这些结果，我们得出结论，SSB 依赖性将 RecOR 招募到复制体对于在 DNA 损伤和复制叉停滞时加载和组织 RecA 进入修复中心是必要的。
