The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks

We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSBCter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSBCter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSBCter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSBCter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.我们通过体内实验探究了枯草芽孢杆菌（Bacillus subtilis）单链DNA结合蛋白（Single-Stranded DNA Binding protein）的羧基末端结构域（SSBCter）作为招募平台，在活性染色体复制叉处招募各类参与基因组维持的蛋白的功能。本研究通过GFP融合蛋白、Tap标签（Tap-tag）纯化及生化分析手段，对该SSBCter的互作组进行了系统解析。该互作组至少包含12种蛋白。此前已有研究证实该互作组包含PriA、RecG与RecQ，本研究通过新增DnaE、SbcC、RarA、RecJ、RecO、XseA、Ung、YpbB及YrrC，进一步拓展了该互作组的成员构成。YpbB向活性复制叉的靶向定位似乎依赖于RecS——一种RecQ的旁系同源蛋白，二者可形成稳定的复合物。这些SSB互作蛋白大多在细菌界中保守存在，而部分蛋白（如必需DNA聚合酶DnaE、YrrC以及YpbB/RecS复合物）则似乎为枯草芽孢杆菌所特有。SSBCter缺失对枯草芽孢杆菌的细胞生长仅产生中度影响，但其会显著损害受损基因组DNA的修复效率以及停滞复制叉的修复能力。ssbΔCter突变体细胞在单链DNA上的RecA加载过程存在缺陷，这一表型解释了其在暴露于遗传毒性试剂后无法有效触发SOS应答的内在原因。综上，本研究结果表明，细菌SSBCter可作为活性染色体复制叉处的基因组维持枢纽，保障复制叉沿基因组的正常推进。