Laboratoire de Microbiologie et Génétique Moléculaires, Université de Toulouse, Centre National de la Recherche Scientifique, LMGM-UMR5100, Toulouse, France.
PLoS Genet. 2010 Dec 9;6(12):e1001238. doi: 10.1371/journal.pgen.1001238.
We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSB(Cter)) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSB(Cter) 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. SSB(Cter) 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 SSB(Cter) acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.
我们研究了枯草芽孢杆菌单链 DNA 结合蛋白（SSB(Cter)）羧基末端结构域在体内作为许多基因组维护机制蛋白在活性染色体叉上募集平台的作用。我们使用 GFP 融合蛋白和 Tap-tag 以及生化分析来探测这个 SSB(Cter)相互作用组。它至少包括 12 种蛋白质。这个相互作用组以前包括 PriA、RecG 和 RecQ，在本研究中通过添加 DnaE、SbcC、RarA、RecJ、RecO、XseA、Ung、YpbB 和 YrrC 进行了扩展。YpbB 靶向活性叉似乎依赖于 RecS，这是 RecQ 的一个旁系同源物，它与 RecS 形成稳定的复合物。这些 SSB 伴侣中的大多数在细菌中是保守的，而其他的，如必需的 DNA 聚合酶 DnaE、YrrC 和 YpbB/RecS 复合物，似乎是枯草芽孢杆菌所特有的。SSB(Cter)缺失对枯草芽孢杆菌细胞生长有中等影响。然而，它显著影响受损基因组 DNA 的修复效率和停滞的复制叉。ssbΔCter 突变体细胞在 ssDNA 上的 RecA 加载似乎存在缺陷，这解释了它们在暴露于遗传毒性物质时触发 SOS 反应的效率低下。总之，我们的发现表明，细菌 SSB(Cter)作为活性染色体叉上的 DNA 维护中心，确保了它们沿着基因组的传播。
