The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.
The C-terminal domain of the bacterial SSB protein acts as a DNA maintenance hub at active chromosome replication forks.
复制标题
DOI：
10.1371/journal.pgen.1001238
复制
发表时间：
2010-12-09
期刊：
影响因子：
4.5
通讯作者：
Polard P
中科院分区：
文献类型：
--
作者：
Costes A;Lecointe F;McGovern S;Quevillon-Cheruel S;Polard P
我们在体内研究了枯草芽孢杆菌单链 DNA 结合蛋白 (SSBCter) 的羧基末端结构域作为基因组维护机制的许多蛋白质在活性染色体分叉处的招募平台的作用。我们使用 GFP 融合以及 Tap-tag 和生化分析探测了这个 SSBCter 相互作用组。它至少包含12种蛋白质。先前显示相互作用组包括 PriA、RecG 和 RecQ，并在本研究中通过添加 DnaE、SbcC、RarA、RecJ、RecO、XseA、Ung、YpbB 和 YrrC 进行扩展。 YpbB 对活跃分叉的靶向似乎依赖于 RecS（RecQ 旁系同源物），它与 RecS 形成稳定的复合物。大多数这些 SSB 伙伴在细菌中是保守的，而其他伙伴，例如必需的 DNA 聚合酶 DnaE、YrrC 和 YpbB/RecS 复合体，似乎是枯草芽孢杆菌特有的。 SSBCter 缺失对枯草芽孢杆菌细胞生长有中等影响。然而，它显着影响受损基因组 DNA 的修复效率并抑制复制叉。 ssbΔCter 突变细胞似乎缺乏 ssDNA 上的 RecA 负载，这解释了它们在暴露于基因毒性剂时无法有效触发 SOS 反应。总之，我们的研究结果表明，细菌 SSBCter 在活性染色体分叉处充当 DNA 维护中心，确保其沿着基因组繁殖。细胞增殖主要依赖于基因组的完整和准确的复制。因此，所有生物体都进化出了多种机制来保护、修复和重新激活 DNA 复制叉。目前大量的研究旨在破译将参与这些救援途径的蛋白质精确引导至染色体复制叉的机制。在这里，我们使用模型细菌枯草芽孢杆菌来证明，活跃的染色体 DNA 复制叉通过与单链 DNA 结合蛋白 (SSB) 的羧基末端 (Cter) 的直接物理相互作用预先配备了许多此类救援效应器。对 SSB Cter (SSBCter) 缺失的活枯草芽孢杆菌突变体的多重缺陷的详细分析揭示了该结构域对于维持基因组完整性和分叉繁殖的重要作用。无法在高温下生长是ssbΔCter突变体的主要缺陷。我们表明，这种致死率可以通过 RecO（SSB 的众多伙伴之一）的过度表达来特异性抑制，显然是通过介导 RecA 重组酶在 ssDNA 上的负载。
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. Cell multiplication relies primarily on the complete and accurate duplication of the genome. Thus, all organisms have evolved multiple mechanisms to protect, repair, and re-activate the DNA replication forks. A large body of research is currently aimed at deciphering the mechanisms that precisely direct the proteins involved in these rescue pathways towards the chromosome replication forks. Here, we have used the model bacterium Bacillus subtilis to demonstrate that the active chromosomal DNA replication forks are pre-equipped with many such rescue effectors via their direct physical interaction with the carboxy-terminal end (Cter) of the Single-Stranded DNA Binding protein (SSB). A detailed analysis of the multiple defects of viable B. subtilis mutants deleted for the Cter of SSB (SSBCter) revealed the vital role of this domain for the maintenance of genome integrity and fork propagation. The inability to grow at high temperature is a major defect of the ssbΔCter mutant. We show that this lethality can be specifically suppressed by overexpression of RecO, one of the numerous partners of SSB, apparently by mediating the loading of the RecA recombinase on ssDNA.
登录
查看更多内容
影响因子：
14.9
作者：
Davidsen T;Beck E;Ganapathy A;Montgomery R;Zafar N;Yang Q;Madupu R;Goetz P;Galinsky K;White O;Sutton G
通讯作者：
Sutton G
影响因子：
64.5
作者：
KONG, XP;ONRUST, R;KURIYAN, J
通讯作者：
KURIYAN, J
影响因子：
56.9
作者：
Dervyn, E;Suski, C;Ehrlich, SD
通讯作者：
Ehrlich, SD
DOI：
10.1073/pnas.0800741105
发表时间：
2008-07-08
影响因子：
11.1
作者：
Lu, Duo;Keck, James L.
通讯作者：
Keck, James L.
影响因子：
4.8
作者：
Liu, J;Marians, KJ
通讯作者：
Marians, KJ