Univ. Paris-Sud 11, CNRS UMR 8621, LRC CEA 42V, Institut de Génétique et Microbiologie, Bâtiment 409, Université Paris-Sud, 91405 Orsay, France.
DNA Repair (Amst). 2011 Dec 10;10(12):1223-31. doi: 10.1016/j.dnarep.2011.09.010. Epub 2011 Oct 2.
The Deinococcus radiodurans bacterium exhibits an extreme resistance to ionizing radiation. Here, we investigated the in vivo role of DdrB, a radiation-induced Deinococcus specific protein that was previously shown to exhibit some in vitro properties akin to those of SSB protein from Escherichia coli but also to promote annealing of single stranded DNA. First we report that the deletion of the C-terminal motif of the DdrB protein, which is similar to the SSB C-terminal motif involved in recruitment to DNA of repair proteins, did neither affect cell radioresistance nor DNA binding properties of purified DdrB protein. We show that, in spite of their different quaternary structure, DdrB and SSB occlude the same amount of ssDNA in vitro. We also show that DdrB is recruited early and transiently after irradiation into the nucleoid to form discrete foci. Absence of DdrB increased the lag phase of the extended synthesis-dependent strand annealing (ESDSA) process, affecting neither the rate of DNA synthesis nor the efficiency of fragment reassembly, as indicated by monitoring DNA synthesis and genome reconstitution in cells exposed to a sub-lethal ionizing radiation dose. Moreover, cells devoid of DdrB were affected in the establishment of plasmid DNA during natural transformation, a process that requires pairing of internalized plasmid single stranded DNA fragments, whereas they were proficient in transformation by a chromosomal DNA marker that integrates into the host chromosome through homologous recombination. Our data are consistent with a model in which DdrB participates in an early step of DNA double strand break repair in cells exposed to very high radiation doses. DdrB might facilitate the accurate assembly of the myriad of small fragments generated by extreme radiation exposure through a single strand annealing (SSA) process to generate suitable substrates for subsequent ESDSA-promoted genome reconstitution.
耐辐射球菌具有极强的抗电离辐射能力。在这里，我们研究了辐射诱导的 Deinococcus 特异蛋白 DdrB 的体内作用，先前的研究表明，它具有类似于大肠杆菌 SSB 蛋白的一些体外特性，但也能促进单链 DNA 的退火。首先，我们报告说，DdrB 蛋白 C 端基序的缺失既不影响细胞的辐射抗性，也不影响纯化 DdrB 蛋白的 DNA 结合特性，该基序类似于参与修复蛋白与 DNA 招募的 SSB C 端基序。我们表明，尽管它们的四级结构不同，DdrB 和 SSB 在体外可以封闭相同数量的 ssDNA。我们还表明，DdrB 在照射后早期和短暂地被招募到核质中形成离散的焦点。尽管 DdrB 缺失增加了扩展合成依赖性链退火（ESDSA）过程的滞后期，但不会影响 DNA 合成的速率或片段重组的效率，这可以通过监测暴露于亚致死电离辐射剂量的细胞中的 DNA 合成和基因组重组来指示。此外，在自然转化过程中，缺乏 DdrB 的细胞会影响质粒 DNA 的建立，这一过程需要内化的质粒单链 DNA 片段的配对，而它们在转化由通过同源重组整合到宿主染色体中的染色体 DNA 标记时则没有受到影响。我们的数据与这样一种模型一致，即在暴露于极高辐射剂量的细胞中，DdrB 参与 DNA 双链断裂修复的早期步骤。DdrB 可能通过单链退火（SSA）过程促进由极端辐射暴露产生的大量小片段的准确组装，从而为随后的 ESDSA 促进的基因组重组生成合适的底物。
