Université Paris-Sud 11, CNRS UMR 8621, LRC CEA 42V, Institut de Génétique et Microbiologie, Orsay, France.
PLoS Genet. 2010 Jan 15;6(1):e1000774. doi: 10.1371/journal.pgen.1000774.
In Deinococcus radiodurans, the extreme resistance to DNA-shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a DeltarecA mutant: DeltarecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to gamma-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, DeltauvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of DeltauvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.
在极端耐辐射球菌中，对 DNA 破碎处理（如电离辐射或干燥）的极端抗性与其从数百个染色体片段重建功能基因组的能力相关。完整基因组的快速重建被认为是通过扩展的合成依赖性链退火过程（ESDSA），随后是 DNA 重组来完成的。在这里，我们研究了该生物体中 RecF 途径的关键成分在 ESDSA 中的作用，该生物体天然缺乏 RecB 和 RecC 蛋白。我们证明，RecJ 核酸外切酶的失活导致细胞致死，表明该蛋白在基因组维护中发挥关键作用。缺乏 RecF、RecO 或 RecR 蛋白的细胞也表现出严重的生长受损和重要的致死性分区，就像缺乏 RecA 蛋白的细菌一样。recFOR 敲除突变体的其他表型与 recA 突变体相似：recFOR 缺失突变体对辐射极其敏感，表现出辐射诱导的染色体片段缓慢组装，不伴有 DNA 合成，并且 DNA 降解减少。缺乏主要涉及大肠杆菌 RecF 途径修复的 RecQ 解旋酶的细胞对 γ 辐射具有抗性，并且具有与缺乏 RecD 解旋酶的细胞相同的野生型 DNA 修复能力；相比之下，DeltauvrD 突变体表现出明显降低的辐射抗性，DNA 双链断裂修复动力学的潜伏期延长，并且片段组装速度较慢，与 DNA 合成速度较慢相关。将 RecQ 或 RecD 缺乏与 UvrD 缺乏结合使用并没有显著加重 DeltauvrD 突变体的表型。总之，RecFOR 蛋白对于通过 ESDSA 的 DNA 双链断裂修复是必不可少的，而 RecJ 蛋白对于细胞活力是必不可少的，UvrD 解旋酶可能参与双链 DNA 末端的加工和/或 ESDSA 的 DNA 合成步骤。
