A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans.
A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans.
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DOI：
10.1371/journal.pgen.1000774
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发表时间：
2010-01-15
期刊：
影响因子：
4.5
通讯作者：
Sommer S
中科院分区：
文献类型：
--
作者：
Bentchikou E;Servant P;Coste G;Sommer S
在耐辐射球菌中，对dna破碎处理（如电离辐射或干燥）的极端抗性与它从数百个染色体片段中重建功能基因组的能力有关。完整基因组的快速重建被认为是通过扩展的合成依赖链退火过程（ESDSA），然后是DNA重组。在此，我们研究了RecF通路的关键组分在这种自然缺乏RecB和RecC蛋白的生物体中的ESDSA中的作用。我们证明了RecJ外切酶的失活会导致细胞死亡，这表明该蛋白在基因组维持中起着关键作用。缺乏RecF、RecO或RecR蛋白的细胞也表现出与缺乏RecA蛋白的细菌一样严重的生长受损和重要的致命分裂。recFOR敲除突变体表型的其他方面与ΔrecA突变体相似：ΔrecFOR突变体对辐射非常敏感，表现出辐射诱导的染色体片段缓慢组装，不伴有DNA合成，DNA降解减少。缺乏RecQ（大肠杆菌中参与通过RecF途径进行修复的主要解旋酶）的细胞对γ辐照具有抗性，并且具有野生型DNA修复能力，这一点在缺乏RecD解旋酶的细胞中也得到了证实；相反，ΔuvrD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增加，片段组装速度慢与DNA合成速度慢相关。将RecQ或RecD缺陷与UvrD缺陷结合并没有显著增强ΔuvrD突变体的表型。综上所述，RecFOR蛋白是通过ESDSA修复DNA双链断裂所必需的，而RecJ蛋白是细胞生存所必需的，UvrD解旋酶可能参与双链DNA末端的加工和/或参与ESDSA的DNA合成步骤。耐辐射球菌细菌是最著名的生物之一，可以抵抗极高的干燥和电离辐射，两者都会导致广泛的DNA双链断裂。由于单个未修复的DNA双链断裂通常是致命的，因此DNA双链断裂被认为是最严重的基因组损伤形式。耐辐射球菌具有极强的抗辐射能力，这与它从数百个染色体片段中重建功能性基因组的能力有关。基因组重组通过两步过程发生：(i)扩展合成依赖链退火过程（ESDSA），将基因组片段组装成长线性中间体，然后（ii）通过重组产生环状染色体。在这里，我们证明了耐辐射球菌RecF通路的关键成分在ESDSA中的重要作用。我们发现(i)仅一种外切酶（RecJ）失活就会导致细胞死亡；（ii）缺乏RecF、RecO或RecR的细胞显示出严重的生长受损；（iii） RecF、RecO或RecR蛋白是通过ESDSA产生辐射抗性所必需的；（iv） UvrD解旋酶在DNA双链断裂ESDSA修复中具有意想不到的关键作用。
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 ΔrecA mutant: ΔrecFOR 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 γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD 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 ΔuvrD 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. Deinococcus radiodurans bacterium is among the best-known organisms found to resist extremely high exposures to desiccation and ionizing radiation, both causing extensive DNA double-strand breaks. Because a single unrepaired DNA double-strand break is usually lethal, DNA double-strand breaks are considered as the most severe form of genomic damage. The extreme radioresistance of D. radiodurans is linked to its ability to reconstruct a functional genome from hundreds of chromosomal fragments. Genome reconstitution occurs through a two step process: (i) an extended synthesis-dependent strand-annealing process (ESDSA) that assembles genomic fragments in long linear intermediates that are then (ii) processed through recombination to generate circular chromosomes. Here, we demonstrate the essential role of key components of the D. radiodurans RecF pathway in ESDSA. We show that (i) inactivation of only one exonuclease (RecJ) results in cell lethality; (ii) cells devoid of RecF, RecO, or RecR display greatly impaired growth; (iii) RecF, RecO, or RecR proteins are essential for radioresistance through ESDSA; and (iv) UvrD helicase has an unexpected crucial function in DNA double-strand-break repair through ESDSA.