Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following γ-radiation-induced damage to pMTa4 DNA in E. coli
Critical role of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of rejoining following γ-radiation-induced damage to pMTa4 DNA in E. coli
复制标题
RecA 和 RecF 蛋白在大肠杆菌中 γ 辐射诱导的 pMTa4 DNA 损伤后链断裂重新连接和维持重新连接保真度中的关键作用
DOI：
--
复制
发表时间：
2007
影响因子：
2.6
通讯作者：
T. Nomura
中科院分区：
文献类型：
--
作者：
R. Sharan;H. Ryo;T. Nomura
目的：本研究旨在了解RecA和RecF蛋白在大肠杆菌暴露后链断裂重接和维持过程保真度中的作用。 大肠杆菌对γ射线的耐受性。材料和方法：将质粒pMTa 4转化到等基因修复活性（野生型）和缺陷型（recF和recA）的E. 大肠杆菌菌株和γ射线高达30戈伊的体内照射。在修复非允许（R-）和允许（R+）条件下分离质粒DNA，并通过凝胶电泳分析单链断裂（SSB）和双链断裂（DSB）的产率及其修复。E.大肠杆菌也被记录。还监测了γ辐照对用野生菌株的无细胞提取物或紫外线（UV）辐照重构的recA的影响。结果：所有菌株均未表现出辐射诱导的氧化性碱基损伤效应。 在R−条件下，野生型和recF突变体中pMTa 4上SSB和DSB的剂量依赖性增加在修复孵育后消失。recA突变体表现出SSB和DSB产量的干扰，沿着形成γ辐射诱导的“梯子”。在野生株无细胞提取物存在下，经修复孵育、UV照射或γ照射后，未观察到“梯状”。recA突变体的生存受到严重影响。结论：野生株、recF株和recA株E. 大肠杆菌能修复γ射线引起的碱基或核苷酸（NT）的氧化损伤。在没有RecA或RecF蛋白的情况下，链的重新连接效率下降; RecA蛋白似乎比RecF更关键。另一方面，链断裂的高保真或正确重新连接似乎需要同时存在RecA和RecF蛋白。
Purpose: This study was undertaken to understand the roles of RecA and RecF proteins in strand break rejoining and maintenance of fidelity of the process following exposure of E. coli to γ-radiation in vivo. Materials and methods: A plasmid DNA construct, pMTa4, was transformed into isogenic repair proficient (wild) and deficient (recF and recA) E. coli strains and γ-irradiated up to 30 Gy in vivo. The plasmid DNA was isolated under repair non-permissive (R−)and permissive (R+) conditions and analyzed by gel electrophoresis for the yields of single strand breaks (SSB) and double strand breaks (DSB) and their repair. The clonogenic survival of the E. coli was also recorded. The effects of γ-irradiation on recA reconstituted with cell free extract of wild strain or ultra-violet (UV)-irradiation were also monitored. Results: None of the strains used in this investigation showed effects of radiation-induced oxidative base damage. The dose dependent increase in SSB and DSB on pMTa4 in wild and recF mutants in R− condition were abolished upon repair incubation. The recA mutant exhibited a disturbed yield of SSB and DSB along with formation of γ-radiation-induced ‘ladder’. The ‘ladder’ was not observed after repair incubation, UV-irradiation or γ-irradiation in presence of cell-free extract of wild strain. The survival of recA mutants was seriously compromised. Conclusions: Wild, recF and recA strains of E. coli could repair γ-irradiation-induced oxidative damage to base or nucleotide (NT) in vivo. In absence of either RecA or RecF proteins, efficiency of rejoining of strand went down; RecA proteins seemed more critical than RecF in this. High fidelity or correct rejoining of strand breaks, on the other hand, seemed to require simultaneous presence of both RecA and RecF proteins.