Jolivet Edmond, Lecointe François, Coste Geneviève, Satoh Katsuya, Narumi Issay, Bailone Adriana, Sommer Suzanne
Institut de Génétique et Microbiologie, CNRS UMR 8621, LRC CEA 42V, Bâtiment 409, Université Paris-Sud, F-91405 Orsay Cedex, France.
Mol Microbiol. 2006 Jan;59(1):338-49. doi: 10.1111/j.1365-2958.2005.04946.x.
To evaluate the importance of RecA in DNA double-strand break (DSB) repair, we examined the effect of low and high RecA concentrations such as 2500 and 100 000 molecules per cell expressed from the inducible Pspac promoter in Deinococcus radiodurans in absence or in presence of IPTG respectively. We showed that at low concentration, RecA has a negligible effect on cell survival after gamma-irradiation when bacteria were immediately plated on TGY agar whereas it significantly decreased the survival to gamma-irradiation of DeltaddrA cells while overexpression of RecA can partially compensate the loss of DdrA protein. In contrast, when cells expressing limited concentration of RecA were allowed to recover in TGY2X liquid medium, they showed a delay in mending DSB, failed to reinitiate DNA replication and were committed to die during incubation. A deletion of irrE resulted in sensitivity to gamma-irradiation and mitomycin C treatment. Interestingly, constitutive high expression of RecA compensates partially the DeltairrE sensitization to mitomycin C. The cells with low RecA content also failed to cleave LexA after DNA damage. However, neither a deletion of the lexA gene nor the expression of a non-cleavable LexA(Ind-) mutant protein had an effect on survival or kinetics of DNA DSB repair compared with their lexA+ counterparts in recA+ as well as in bacteria expressing limiting concentration of RecA, suggesting an absence of relationship between the absence of LexA cleavage and the loss of viability or the delay in the kinetics of DSB repair. Thus, LexA protein seems to play no major role in the recovery processes after gamma-irradiation in D. radiodurans.
为评估RecA在DNA双链断裂（DSB）修复中的重要性，我们分别在有无异丙基-β-D-硫代半乳糖苷（IPTG）的情况下，检测了由耐辐射球菌中可诱导的Pspac启动子表达的低浓度和高浓度RecA（如每细胞2500和100000个分子）的影响。我们发现，在低浓度时，当细菌立即接种于TGY琼脂平板上时，RecA对γ射线照射后的细胞存活影响可忽略不计，而在ΔddrA细胞中，它显著降低了γ射线照射后的存活率，同时RecA的过表达可部分补偿DdrA蛋白的缺失。相反，当表达有限浓度RecA的细胞在TGY2X液体培养基中恢复时，它们在修复DSB方面出现延迟，未能重新启动DNA复制，并在培养过程中死亡。irrE基因的缺失导致对γ射线照射和丝裂霉素C处理敏感。有趣的是，RecA的组成型高表达部分补偿了ΔirrE对丝裂霉素C的敏感性。RecA含量低的细胞在DNA损伤后也无法切割LexA。然而，与recA+中的lexA+对应物以及表达有限浓度RecA的细菌相比，lexA基因的缺失或不可切割的LexA（Ind-）突变蛋白的表达对DNA DSB修复的存活或动力学均无影响，这表明LexA切割的缺失与活力丧失或DSB修复动力学延迟之间不存在关联。因此，LexA蛋白似乎在耐辐射球菌γ射线照射后的恢复过程中不起主要作用。
