Limited concentration of RecA delays DNA double-strand break repair in R1

低濃度RecAはデイノコッカス・ラジオデュランスR1株のDNA2本鎖切断修復を遅延させる

Jolivet, E.*; Lecointe, F.*; Coste, G.*; 佐藤 勝也*; 鳴海 一成; Bailone, A.*; Sommer, S.*

Jolivet, E.*; Lecointe, F.*; Coste, G.*; Sato, Katsuya*; Narumi, Issei; Bailone, A.*; Sommer, S.*

放射線抵抗性細菌のDNA2本鎖切断修復における組換え修復タンパク質RecAの重要性を評価するために、遺伝子発現が調節可能なPプロモーターを用いて、RecA濃度変化による細胞への影響を調べた。野生株RecA低濃度細胞の線照射後の生存率は、RecA高濃度細胞と同様であったが、あるいは遺伝子破壊株をRecA低濃度状態にすると線高感受性になり、遺伝子破壊株をRecA高濃度状態にすると線耐性が部分的に復帰した。また、野生株RecA低濃度細胞を線照射後に液体培地で培養すると、DNA2本鎖切断修復の遅延が起こり、DNA複製の再開ができないことが明らかになった。さらに、RecA低濃度細胞ではDNA損傷後のLexAタンパク質分解が起こらないこともわかった。しかしながら、遺伝子破壊株及びLexA非分解変異タンパク質産生株における線照射後の生存率とDNA2本鎖切断修復の速度は、野生株と比べて変化がなく、RecAの細胞内濃度にも依存していなかった。このことから、放射線抵抗性細菌のLexAタンパク質は線照射後のDNA修復過程に関与していないと考えられた。

To evaluate the importance of RecA in DNA double strand break (DSB) repair, we examined the effect of low and high RecA concentration such as 2,500 and 100,000 molecules per cell from the inducible P promoter in in absence or in presence of IPTG, respectively. We showed that at low concentration, RecA has a negligible effect on cell survival after -irradiation when bacteria were immediately plated on TGY agar whereas it significantly decreased the survival to -irradiation of 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 TGY liquid medium, they showed a delay in mending DSB, failed to reinitiate DNA replication and were committed to die during incubation in liquid medium. A deletion of resulted in sensitivity to -irradiation and mitomycin C treatment. Interestingly, constitutive high expression of RecA compensates partially the sensitization to mitomycin C. The cells with low RecA content also failed to cleave LexA after DNA damage. However, neither a deletion of the gene nor the expression of a non cleavable LexA(Ind) mutant protein, had an effect on survival or kinetics of DNA DSB repair compared to their counterparts in as well as in bacteria expressing limiting concentration of RecA, suggesting an absence of relationship between the loss of viability, the delay in the kinetic of DSB repair, and the absence of LexA cleavage. Thus, LexA protein seems to play no major role in the recovery processes after -irradiation in .