大肠杆菌DNA损伤修复基因recA、recB、ruvC的实时定量PCR分析

Real-Time quantitative PCR Analysis of DNA Damage Repair genes recA,recB and ruvC in Escherichia Coli

大肠杆菌重组酶RecA能够结合单链DNA,介导链交联,使停滞的复制叉回退。RecB参与形成RecBCD复合体,RecBCD处理DNA产生3’-overhang结构,此结构是RecA介导的同源重组(Homologous Recombination,HR)所必需的。RuvC参与形成RuvABC复合体,RuvABC处理Holliday junction(Hj)结构,进而通过同源重组途径完成DNA损伤修复。本研究表明甲基磺酸甲酯(Methyl Methanesulfonate,MMS)对细胞生长、克隆形成能力及细胞形态有明显影响。实时定量PCR分析发现MMS引起recA上调,recB和ruvC下调。recA在MMS处理后6小时达到最高,之后回落,说明需要大量的RecA处理受损的DNA。recB和ruvC下调后随着时间推移有所恢复,说明细胞正在逐步完成DNA损伤的修复。细胞可能通过同源重组修复受损的DNA。

The E.coli recombinase RecA can bind to single-stranded DNA and promotes DNA strand exchange,which contributes to fork regression.RecB is involved in the formation of RecBCD,and RecBCD can generate a 3’-overhang needed for RecA-mediated homologous recombination(HR).RuvC can form RuvABC complex,which process the Holliday junction(HJ)structure,and subsequently accomplish DNA repair via HR.This study revealed that methyl methanesulfonate(MMS)exhibited significant influences on cell proliferation,colony formation ability and cell morphology.Real-time quantitative PCR analysis indicated that MMS treatment induced up-regulation of recA,as well as down-regulation of recB and ruvC.Six hours after MMS treatment,the expression of recA reached the highest level and then declined,indicating that a large amount of RecA was required to process the damaged DNA.The down-regulation of recB and ruvC slightly recovered over time,indicating that the cells are gradually completing DNA damage repair.Therefore,the cells may repair damaged DNA via HR.