DNA末端构型调控RecJ核酸外切酶活性

DNA End Configurations Regulate RecJ Exonuclease Activity

目的核酸酶介导的DNA双链末端切割对同源重组修复至关重要。然而,DNA末端构型对RecJ 5’-3’核酸外切酶活性的调控尚不清楚。本研究旨在探究DNA3’端和5’端构型对RecJ核酸外切酶活性的影响及其机制。方法为探究DNA3’端构型对RecJ核酸外切酶活性的影响,使用含有Mg;的体系,对具有不同3’突出末端长度(9 nt与18 nt)和3’突出末端修饰(磷酸化和硫代磷酸酯修饰)的单链DNA分别进行RecJ核酸酶活性检测。为揭示DNA 3’端构型对RecJ外切酶活性的调控机制,在Mg;缺失的体系中,使RecJ与底物结合后进行凝胶迁移实验(EMSA)。为探索其他调控因子与DNA3’端构型对RecJ的协同作用,分别检测5’端磷酸化修饰和单链DNA结合蛋白(SSB)对DNA3’突出末端修饰的影响。结果DNA3’端构型包括突出末端的长度和修饰(磷酸化和硫代磷酸酯修饰)均会抑制RecJ外切酶活性。DNA 3’端磷酸化和硫代磷酸酯修饰通过重塑RecJ-DNA的结合模式抑制RecJ外切酶活性。DNA 5’端磷酸化修饰可增强RecJ对具有不同3’端修饰底物的核酸外切酶活性,并改变RecJ-DNA的结合模式。此外,SSB通过增强RecJ-DNA结合可部分克服DNA3’端修饰介导的抑制作用。结论DNA3’与5’端构型协同调控RecJ核酸外切酶的活性。

Objective DNA end resection is a common mechanism for the formation of 3’-ssDNA tails in homologous recombination(HR)and is mainly mediated by 5’-3’exonuclease.However,whether DNA end configurations directly regulate 5’-3’exonuclease activity remains unclear.In this study,we explored the regulation and mechanisms of DNA end configurations on RecJ,the only 5’-3’exonuclease of RecF recombination pathway in Escherichia coli.Methods To investigate the regulation of DNA 3’-end configurations on RecJ exonuclease,single-stranded DNAs(ssDNAs)containing different lengths of 3’-ssDNA overhangs(9 nt and 18 nt)and 3’-end modifications(phosphorylation and phosphorothioation)were used for exonuclease assays in the presence of Mg;.To elucidate the mechanisms,RecJ was incubated with substrates containing different 3’-end configurations in the absence of Mg;and analyzed by electrophoretic mobility shift assays(EMSA).Furthermore,the coordination of DNA 3’end configurations and two other RecJ regulatory factors,DNA 5’-end phosphorylation and single-stranded DNA binding protein(SSB),were determined by exonuclease assays and EMSA on substrates with different 3’-end configurations respectively.Results DNA 3’-end configurations inhibited the RecJ exonuclease activity,including DNA 3’-overhang length and 3’-end modifications(phosphorylation and phosphorothioation).3’-End phosphorylation and phosphorothioation of DNA reshaped the RecJ-DNA binding patterns to inhibit RecJ exonuclease activity.DNA 5’-end phosphorylation overcame RecJ inhibition of 3’-end modifications and remodeled the RecJDNA binding patterns.In addition,SSB partially overcame the 3’-end modifications mediated inhibition by enhancing RecJ-DNA binding.Conclusion The RecJ exonuclease activity was regulated and orchestrated by the DNA configurations of 3’and 5’ends.