DNA末端构型调控RecJ核酸外切酶活性

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Mg²⁺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Mg²⁺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...

DNA End Configurations Regulate RecJ Exonuclease Activity

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 Mg²⁺. To elucidate the mechanisms, RecJ was incubated with substrates containing different 3"-end configurations in the absence of Mg²⁺ 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 RecJ-DNA 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.