Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
Nucleic Acids Res. 2020 Jul 9;48(12):6640-6653. doi: 10.1093/nar/gkaa442.
G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.
四链体 (G4) DNA 结构可以在基因组内形成物理屏障，必须解开这些结构以确保细胞基因组的完整性。在这里，我们报告了大肠杆菌 Rep 解旋酶和 RecA 重组酶在体内耐受 G4 稳定配体诱导的毒性方面的意外作用。我们证明 Rep 和 Rep-X（Rep 的增强版本）在体外显示出比密切相关的 UvrD 解旋酶更高的 G4 解旋活性。Rep 介导的 G4 解旋涉及 G4 展开和折叠的重复循环，由 ATP 水解提供动力。Rep-X 和 Rep 还会置换 G4 稳定配体，这与我们体内 G4-配体敏感性结果一致。我们进一步证明 RecA 丝在体外破坏 G4 结构并去除 G4 配体，这与其在对抗细胞毒性 G4 稳定配体中的作用一致。总之，我们的研究揭示了 Rep 和 RecA 在解决有害 G4 结构方面的新型基因组维护功能。
