biostudies-literaturePaul TNational Science Foundation Physics Frontiers Center ProgramHoward Hughes Medical InstituteCenter for the Physics of Living CellsNCI NIH HHSNational Institutes of HealthNIGMS NIH HHSNIH HHS6640-6653https://www.ebi.ac.uk/biostudies/studies/S-EPMC7337899biostudies-literatureUnknown48(12)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, consistNucleic acids researchE. coli Rep helicase and RecA recombinase unwind G4 DNA and are important for resistance to G4-stabilizing ligands.PMC7337899T32 GM0086920822613RM1 GM130450R01 GM1156311R01GM115631-01A1R01 GM098885F30 CA210465S10 OD021567Keck JLVoter AFCueny RRMyong SPaul TGavrilov MHa TfalseE. coli Rep helicase and RecA recombinase unwind G4 DNA and are important for resistance to G4-stabilizing ligands.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, consist2020-01-01T00:00:00Z2020 Jul2025-04-19T18:00:29.365Z2025-04-19T18:00:29.365ZS-EPMC73378993244993010.1093/nar/gkaa442