E. coli Rep helicase and RecA recombinase unwind G4 DNA and are important for resistance to G4-stabilizing ligands.

Andrew F Voter,Taekjip Ha,Rachel R Cueny,Tapas Paul,Momčilo Gavrilov,James L Keck,Sua Myong

doi:10.1093/nar/gkaa442

Abstract

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.

Highlights

Guanine-rich nucleic acid sequences have strong propensities to form four-stranded G-quadruplex (G4) structures under physiological conditions [1]
We tested the ability of mutant strains with deletions in rep::kan, uvrD::kan, recA::kan, recQ::kan to grow on LB plates containing two structurally distinct G4 stabilizers, N-methyl mesoporphyrin IX (NMM) or BRACO-19
To determine the G4 stabilizer concentrations for these experiments, the sensitivities of E. coli MG1655 and of an imp-4213 strain were tested using a range of NMM and BRACO-19 concentrations

Summary

Introduction

Guanine-rich nucleic acid sequences have strong propensities to form four-stranded G-quadruplex (G4) structures under physiological conditions [1]. In these structures, four guanine bases are cyclically coordinated through Hoogsteen hydrogen bonds to form a G-quartet or tetrad ring, which is further stabilized by stacking interaction with other ring layers in the presence of monovalent cation [1,2,3]. Stable G4 structures can act as roadblocks to numerous cellular processes such as replication, transcription, and translation [12,13,14]. G4 ligands (e.g. BRACO-19 and Nmethyl mesoporphyrin IX (NMM)) can further enhance the stability of G4 structures, disrupting critical cellular pathways and thereby inducing toxicity in cells [13,15]. Dedicated cellular machineries have evolved to resolve G4 structures

Methods

Results

Conclusion