Resolving Toxic DNA repair intermediates in every E.coli replication cycle: critical roles for RecG, Uup and RadD.

Elizabeth A Wood,Antoine van Oijen,Thomas Armstrong,Stefanie H Chen,Zachary J Romero,Andrew Robinson,Michael M Cox

doi:10.1093/nar/gkaa579

Abstract

DNA lesions or other barriers frequently compromise replisome progress. The SF2 helicase RecG is a key enzyme in the processing of postreplication gaps or regressed forks in Escherichia coli. A deletion of the recG gene renders cells highly sensitive to a range of DNA damaging agents. Here, we demonstrate that RecG function is at least partially complemented by another SF2 helicase, RadD. A ΔrecGΔradD double mutant exhibits an almost complete growth defect, even in the absence of stress. Suppressors appear quickly, primarily mutations that compromise priA helicase function or recA promoter mutations that reduce recA expression. Deletions of uup (encoding the UvrA-like ABC system Uup), recO, or recF also suppress the ΔrecGΔradD growth phenotype. RadD and RecG appear to avoid toxic situations in DNA metabolism, either resolving or preventing the appearance of DNA repair intermediates produced by RecA or RecA-independent template switching at stalled forks or postreplication gaps. Barriers to replisome progress that require intervention by RadD or RecG occur in virtually every replication cycle. The results highlight the importance of the RadD protein for general chromosome maintenance and repair. They also implicate Uup as a new modulator of RecG function.

Highlights

The replication of genomic DNA is an essential process that is carried out by a highly complex and regulated assembly of proteins called the replisome
We previously showed that removing both RadD and RadA function did not affect growth under standard conditions
To further investigate the relationship between Uup and RecG, we explored a process with which RecG is closely associated, stable DNA replication or SDR

Summary

Introduction

The replication of genomic DNA is an essential process that is carried out by a highly complex and regulated assembly of proteins called the replisome. Exogenous damage from the environment, protein–DNA complexes, reactive oxygen species (ROS), and genotoxic agents can cause replisome stalling and fork collapse. Mutagenesis in turn can give rise to human disease. Replication rarely, if ever, completes uninterrupted [1,2,3]. The potential biological consequences and frequency of replication conflicts underscores the importance of understanding DNA repair and replication enzymes

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Conclusion