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

Abstract

<jats:title>Abstract</jats:title> <jats:p>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.</jats:p>

RecA‐independent recombination: Dependence on the Escherichia coli RarA protein

“…Stalling, particularly when it involves an encounter with a template strand break, may lead to a double strand break, fork collapse, and replisome dissociation (Cox, 2001; Cox et al, 2000; Heller & Marians, 2006; Klein & Kreuzer, 2002; Kowalczykowski, 2000; Kuzminov, 1999; Kuzminov, 2001; Lopes et al, 2001; Merrikh et al, 2012; Michel, 2000; Michel et al, 2007). Although estimates vary, replication forks in bacteria may stall or collapse as often as once per cell generation during normal growth conditions (Courcelle et al, 2015; Cox, 2001; Cox, 2002; Cox et al, 2000; Kuzminov, 1995; Mangiameli et al, 2017; McCool et al, 2004; Michel et al, 2001; Michel et al, 2004; Romero et al, 2019; Romero et al, 2020; Syeda et al, 2014). Most of the adverse replication‐fork encounters are resolved using a variety of pathways that do not introduce mutations, particularly recombinational DNA repair (Aguilera & Garcia‐Muse, 2013; Cox, 2001; Cox, 2002; Cox et al, 2000; Heller & Marians, 2006; Klein & Kreuzer, 2002; Kowalczykowski, 2000; Kuzminov, 1999; Kuzminov, 2001; Lopes et al, 2001; Merrikh et al, 2012; Michel, 2000; Michel et al, 2007; Mirkin & Mirkin, 2007).…”

“…In at least some cases, the replisome skips over lesions, leaving them behind in what is termed a postreplication gap. In bacteria, the postreplication gap is closed by either translesion DNA synthesis, template switching, or by recombinational DNA repair via the RecFOR pathway (Fuchs, 2016; Fujii et al, 2006; Laranjo et al, 2017; Lovett, 2017; Naiman et al, 2016; Romero et al, 2019; Romero et al, 2020). Failure to close a gap inevitably leads to a double strand break when the gap is encountered in the next replication cycle (Cox et al, 2000).…”

Effects of Conserved Wedge Domain Residues on DNA Binding Activity of Deinococcus radiodurans RecG Helicase

“…RecG provides a more general defense against pathological DNA replication, e.g., the rescue of stalled or damaged replication forks, and is associated with a number of additional cellular processes ( Rudolph et al, 2010 ; Azeroglu et al, 2016 ; Lloyd and Rudolph, 2016 ; Romero et al, 2020 ). Recently, it has become increasingly clear that RecG is an enzyme responsible for regression of stalled DNA replication forks that can be induced by various types of lesions, including single-strand breaks and DSB.…”

ABC‐F translation factors: from antibiotic resistance to immune response

“…Uup binds to branched DNAs in vitro and can prevent nucleoid mis‐segregation during DNA repair [62]. A recent publication [63] proposes based on genetic results that uup is implicated in the stabilization of possible toxic branched DNA intermediates produces in a strain deleted of two gene DNA repair genes radD and recG . Deletion of uup restores the defect of growth of the strain deleted of radD and recG .…”