Promoting and Avoiding Recombination: Contrasting Activities of the Escherichia coli RuvABC Holliday Junction Resolvase and RecG DNA Translocase

Abstract

RuvABC and RecG are thought to provide alternative pathways for the late stages of recombination in Escherichia coli. Inactivation of both blocks the recovery of recombinants in genetic crosses. RuvABC resolves Holliday junctions, with RuvAB driving branch migration and RuvC catalyzing junction cleavage. RecG also drives branch migration, but no nuclease has been identified that might act with RecG to cleave junctions, apart from RusA, which is not normally expressed. We searched for an alternative nuclease using a synthetic lethality assay to screen for mutations causing inviability in the absence of RuvC, on the premise that a strain without any ability to cut junctions might be inviable. All the mutations identified mapped to polA, dam, or uvrD. None of these genes encodes a nuclease that cleaves Holliday junctions. Probing the reason for the inviability using the RusA Holliday junction resolvase provided strong evidence in each case that the RecG pathway is very ineffective at removing junctions and indicated that a nuclease component most probably does not exist. It also revealed new suppressors of recG, which were located to the ssb gene. Taken together with the results from the synthetic lethality assays, the properties of the mutant SSB proteins provide evidence that, rather than promoting recombination, a major function of RecG is to curb potentially pathological replication initiated via PriA protein at sites remote from oriC.

The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis

“…RecG is associated with later stages of repair by recombination because of its ability to catalyze branch migration of HJs, as does RuvAB. But RecG is not redundant with RuvAB, since it has no associated nuclease activity that could resolve HJs, whereas RuvAB recruits the RuvC nuclease (Zhang et al, 2010). Analysis of recombination intermediates also revealed independent effects of ruvAB and recG mutations in the resolution of HJs (Mawer and Leach, 2014).…”

“…The two helicase domains are in green and light blue, with complexed ADP and Mg 2+ (green ball). The three sequences specific to plant RecGs (see alignment in Supplemental Figure 2 whether the Arabidopsis RECG1 could complement a deficiency in RuvAB because both RecG and RuvAB are translocases that drive branch migration and have been described as either partially redundant or complementary in the resolution of HJ (Zhang et al, 2010;Mawer and Leach, 2014). In contrast to ΔrecG, the ΔruvA mutation could not be rescued by RECG1 (Figure 2A).…”

Regression of Replication Forks Stalled by Leading-strand Template Damage

“…The apparent cooperation between RecG and RuvC that we describe may simply be a function of a reduced processivity of RecG in an RFR reaction, as has been reported by Cox (34). However, it is interesting that a synthetic lethal screen designed to uncover an alternative to RuvC that would act as the HJ resolvase coupled to RecG-catalyzed branch migration yielded only mutations in polA, dam, and uvrD (67).…”

Homologous Recombination—Experimental Systems, Analysis, and Significance

“…The prediction is that, if performed in the recBC sbcA or recBC sbcBC backgrounds, such a screen should yield all other known rec mutants, as well as (hopefully) some still unknown ones. As an approach, identification of synthetic lethal mutant combinations shows its power in unraveling complicated pathways of the DNA metabolism around recombinational repair (270, 357), although care should be exercised with interpretation of quadruple and quintuple mutants, as the comfort zone of typical human thinking is limited to three dimensions.…”