Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

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 early stages of genetic recombination in Escherichia coli associated with initiation of homologous DNA pairing and with strand exchange are well established and can be described in terms of enzymology and reaction pathways (Dillingham and Kowalczykowski 2000, 2008; Singleton et al. 2004; Cox 2007a,b). However, later stages associated with resolution of Holliday junction intermediates have proven more difficult to pin down because of what appears at first sight to be a functional overlap between the RuvABC and RecG proteins. RecG also appears to have multiple roles in DNAmetabolismthat obscure the nature and extent of its involvement in recombination.

The RuvA and RuvB proteins together catalyze branch migration of Holliday junction intermediates and form a complex with RuvC protein that enables the latter to resolve these intermediates by a dual strand cleavage reaction (van Gool et al. 1998). RecG is a dsDNA translocase and, like RuvAB, catalyses branch migration of Holliday junctions (Lloyd and Sharples 1993; McGlynn and Lloyd 2001; Singleton et al. 2001). Its elimination from ruv mutants blocks the recovery of recombinants in genetic crosses and confers extreme sensitivity to genotoxic agents (Lloyd 1991). The strong synergism observed led to the idea that RuvABC and RecG provide partially overlapping pathways for the resolution of Holliday junctions. However, RecG proved to have no intrinsic ability to cleave junctions (Lloyd and Sharples 1993), which raised the possibility that some unidentified nuclease could act with RecG to promote Holliday junction resolution in the way RuvC acts with RuvAB. The RusA protein was a possible candidate (Sharples et al. 1994). This homodimeric endonuclease resolves Holliday junctions by a dual strand cleavage mechanism that targets specific DNA sequences (Sharples et al. 1994; Bolt and Lloyd 2002). Its expression compensates very effectively for the absence of RuvABC and in a RecG-dependent manner (Mandal et al. 1993; Mahdi et al. 1996). However, RusA is encoded by a cryptic prophage gene (rusA) and is not normally expressed because the gene lacks a promoter. Furthermore, its deletion does not reduce recombination in ruv mutant strains (Mahdi et al. 1996). Therefore, RusA cannot be the resolvase that operates in strains lacking RuvABC, although it can act as such when activated by a promoter inserted upstream of rusA (Mandal et al. 1993; Mahdi et al. 1996).

To date, our screens for mutations blocking recombination in ruv mutants failed to identify an alternative nuclease that could act with RecG, revealing only knockouts of the RecA, RecB, or RecC proteins needed to initiate exchanges or of RecG (our unpublished work). …

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