ruvA Mutants That Resolve Holliday Junctions but Do Not Reverse Replication Forks

Baharoglu, Zeynep; Bradley, Alison S.; Masson, Marie; Tsaneva, Irina R.; Michel, Bertrand

doi:10.1371/journal.pgen.1000012

Cited by 25 publications

(48 citation statements)

References 62 publications

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“…2B). The inability of RuvAz mutants to protect the HJ from RuvC cleavage confirms that they form unstable complex II (29). These results also suggest that a tetramer of RuvA bound to the HJ (complex I) does not inhibit HJ cleavage by RuvC, and it is possible that both RuvA and RuvC bind together to the junction.…”

Section: Ruva2 Kap Binds Efficiently To Holliday Junctions As a Singlementioning

confidence: 58%

“…Stability of RuvA2 KaP Complex II in Solution-In vivo RuvC cannot function without RuvAB (7,8,29,30); however, it has been shown that formation of RuvA complex II occludes the HJ and prevents RuvC-mediated cleavage in vitro (26,31,32). To assess the stability of the RuvA2 KaP complex II in the presence of Mg 2ϩ , we compared the ability of both wild type and mutant proteins to protect a HJ from cleavage by RuvC (Fig.…”

Section: Ruva2 Kap Binds Efficiently To Holliday Junctions As a Singlementioning

confidence: 99%

“…The ability of RuvA2 KaP to process HJs in vivo was additionally tested during the DNA single strand gap and double strand break repair, by assessing whether the mutant RuvA2 KaP was able to suppress the UV or mitomycin C (MMC) sensitivity of the mutant strain JJC2971 (⌬ruvA100::cat) (29). JJC2971 was transformed with pGB-ruvA, pGB-ruvAB, pGB-ruvA2 KaP , and pGB-ruvA2 KaP B, and the cells were exposed to increasing doses of UV light.…”

Section: Binding and Processing Of Synthetic Replication Forks By Ruva2mentioning

confidence: 99%

“…Recently, two ruvA mutants called ruvAz3 and ruvAz87 (H29R/K129E/F140S and N79D/N100D, respectively) were isolated and characterized as separation-of-function mutants that can process Holliday junctions but cannot reverse replication forks (29). The RuvAz proteins contain several mutations in different domains of the proteins, making it difficult to ascertain the molecular cause for their phenotypes.…”

mentioning

confidence: 99%

“…Measurement of Recombinational DNA Repair-UV irradiation was performed as described previously (29). The survival ratio was a comparison of colonies that grew on a replica control plate compared with colonies that grew on a plate that was irradiated.…”

mentioning

confidence: 99%

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Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

Bradley

Baharoglu

Niewiarowski

et al. 2011

Journal of Biological Chemistry

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In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2 KaP , specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2 KaP is fully active for homologous recombination in vivo. RuvA2 KaP is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2 KaP for forks is decreased compared with wild type. Accordingly, RuvA2 KaP is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2 KaP is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2 KaP is defective for stimulation of RuvB activity and stability of HJ⅐RuvA⅐RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.The RuvAB complex is a highly sophisticated molecular machine, which carries out branch migration of Holliday junctions during homologous recombination. RuvA binds specifically to four-armed Holliday junctions (HJ) 4 and guides the assembly of two RuvB hexameric rings onto diametrically opposite arms of the HJ. RuvB, an AAA ϩ ATPase (1), is the motor that drives branch migration of the crossover point (1-3). After branch migration, a dimer of RuvC resolves the HJ by making two sequence-specific symmetrical cuts, producing either patched or spliced linear products (4 -6). Genetic studies showed that RuvC cannot function in vivo in the absence of RuvAB (7, 8), and it has been proposed that an RuvABC complex, known as the resolvasome, allows RuvC to scan for cleavable sequences (3, 9 -11). RuvA binds to HJs in vitro as one tetramer (complex I) or two tetramers that sandwich the junction (complex II) in a concentration-dependent manner; however, it is not clear whether the RuvAB complex contains one or two tetramers of RuvA in vivo (12-21). A RuvAB branch migration complex made of two RuvA tetramers would prevent access of RuvC to the Holliday junction. Whether to form the resolvasome the RuvC dimer displaces one of the two RuvA tetramers present in the RuvAB complex, or whether the RuvC dimer simply binds opposite a single RuvA tetramer present in the complex is a currently unanswered question.In addition to ...

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Section: Ruva2 Kap Binds Efficiently To Holliday Junctions As a Singlementioning

confidence: 58%

Section: Ruva2 Kap Binds Efficiently To Holliday Junctions As a Singlementioning

confidence: 99%

Section: Binding and Processing Of Synthetic Replication Forks By Ruva2mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

Bradley

Baharoglu

Niewiarowski

et al. 2011

Journal of Biological Chemistry

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25 years on and no end in sight: a perspective on the role of RecG protein

Lloyd

Rudolph

2016

Curr Genet

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The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched substrates in vitro. Although initially associated with homologous recombination and DNA repair, studies of cells lacking RecG over the past 25 years have led to the suggestion that the protein might be multi-functional and associated with a number of additional cellular processes, including initiation of origin-independent DNA replication, the rescue of stalled or damaged replication forks, replication restart, stationary phase or stress-induced ‘adaptive’ mutations and most recently, naïve adaptation in CRISPR-Cas immunity. Here we discuss the possibility that many of the phenotypes of recG mutant cells that have led to this conclusion may stem from a single defect, namely the failure to prevent re-replication of the chromosome. We also present data indicating that this failure does indeed contribute substantially to the much-reduced recovery of recombinants in conjugational crosses with strains lacking both RecG and the RuvABC Holliday junction resolvase.Electronic supplementary materialThe online version of this article (doi:10.1007/s00294-016-0589-z) contains supplementary material, which is available to authorized users.

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Global transcriptomic and proteomics analysis of Lactobacillus plantarum Y44 response to 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) stress

Gao

Liu

et al. 2020

Journal of Proteomics

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ruvA Mutants That Resolve Holliday Junctions but Do Not Reverse Replication Forks

Cited by 25 publications

References 62 publications

Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for Replication Fork Reversal in Vivo

25 years on and no end in sight: a perspective on the role of RecG protein

Global transcriptomic and proteomics analysis of Lactobacillus plantarum Y44 response to 2,2-azobis(2-methylpropionamidine) dihydrochloride (AAPH) stress

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