Holliday Junction Resolvases Encoded by HomologousrusAGenes inEscherichia coliK-12 and Phage 82

Mahdi, Akeel A.; Sharples, Gary J.; Mandal, Tikshna N.; Lloyd, Robert G.

doi:10.1006/jmbi.1996.0185

Cited by 140 publications

(120 citation statements)

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“…However, the clones obtained had suffered substantial deletions, indicating that this combination is highly deleterious. Similarly, we were unable to maintain pCB559 (RuvAB) and pCB564 (RecU) jointly in a strain lacking ruvAC (effectively a ruvABC mutant) and the cryptic HJ resolvase rusA (Mahdi et al 1996; data not shown). It seems likely that too much RuvAB and RecU generates frequent lethal DSBs at regressed replication forks or additionally blocks their processing even in the absence of DNA-damaging agents.…”

Section: Resultsmentioning

confidence: 90%

“…coli K12 ruv mutants strains, SR2210 (ruvA200), N1057 (ruvB4), N2057 (ruvAB60TTn10), GS1481 (DruvCTkan), CS85 (ruvC53 edaTTn10), AM888 (DruvAC65 DrusATkan), and N4454 (DruvABCTcat) are derivatives of the ruv 1 wild-type strain, AB1157 (Sargentini and Smith 1989;Sharples et al 1990;Mandal et al 1993;Mahdi et al 1996;Seigneur et al 1998). Plasmid pCB564 was constructed by transferring the 1.2-kb BspEI-BamHI DNA segment containing the recU gene into pHP13 (pRecU).…”

Section: Methodsmentioning

confidence: 99%

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The RuvAB Branch Migration Translocase and RecU Holliday Junction Resolvase Are Required for Double-Stranded DNA Break Repair in Bacillus subtilis

et al. 2005

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In models of Escherichia coli recombination and DNA repair, the RuvABC complex directs the branch migration and resolution of Holliday junction DNA. To probe the validity of the E. coli paradigm, we examined the impact of mutations in DruvAB and DrecU (a ruvC functional analog) on DNA repair. Under standard transformation conditions we failed to construct DruvAB DrecG, DrecU DruvAB, DrecU DrecG, or DrecU DrecJ strains. However, DruvAB could be combined with addAB (recBCD), recF, recH, DrecS, DrecQ, and DrecJ mutations. The DruvAB and DrecU mutations rendered cells extremely sensitive to DNA-damaging agents, although less sensitive than a DrecA strain. When damaged cells were analyzed, we found that RecU was recruited to defined double-stranded DNA breaks (DSBs) and colocalized with RecN. RecU localized to these centers at a later time point during DSB repair, and formation was dependent on RuvAB. In addition, expression of RecU in an E. coli ruvC mutant restored full resistance to UV light only when the ruvAB genes were present. The results demonstrate that, as with E. coli RuvABC, RuvAB targets RecU to recombination intermediates and that all three proteins are required for repair of DSBs arising from lesions in chromosomal DNA.

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Section: Resultsmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

The RuvAB Branch Migration Translocase and RecU Holliday Junction Resolvase Are Required for Double-Stranded DNA Break Repair in Bacillus subtilis

et al. 2005

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“…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%

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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“…Also common was ORF 78, with high sequence similarity to RusA, an enzyme thought to have phage origins (Sharples et al, 2002). RusA is a DNA endonuclease that resolves Holliday junctions in DNA replication, recombination and repair (Mahdi et al, 1996).…”

Section: Mosaic Structure Of Wc2 Genomementioning

confidence: 99%

The complete genome sequence of Clostridium difficile phage ϕC2 and comparisons to ϕCD119 and inducible prophages of CD630

et al. 2007

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The complete genome sequence of Clostridium difficile phage wC2 and comparisons to wCD119 and inducible prophages of CD630 The complete genomic sequence of a previously characterized temperate phage of Clostridium difficile, wC2, is reported. The genome is 56 538 bp and organized into 84 putative ORFs in six functional modules. The head and tail structural proteins showed similarities to that of C. difficile phage wCD119 and Streptococcus pneumoniae phage EJ-1, respectively. Homologues of structural and replication proteins were found in prophages 1 and 2 of the sequenced C. difficile CD630 genome. A putative holin appears unique to the C. difficile phages and was functional when expressed in Escherichia coli. Nucleotide sequence comparisons of wC2 to wCD119 and the CD630 prophage sequences showed relatedness between wC2 and the prophages, but less so to wCD119. wC2 integrated into a gene encoding a putative transcriptional regulator of the gntR family. wC2, wCD119 and CD630 prophage 1 genomes had a Cdu1-attP-integrase arrangement, suggesting that the pathogenicity locus (PaLoc) of C. difficile, flanked by cdu1, has phage origins. The attP sequences of wC2, wCD119 and CD630 prophages were dissimilar. wC2-related sequences were found in 84 % of 37 clinical C. difficile isolates and typed reference strains. INTRODUCTIONClostridium difficile has emerged as an important intestinal pathogen since the 1970s, continuing to plague hospital settings worldwide and causing recent epidemics in the USA and Canada (Loo et al., 2005;Warny et al., 2005). The major virulence factors of C. difficile are toxins A and B encoded by tcdA and tcdB respectively, which are located on a 19 kb genomic region termed the pathogenicity locus (PaLoc) (Braun et al., 1996;Hammond & Johnson, 1995). The toxins are positively regulated by TcdR (Mani & Dupuy, 2001;Rupnik et al., 2005) and are negatively regulated by TcdC (Hundsberger et al., 1997;Matamouros et al., 2006); they are encoded by tcdR and tcdC respectively, also on the PaLoc. Another toxin-associated gene, tcdE, appears phage related but its function is unknown (Tan et al., 2001). C. difficile acquires antibiotic resistance and virulence genes through plasmids and transposons (Bruggemann, 2005) shown to significantly contribute to genome plasticity (Sebaihia et al., 2006). It is possible that phages also contribute to variance in virulence-associated genes (Lemee et al., 2005) and to the emergence of outbreak strains . However, the prevalence of phage genes within C. difficile genomes is not known. In comparison to phages of Escherichia coli, Staphylococcus aureus and Lactobacillus species, the study of clostridial phages is in its infancy. Only one phage specific for C. difficile, temperate phage wCD119, has been sequenced , while two putative prophage sequences were detected in the recently sequenced genome of C. difficile CD630 (Sebaihia et al., 2006).

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Holliday Junction Resolvases Encoded by HomologousrusAGenes inEscherichia coliK-12 and Phage 82

Cited by 140 publications

References 0 publications

The RuvAB Branch Migration Translocase and RecU Holliday Junction Resolvase Are Required for Double-Stranded DNA Break Repair in Bacillus subtilis

The RuvAB Branch Migration Translocase and RecU Holliday Junction Resolvase Are Required for Double-Stranded DNA Break Repair in Bacillus subtilis

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

The complete genome sequence of Clostridium difficile phage ϕC2 and comparisons to ϕCD119 and inducible prophages of CD630

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