The Role of RuvA Octamerization for RuvAB Function in Vitro and in Vivo

Privezentzev, Cyril V.; Keeley, Anthony; Sigala, Barbara; Tsaneva, Irina R.

doi:10.1074/jbc.m409256200

Cited by 25 publications

(30 citation statements)

References 59 publications

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“…RuvA3m, which was used in previous work (26), is inactive in vivo and displays significant nonspecific DNA binding, which could explain some discrepancies compared with studies of RuvA(DK) (28). In contrast, RuvA2 KaP was designed to only disrupt tetramer-tetramer interactions.…”

mentioning

confidence: 69%

“…A protocol for producing RuvBD113E was modified to produce wild type RuvB (26). RuvB was overexpressed from plasmid pET21a in BL21-GOLD(DE3) cells.…”

Section: Methodsmentioning

confidence: 99%

“…The suspension was further spun for 10 min at 4000 relative centrifugal force at 4°C, and the resulting pellets were frozen at Ϫ20°C. Purification was carried out as described previously (26). RuvC was expressed and purified using a protocol as described previously (5).…”

Section: Methodsmentioning

confidence: 99%

“…The role of RuvA octamers for efficient branch migration has been investigated using RuvA mutants designed to disrupt the tetramer-tetramer interface and prevent complex II formation. A triple Escherichia coli RuvA mutant, RuvA3m, was unable to form complex II at RuvA concentrations of up to 2 M and was deficient in processing synthetic HJs in vitro and in vivo (26). Unexpectedly, RuvA3m helicase activity and branch migration of Y-junctions in vitro appeared unaffected, and it was proposed that complex I was able to support one RuvB hexameric ring, but complex II was needed to assemble two hexameric rings on the Holliday junction (26).…”

mentioning

confidence: 99%

“…A triple Escherichia coli RuvA mutant, RuvA3m, was unable to form complex II at RuvA concentrations of up to 2 M and was deficient in processing synthetic HJs in vitro and in vivo (26). Unexpectedly, RuvA3m helicase activity and branch migration of Y-junctions in vitro appeared unaffected, and it was proposed that complex I was able to support one RuvB hexameric ring, but complex II was needed to assemble two hexameric rings on the Holliday junction (26). A Thermus thermophilus "tetramer-only" RuvA mutant, RuvA(DK) (L125D and E126K) was also studied.…”

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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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mentioning

confidence: 69%

“…A protocol for producing RuvBD113E was modified to produce wild type RuvB (26). RuvB was overexpressed from plasmid pET21a in BL21-GOLD(DE3) cells.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

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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Branch migration of Holliday junction in RuvA tetramer complex studied by umbrella sampling simulation using a path‐search algorithm

Ishida

2010

J Comput Chem

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Branch migration of the Holliday junction takes place at the center of the RuvA tetramer. To elucidate how branch migration occurs, umbrella sampling simulations were performed for complexes of the RuvA tetramer and Holliday junction DNA. Although conventional umbrella sampling simulations set sampling points a priori, the umbrella sampling simulation in this study set the sampling points one by one in order to search for a realistic path of the branch migration during the simulations. Starting from the X-ray structure of the complex, in which the hydrogen bonds between two base-pairs were unformed, the hydrogen bonds between the next base-pairs of the shrinking stems were observed to start to disconnect. At the intermediate stage, three or four of the eight unpaired bases interacted closely with the acidic pins from RuvA. During the final stage, these bases moved away from the pins and formed the hydrogen bonds of the new base-pairs of the growing stems. The free-energy profile along this reaction path showed that the intermediate stage was a meta-stable state between two free-energy barriers of about 10 to 15 kcal/mol. These results imply that the pins play an important role in stabilizing the interactions between the pins and the unpaired base-pairs.

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Survey of the year 2005 commercial optical biosensor literature

2006

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We identified 1113 articles (103 reviews, 1010 primary research articles) published in 2005 that describe experiments performed using commercially available optical biosensors. While this number of publications is impressive, we find that the quality of the biosensor work in these articles is often pretty poor. It is a little disappointing that there appears to be only a small set of researchers who know how to properly perform, analyze, and present biosensor data. To help focus the field, we spotlight work published by 10 research groups that exemplify the quality of data one should expect to see from a biosensor experiment. Also, in an effort to raise awareness of the common problems in the biosensor field, we provide side-by-side examples of good and bad data sets from the 2005 literature.

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The Role of RuvA Octamerization for RuvAB Function in Vitro and in Vivo

Cited by 25 publications

References 59 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

Branch migration of Holliday junction in RuvA tetramer complex studied by umbrella sampling simulation using a path‐search algorithm

Survey of the year 2005 commercial optical biosensor literature

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