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Stephen C West - One of the best experts on this subject based on the ideXlab platform.
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Holliday junction Branch Migration and resolution assays.
Methods in molecular biology (Clifton N.J.), 2004Co-Authors: Angelos Constantinou, Stephen C WestAbstract:Holliday junctions are central intermediates in the process of genetic recombination; they form as a consequence of a reciprocal exchange of strands between paired DNA molecules. Enzymes that specifically recognize and process these junctions are necessary for the formation of recombinant products. In the methods described here, we detail the in vitro construction of two types of Holliday junction: (1) a small synthetic junction formed by the annealing of partially complementary oligonucleotides; and (2) a true recombination intermediate structure formed by RecA protein-mediated strand exchange. The use of these substrates in assays designed to detect Holliday junction Branch Migration and resolution activities is described.
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Branch Migration and Holliday Junction Resolution Catalyzed by Activities from Mammalian Cells
Cell, 2001Co-Authors: Angelos Constantinou, Adelina A. Davies, Stephen C WestAbstract:During homologous recombination, DNA strand exchange leads to Holliday junction formation. The movement, or Branch Migration, of this junction along DNA extends the length of the heteroduplex joint. In prokaryotes, Branch Migration and Holliday junction resolution are catalyzed by the RuvA and RuvB proteins, which form a complex with RuvC resolvase to form a "resolvasome". Mammalian cell-free extracts have now been fractionated to reveal analogous activities. An ATP-dependent Branch Migration activity, which migrates junctions through >2700 bp, cofractionates with the Holliday junction resolvase during several chromatographic steps. Together, the two activities promote concerted Branch Migration/resolution reactions similar to those catalyzed by E. coli RuvABC, highlighting the preservation of this essential pathway in recombination and DNA repair from prokaryotes to mammals.
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RuvAB-mediated Branch Migration does not involve extensive DNA opening within the RuvB hexamer.
Current biology : CB, 2000Co-Authors: Helen George, Isao Kuraoka, David A. Nauman, William R. Kobertz, Richard D. Wood, Stephen C WestAbstract:Abstract The Escherichia coli RuvA and RuvB proteins promote the Branch Migration of Holliday junctions during the late stages of homologous recombination and DNA repair (reviewed in [1]). Biochemical and structural studies of the RuvAB–Holliday junction complex have shown that RuvA binds directly to the Holliday junction [2–6] and acts as a specificity factor that promotes the targeting of RuvB [7,8], a hexameric ring protein that drives Branch Migration [9–11]. Electron microscopic visualisation of the RuvAB complex revealed that RuvA is flanked by two RuvB hexamers, which bind DNA arms that lie diametrically opposed across the junction [8]. ATP-dependent Branch Migration occurs as duplex DNA is pumped out through the centre of each ring. Because RuvB possesses well-conserved helicase motifs and RuvAB exhibits a 5′–3′ DNA helicase activity in vitro [12], the mechanism of Branch Migration is thought to involve DNA opening within the RuvB ring, which provides a single strand for the unidirectional translocation of the protein along DNA. We have investigated whether the RuvB ring can translocate along duplex DNA containing a site-directed interstrand psoralen crosslink. Surprisingly, we found that the crosslink failed to inhibit Branch Migration. We interpret these data as evidence against a base-by-base tracking model and suggest that extensive DNA opening within the RuvB ring is not required for DNA translocation by RuvB.
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Helicase-defective RuvBD113E promotes RuvAB-mediated Branch Migration in Vitro
Journal of molecular biology, 1999Co-Authors: Helen George, Christine Mézard, Andrzej Stasiak, Stephen C WestAbstract:Abstract In Escherichia coli , the RuvA and RuvB proteins interact at Holliday junctions to promote Branch Migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent Branch Migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro , we have analysed the role of DNA unwinding in relation to Branch Migration. A mutant RuvB protein, RuvB D113E , mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes Branch Migration in the presence of RuvA. However, RuvB D113E exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated Branch Migration that invoke only limited DNA unwinding activity are proposed.
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functional interactions between the holliday junction resolvase and the Branch Migration motor of escherichia coli
The EMBO Journal, 1998Co-Authors: Alain J Van Gool, Christine Mézard, Rajvee Shah, Stephen C WestAbstract:Homologous recombination generates genetic diversity and provides an important cellular pathway for the repair of double-stranded DNA breaks. Two key steps in this process are the Branch Migration of Holliday junctions followed by their resolution into mature recombination products. In E.coli, Branch Migration is catalysed by the RuvB protein, a hexameric DNA helicase that is loaded onto the junction by RuvA, whereas resolution is promoted by the RuvC endonuclease. Here we provide direct evidence for functional interactions between RuvB and RuvC that link these biochemically distinct processes. Using synthetic Holliday junctions, RuvB was found to stabilize the binding of RuvC to a junction and to stimulate its resolvase activity. Conversely, RuvC facilitated interactions between RuvB and the junction such that RuvBC complexes catalysed Branch Migration. The observed synergy between RuvB and RuvC provides new insight into the structure and function of a RuvABC complex that is capable of facilitating Branch Migration and resolution of Holliday junctions via a concerted enzymatic mechanism.
Peggy Hsieh - One of the best experts on this subject based on the ideXlab platform.
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Branch Migration through DNA sequence heterology
Journal of molecular biology, 1998Co-Authors: Indranil Biswas, Akira Yamamoto, Peggy HsiehAbstract:Branch Migration of a DNA Holliday junction is a key step in genetic recombination. Previously, it was shown that a single base-pair heterology between two otherwise identical DNA sequences is a substantial barrier to passage of a Holliday junction during spontaneous Branch Migration. Here, we exploit this inhibitory effect of sequence heterology to estimate the step size of Branch Migration. We also devise a simulation of Branch Migration through mismatched base-pairs to arrive at the underlying molecular basis for the block to Branch Migration imposed by sequence heterology. Based on the observation that two adjacent sequence heterologies exert their effects on Branch Migration more or less independently, we conclude that the step size of Branch Migration is quite small, of the order of one or two base-pairs per migratory step. Comparison of Branch Migration experiments through a single base-pair heterology with simulations of a random walk through sequence heterology suggests that the inhibition of Branch Migration is largely attributable to a thermodynamic barrier arising from the formation of unpaired or mispaired bases in heteroduplex DNAs.
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A histone octamer blocks Branch Migration of a Holliday junction.
Molecular and Cellular Biology, 1997Co-Authors: Mikhail Grigoriev, Peggy HsiehAbstract:The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous Branch Migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted Branch Migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch Migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with Branch Migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can Branch migrate in regions free of histone octamers. Our findings suggest that Migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of Branch Migration but also to facilitate the passage of the Holliday junction through a nucleosome.
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A pivotal role for the structure of the Holliday junction in DNA Branch Migration.
The EMBO journal, 1995Co-Authors: Igor G. Panyutin, I. Biswas, Peggy HsiehAbstract:Abstract Branch Migration of a DNA Holliday junction is a key step in genetic recombination that affects the extent of transfer of genetic information between homologous DNA sequences. We previously observed that the rate of spontaneous Branch Migration is exceedingly sensitive to metal ions and postulated that the structure of the cross-over point might be one critical determinant of the rate of Branch Migration. Other investigators have shown that in the presence of divalent metal ions like magnesium, the Holliday junction assumes a folded conformation in which base stacking is retained through the cross-over point. This base stacking is disrupted in the absence of magnesium. Here we measure the rate of Branch Migration as a function of Mg2+ concentration. The rate of Branch Migration increases dramatically at MgCl2 concentrations below 500 microM, with the steepest acceleration occurring between 300 and 100 microM MgCl2. This increase in the rate of Branch Migration coincides with the loss of base stacking in the four-way junction over this same interval of magnesium concentration, as measured by the susceptibility of junction residues to modification by osmium tetroxide and diethyl pyrocarbonate. We conclude that at physiological concentrations of intracellular Mg2+, base stacking in the Holliday junction constitutes one kinetic barrier to Branch Migration and that disruption of base stacking at the cross-over relieves this constraint.
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The kinetics of spontaneous DNA Branch Migration.
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Igor G. Panyutin, Peggy HsiehAbstract:Abstract An important step in genetic recombination is DNA Branch Migration, the movement of the Holliday junction or exchange point between two homologous duplex DNAs. We have determined kinetic parameters of spontaneous Branch Migration as a function of temperature and ionic conditions. The Branch Migration substrates consist of two homologous duplex DNAs each having two single-strand tails at one end that are complementary to the corresponding single-strand tails of the other duplex. Upon rapid annealing of the two duplex DNAs, a four-stranded intermediate is formed that has a Holliday junction at one end of the duplexes. Branch Migration to the opposite end of the duplexes results in complete strand exchange and formation of two duplex products. The rate of Branch Migration is exceedingly sensitive to the type of metal ions present. In magnesium, Branch Migration is quite slow with a step time, tau, equal to 300 msec at 37 degrees C. Surprisingly, Branch Migration in the absence of magnesium was 1000 times faster. Despite this difference in rates, apparent activation energies for the Branch Migration step in the presence and absence of magnesium are similar. Since metal ions have a profound effect on the structure of the Holliday junction, it appears that the structure of the Branch point plays a key role in determining the rate of spontaneous DNA Branch Migration. We discuss the role of proteins in promoting the Branch Migration step during homologous recombination.
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Formation of a single base mismatch impedes spontaneous DNA Branch Migration.
Journal of molecular biology, 1993Co-Authors: Igor G. Panyutin, Peggy HsiehAbstract:Abstract DNA Branch Migration, a process whereby two homologous DNA duplexes exchange strands, is an essential component of genetic recombination. Models for homologous recombination have invoked spontaneous Branch Migration as one mechanism for the generation of large regions of heteroduplex DNA. During recombination, two homologous parental duplexes that contain similar, but not identical, sequences are paired and undergo strand exchange. An important issue is whether spontaneous Branch Migration is capable of traversing sequence heterology such as mismatches, insertions and deletions. We use a model four strand system to examine the effect of mispaired or unpaired bases on Branch Migration. The assay consists of annealing two short duplexes having defined sequence heterologies. Following annealing, a Holliday junction is formed that is free to Branch migrate. Our results demonstrate that a single base mismatch, insertion or deletion is sufficient to pose a substantial barrier to spontaneous Branch Migration. In the presence of magnesium, Branch Migration through such sequence heterologies is almost completely blocked. Others have shown that non-mobile four-way junctions undergo a dramatic shift in conformation in the presence of magnesium. Our data suggest that a similar transition occurs for the mobile Holliday junction. We also discuss how proteins may facilitate Branch Migration through sequence heterologies in vivo.
Irina R. Tsaneva - One of the best experts on this subject based on the ideXlab platform.
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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.
The Journal of biological chemistry, 2011Co-Authors: Alison S. Bradley, Zeynep Baharoglu, Andrew Niewiarowski, Bénédicte Michel, Irina R. TsanevaAbstract:Abstract 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, RuvA2KaP, 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, RuvA2KaP is fully active for homologous recombination in vivo. RuvA2KaP is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2KaP for forks is decreased compared with wild type. Accordingly, RuvA2KaP is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2KaP is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2KaP is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB 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.
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Formation of a Stable RuvA Protein Double Tetramer Is Required for Efficient Branch Migration in Vitro and for
2011Co-Authors: Alison S. Bradley, Zeynep Baharoglu, Andrew Niewiarowski, Irina R. TsanevaAbstract: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, RuvA2KaP, 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, RuvA2KaP is fully active for homologous recombination in vivo. RuvA2KaP is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2KaP for forks is decreased compared with wild type. Accordingly, RuvA2KaP is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2KaP is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2KaP is defective for stimulation of RuvB activity and stability of
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The directionality of RuvAB-mediated Branch Migration: in vitro studies with three-armed junctions
Genes to cells : devoted to molecular & cellular mechanisms, 1996Co-Authors: Kevin Hiom, Irina R. Tsaneva, Stephen C WestAbstract:Background: The Escherichia coli RuvA and RuvB proteins promote the Branch Migration of 4-way (Holliday) junctions during genetic recombination. The active complex is a tripartite structure in which RuvA protein is bound to the crossover and is sandwiched between two hexameric rings of RuvB. Branch Migration requires ATP hydrolysis and occurs as the DNA passes through each RuvB ring. Results: In this work, we have investigated the mechanism by which RuvAB catalyses the Branch Migration of a three-armed (Y) junction. Using synthetic DNA structures, we observed the formation of DNA products, a partial duplex DNA molecule and a single-stranded oligonucleotide, indicative of a Branch Migration reaction that occurred with unique polarity. Analysis of the RuvAB-junction complex by DNase footprinting showed that RuvA bound asymmetrically to the junction and targeted a single hexameric RuvB ring to one arm of DNA. Conclusion: Branch Migration of a three-armed junction occurs in a unidirectional manner that is determined by the assembly of a single RuvB ring onto one arm of the DNA. The asymmetry of the complex and observed directionality of Branch Migration indicate that strand passage occurs as the DNA is pulled into the RuvB ring structure, a reaction likely to be coupled with DNA unwinding.
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the escherichia coli ruvb Branch Migration protein forms double hexameric rings around dna
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Andrzej Stasiak, Stephen C West, Irina R. Tsaneva, Catherine J B Benson, Edward H. EgelmanAbstract:Abstract The RuvB protein is induced in Escherichia coli as part of the SOS response to DNA damage. It is required for genetic recombination and the postreplication repair of DNA. In vitro, the RuvB protein promotes the Branch Migration of Holliday junctions and has a DNA helicase activity in reactions that require ATP hydrolysis. We have used electron microscopy, image analysis, and three-dimensional reconstruction to show that the RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry. Although helicases are ubiquitous and essential for many aspects of DNA repair, replication, and transcription, three-dimensional reconstruction of a helicase has not yet been reported, to our knowledge. The structural arrangement that is seen may be common to other helicases, such as the simian virus 40 large tumor antigen.
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Structural analysis of the E. coli RUVB Branch Migration protein by EM and image analysis
Proceedings annual meeting Electron Microscopy Society of America, 1994Co-Authors: K. Benson, Stephen C West, Andrzej Stasiak, Irina R. Tsaneva, Edward H. EgelmanAbstract:We have been interested in the structure and function of proteins involved in genetic recombinaton. The ruv locus on the E. coli chromosome contains three genes (ruvA, ruvB and ruvC) that are important for genetic recombination and DNA repair. The ruvA and ruvB genes form part of the SOS response to DNA damage and encode the RuvA and RuvB proteins. Together, RuvA and RuvB promote the Branch Migration of Holliday junctions in a reaction that requires ATP hydrolysis. Each protein plays a defined role, with RuvA responsible for DNA binding (and, in particular, junction recognition), whereas the RuvB ATPase provides the motor for Branch Migration. Sequence analysis has identified RuvB as a member of a superfamily of helicases, and experimentally it has been shown that RuvB, in the presence of RuvA, acts as an ATP-dependent helicase.When purified RuvB protein was incubated (in the presence of the ATP analog, ATP-γ-S) with covalently closed, relaxed dsDNA, double-ringed structures were observed on the DNA in the electron microscope (Fig. 1). The DNA must be passing through the center of these rings, since the rings are always aligned along a common axis.
Hiroshi Iwasaki - One of the best experts on this subject based on the ideXlab platform.
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Biochemical Analysis of RuvA-RuvB Complex Formation During Branch Migration of Holliday Junction DNA
Biophysical Journal, 2010Co-Authors: Yong-woon Han, Hiroshi Iwasaki, Hideo Shinagawa, Masahito Hayashi, Takashi Hishida, Hiroaki Yokota, Yoshie HaradaAbstract:Escherichia coli RuvA-RuvB protein complex promotes Holliday junction Branch Migration during homologous recombination and recombination repair. RuvA forms tetramer and the two tetramers sandwich planer Holliday junction. RuvB is a member of AAA+ ATPase superfamily and forms a hexameric ring, which acts as a motor protein. The two rings flank the junction by interacting RuvA octameric core and promote Branch Migration by pumping out DNA duplex through their central cavities. Two models are conceived to explain how the DNA double helices are pulled out through the cavities of the rings. (i) RuvB hexameric rings rotate against RuvA octameric core and the duplexes are moved by interacting with inner surfaces of the rotating RuvB rings. (ii) RuvB hexamric rings are fixed to the RuvA octameric core and the duplexes are moved by interaction with RuvB subunits which undergo sequential conformational changes. Previously, we showed that I150T-RuvB mutant was defective in interaction with RuvA. Here, we show the detailed analysis of the heterooligomer composed of wild type and the mutant I150T RuvB proteins in vitro to clarify which mechanism is employed for the RuvA-RuvB directed Branch Migration of Holliday junction. In this study, we would like to discuss how RuvA-RuvB promote Branch Migration of Holliday junction.
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Formation and Branch Migration of Holliday junctions mediated by eukaryotic recombinases
Nature, 2008Co-Authors: Yasuto Murayama, Kouta Mayanagi, Yumiko Kurokawa, Hiroshi IwasakiAbstract:Holliday junctions (HJs) are key intermediates in homologous recombination and are especially important for the production of crossover recombinants. Bacterial RecA family proteins promote the formation and Branch Migration of HJs in vitro by catalysing a reciprocal DNA-strand exchange reaction between two duplex DNA molecules, one of which contains a single-stranded DNA region that is essential for initial nucleoprotein filament formation. This activity has been reported only for prokaryotic RecA family recombinases, although eukaryotic homologues are also essential for HJ production in vivo. Here we show that fission yeast (Rhp51) and human (hRad51) RecA homologues promote duplex-duplex DNA-strand exchange in vitro. As with RecA, a HJ is formed between the two duplex DNA molecules, and reciprocal strand exchange proceeds through Branch Migration of the HJ. In contrast to RecA, however, strand exchange mediated by eukaryotic recombinases proceeds in the 3'-->5' direction relative to the single-stranded DNA region of the substrate DNA. The opposite polarity of Rhp51 makes it especially suitable for the repair of DNA double-strand breaks, whose repair is initiated at the processed ends of breaks that have protruding 3' termini.
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direct observation of dna rotation during Branch Migration of holliday junction dna by escherichia coli ruva ruvb protein complex
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Yong-woon Han, Hiroshi Iwasaki, Hideo Shinagawa, Tomomi Tani, Masahito Hayashi, Takashi Hishida, Yoshie HaradaAbstract:The Escherichia coli RuvA–RuvB complex promotes Branch Migration of Holliday junction DNA, which is the central intermediate of homologous recombination. Like many DNA motor proteins, it is suggested that RuvA–RuvB promotes Branch Migration by driving helical rotation of the DNA. To clarify the RuvA–RuvB-mediated Branch Migration mechanism in more detail, we observed DNA rotation during Holliday junction Branch Migration by attaching a bead to one end of cruciform DNA that was fixed to a glass surface at the opposite end. Bead rotation was observed when RuvA, RuvB, and ATP were added to the solution. We measured the rotational rates of the beads caused by RuvA–RuvB-mediated Branch Migration at various ATP concentrations. The data provided a Km value of 65 μM and a Vmax value of 1.6 revolutions per second, which corresponds to 8.3 bp per second. This real-time observation of the DNA rotation not only allows us to measure the kinetics of the RuvA–RuvB-mediated Branch Migration, but also opens the possibility of elucidating the Branch Migration mechanism in detail.
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Uncoupling of the ATPase activity from the Branch Migration activity of RuvAB protein complexes containing both wild-type and ATPase-defective RuvB proteins
Genes to cells : devoted to molecular & cellular mechanisms, 2003Co-Authors: Takashi Hishida, Yong-woon Han, Hiroshi Iwasaki, Takayuki Ohnishi, Hideo ShinagawaAbstract:Background: Escherichia coli RuvAB promotes Branch Migration of Holliday junctions during recombination repair and homologous recombination. RuvB forms a hexameric ring through which duplex DNA passes and is translocated in an ATP-dependent manner. ATPase-deficient RuvB mutant K68A has a mutation in the Walker A motif and exerts a dominant-negative effect on in vivo repair of UV-induced DNA damage. In this study, we examined RuvAB-dependent Branch Migration in the presence of a mutant RuvB, K68A. Results: Mixing K68A with wild-type RuvB resulted in the formation of heterohexamers that showed unique properties of DNA binding, ATPase, and Branch Migration activities different from those of either wild-type or mutant homohexamers. RuvB heterohexamers inhibited Branch Migration and caused Holliday junctions to accumulate during RecA-mediated strand exchange. In the presence of RuvA, RuvB heterohexamers had Holliday junction-dependent ATPase activity, but did not promote Branch Migration. Conclusions: These results suggest that functional cooperation among the subunits in the hexamers is required for Branch Migration, but inclusion of inactive subunits is tolerated for ATP hydrolysis. Therefore, we propose that an essential ATP hydrolysis-dependent functional cooperation is induced in RuvB hexamer subunits during RuvAB-mediated Branch Migration.
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p53 blocks RuvAB promoted Branch Migration and modulates resolution of Holliday junctions by RuvC.
Journal of molecular biology, 2002Co-Authors: Vidya P Prabhu, Hiroshi Iwasaki, Amanda M. Simons, Dahai Gai, Daniel T. Simmons, Junghuei ChenAbstract:The Holliday junction is the central intermediate in homologous recombination. Branch Migration of this four-stranded DNA structure is a key step in genetic recombination that affects the extent of genetic information exchanged between two parental DNA molecules. Here, we have constructed synthetic Holliday junctions to test the effects of p53 on both spontaneous and RuvAB promoted Branch Migration as well as the effect on resolution of the junction by RuvC. We demonstrate that p53 blocks Branch Migration, and that cleavage of the Holliday junction by RuvC is modulated by p53. These findings suggest that p53 can block Branch Migration promoted by proteins such as RuvAB and modulate the cleavage by Holliday junction resolution proteins such as RuvC. These results suggest that p53 could have similar effects on eukaryotic homologues of RuvABC and thus have a direct role in recombinational DNA repair.
Hideo Shinagawa - One of the best experts on this subject based on the ideXlab platform.
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Biochemical Analysis of RuvA-RuvB Complex Formation During Branch Migration of Holliday Junction DNA
Biophysical Journal, 2010Co-Authors: Yong-woon Han, Hiroshi Iwasaki, Hideo Shinagawa, Masahito Hayashi, Takashi Hishida, Hiroaki Yokota, Yoshie HaradaAbstract:Escherichia coli RuvA-RuvB protein complex promotes Holliday junction Branch Migration during homologous recombination and recombination repair. RuvA forms tetramer and the two tetramers sandwich planer Holliday junction. RuvB is a member of AAA+ ATPase superfamily and forms a hexameric ring, which acts as a motor protein. The two rings flank the junction by interacting RuvA octameric core and promote Branch Migration by pumping out DNA duplex through their central cavities. Two models are conceived to explain how the DNA double helices are pulled out through the cavities of the rings. (i) RuvB hexameric rings rotate against RuvA octameric core and the duplexes are moved by interacting with inner surfaces of the rotating RuvB rings. (ii) RuvB hexamric rings are fixed to the RuvA octameric core and the duplexes are moved by interaction with RuvB subunits which undergo sequential conformational changes. Previously, we showed that I150T-RuvB mutant was defective in interaction with RuvA. Here, we show the detailed analysis of the heterooligomer composed of wild type and the mutant I150T RuvB proteins in vitro to clarify which mechanism is employed for the RuvA-RuvB directed Branch Migration of Holliday junction. In this study, we would like to discuss how RuvA-RuvB promote Branch Migration of Holliday junction.
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direct observation of dna rotation during Branch Migration of holliday junction dna by escherichia coli ruva ruvb protein complex
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Yong-woon Han, Hiroshi Iwasaki, Hideo Shinagawa, Tomomi Tani, Masahito Hayashi, Takashi Hishida, Yoshie HaradaAbstract:The Escherichia coli RuvA–RuvB complex promotes Branch Migration of Holliday junction DNA, which is the central intermediate of homologous recombination. Like many DNA motor proteins, it is suggested that RuvA–RuvB promotes Branch Migration by driving helical rotation of the DNA. To clarify the RuvA–RuvB-mediated Branch Migration mechanism in more detail, we observed DNA rotation during Holliday junction Branch Migration by attaching a bead to one end of cruciform DNA that was fixed to a glass surface at the opposite end. Bead rotation was observed when RuvA, RuvB, and ATP were added to the solution. We measured the rotational rates of the beads caused by RuvA–RuvB-mediated Branch Migration at various ATP concentrations. The data provided a Km value of 65 μM and a Vmax value of 1.6 revolutions per second, which corresponds to 8.3 bp per second. This real-time observation of the DNA rotation not only allows us to measure the kinetics of the RuvA–RuvB-mediated Branch Migration, but also opens the possibility of elucidating the Branch Migration mechanism in detail.
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Uncoupling of the ATPase activity from the Branch Migration activity of RuvAB protein complexes containing both wild-type and ATPase-defective RuvB proteins
Genes to cells : devoted to molecular & cellular mechanisms, 2003Co-Authors: Takashi Hishida, Yong-woon Han, Hiroshi Iwasaki, Takayuki Ohnishi, Hideo ShinagawaAbstract:Background: Escherichia coli RuvAB promotes Branch Migration of Holliday junctions during recombination repair and homologous recombination. RuvB forms a hexameric ring through which duplex DNA passes and is translocated in an ATP-dependent manner. ATPase-deficient RuvB mutant K68A has a mutation in the Walker A motif and exerts a dominant-negative effect on in vivo repair of UV-induced DNA damage. In this study, we examined RuvAB-dependent Branch Migration in the presence of a mutant RuvB, K68A. Results: Mixing K68A with wild-type RuvB resulted in the formation of heterohexamers that showed unique properties of DNA binding, ATPase, and Branch Migration activities different from those of either wild-type or mutant homohexamers. RuvB heterohexamers inhibited Branch Migration and caused Holliday junctions to accumulate during RecA-mediated strand exchange. In the presence of RuvA, RuvB heterohexamers had Holliday junction-dependent ATPase activity, but did not promote Branch Migration. Conclusions: These results suggest that functional cooperation among the subunits in the hexamers is required for Branch Migration, but inclusion of inactive subunits is tolerated for ATP hydrolysis. Therefore, we propose that an essential ATP hydrolysis-dependent functional cooperation is induced in RuvB hexamer subunits during RuvAB-mediated Branch Migration.
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Identification and characterization of Thermus thermophilus HB8 RuvA protein, the subunit of the RuvAB protein complex that promotes Branch Migration of Holliday junctions.
Genes & genetic systems, 2000Co-Authors: Takayuki Ohnishi, Hiroshi Iwasaki, Yoshizumi Ishino, Seiki Kuramitsu, Atsuo Nakata, Hideo ShinagawaAbstract:The Escherichia coli ruvA and ruvB genes constitute an SOS-regulated operon. The products of these genes form a protein complex that promotes Branch Migration of the Holliday junction, an intermediate of homologous recombination. RuvA protein binds specifically to the Holliday junction and recruits RuvB protein to the junction. RuvB is an ATP-driven motor protein involved in Branch Migration. We previously cloned the ruvB gene of the thermophilic bacterium Thermus thermophilus HB8 (Tth) and found that, in contrast to the operon structure in most mesothermic bacteria, the ruvA gene is absent from the vicinity of ruvB. In this work, we cloned the ruvA gene from T. thermophilus HB8 and analyzed its nucleotide sequence. Tth RuvA is a protein of 20,414 Da consisting of 191 amino acid residues, and is 37% identical in amino acid sequence to E. coli RuvA. Tth ruvA complemented the DNA repair defect of E. coli ΔruvA mutants. The purified Tth RuvA protein stimulated Tth RuvB activities, such as hydrolysis of ATP and promotion of Branch Migration of the Holliday junction, in a manner similar to the RuvA-RuvB interactions observed in E. coli. In addition, Tth RuvA stimulated the E. coli RuvB activities in vitro, which was well consistent with the results of in vivo hetero-complementation experiments.
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Role of Walker Motif A of RuvB Protein in Promoting Branch Migration of Holliday Junctions WALKER MOTIF A MUTATIONS AFFECT ATP BINDING, ATP HYDROLYZING, AND DNA BINDING ACTIVITIES OF RuvB
Journal of Biological Chemistry, 1999Co-Authors: Takashi Hishida, Hiroshi Iwasaki, Toshihiro Yagi, Hideo ShinagawaAbstract:Abstract Escherichia coli RuvB protein, an ATP-dependent hexameric DNA helicase, acts together with RuvA protein to promote Branch Migration of Holliday junctions during homologous recombination and recombinational repair. To elucidate the role of the Walker motif A of RuvB (GXGKT; Xindicates a nonconserved residue) in ATP hydrolysis and Branch Migration activities, we constructed four ruvB mutant genes by site-directed mutagenesis, altering the highly conserved Lys68 and Thr69. K68R, K68A, and T69A mutants except T69S failed to complement UV-sensitive phenotype of theruvB strain. These three mutant proteins, when overexpressed, made the wild-type strain UV-sensitive to varying degrees. K68R, K68A, and T69A were defective in ATP hydrolysis and Branch Migration activities in vitro. In the presence of Mg2+, K68R showed markedly reduced affinity for ATP, while K68A and T69A showed only mild reduction. K68A and T69A could form hexamers in the presence of Mg2+ and ATP, while K68R failed to form hexamers and existed instead as a higher oligomer, probably a dodecamer. In contrast to wild-type RuvB, K68R, K68A, and T69A by themselves were defective in DNA binding. However, RuvA could facilitate binding of K68A and T69A to DNA, whereas it could not promote binding of K68R to DNA. All of the three mutant RuvBs could physically interact with RuvA. These results indicate the direct involvement in ATP binding and ATP hydrolysis of the invariant Lys68 and Thr69 residues of Walker motif A of RuvB and suggest that these residues play key roles in interrelating these activities with the conformational change of RuvB, which is required for the Branch Migration activity.