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Stephen C West - One of the best experts on this subject based on the ideXlab platform.
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spatial control of the gen1 Holliday Junction resolvase ensures genome stability
Nature Communications, 2014Co-Authors: Ying Wai Chan, Stephen C WestAbstract:The human Holliday Junction resolvase GEN1 functions during anaphase to eliminate recombination intermediates that block proper chromosome segregation. Here, the authors demonstrate that GEN1 activity is regulated independently of its phosphorylation status and relies on its active exclusion from the nucleus.
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Aberrant chromosome morphology in human cells defective for Holliday Junction resolution
Nature, 2011Co-Authors: Thomas Wechsler, Scott Newman, Stephen C WestAbstract:In somatic cells, Holliday Junctions can be formed between sister chromatids during the recombinational repair of DNA breaks or after replication fork demise. A variety of processes act upon Holliday Junctions to remove them from DNA, in events that are critical for proper chromosome segregation. In human cells, the BLM protein, inactivated in individuals with Bloom's syndrome, acts in combination with topoisomerase IIIα, RMI1 and RMI2 (BTR complex) to promote the dissolution of double Holliday Junctions. Cells defective for BLM exhibit elevated levels of sister chromatid exchanges (SCEs) and patients with Bloom's syndrome develop a broad spectrum of early-onset cancers caused by chromosome instability. MUS81-EME1 (refs 4-7), SLX1-SLX4 (refs 8-11) and GEN1 (refs 12, 13) also process Holliday Junctions but, in contrast to the BTR complex, do so by endonucleolytic cleavage. Here we deplete these nucleases from Bloom's syndrome cells to analyse human cells compromised for the known Holliday Junction dissolution/resolution pathways. We show that depletion of MUS81 and GEN1, or SLX4 and GEN1, from Bloom's syndrome cells results in severe chromosome abnormalities, such that sister chromatids remain interlinked in a side-by-side arrangement and the chromosomes are elongated and segmented. Our results indicate that normally replicating human cells require Holliday Junction processing activities to prevent sister chromatid entanglements and thereby ensure accurate chromosome condensation. This phenotype was not apparent when both MUS81 and SLX4 were depleted from Bloom's syndrome cells, suggesting that GEN1 can compensate for their absence. Additionally, we show that depletion of MUS81 or SLX4 reduces the high frequency of SCEs in Bloom's syndrome cells, indicating that MUS81 and SLX4 promote SCE formation, in events that may ultimately drive the chromosome instabilities that underpin early-onset cancers associated with Bloom's syndrome.
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The human Holliday Junction resolvase GEN1 rescues the meiotic phenotype of a Schizosaccharomyces pombe mus81 mutant
Nucleic acids research, 2009Co-Authors: Alexander Lorenz, Stephen C West, Matthew C. WhitbyAbstract:A key step in meiotic recombination involves the nucleolytic resolution of Holliday Junctions to generate crossovers. Although the enzyme that performs this function in human cells is presently unknown, recent studies led to the identification of the XPG-family endonuclease GEN1 that promotes Holliday Junction resolution in vitro, suggesting that it may perform a related function in vivo. Here, we show that ectopic expression of GEN1 in fission yeast mus81Δ strains results in Holliday Junction resolution and crossover formation during meiosis.
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The search for a human Holliday Junction resolvase.
Biochemical Society transactions, 2009Co-Authors: Stephen C WestAbstract:Four-way DNA intermediates, known as Holliday Junctions, are formed during mitotic and meiotic recombination, and their efficient resolution is essential for proper chromosome segregation. Bacteria, bacteriophages and archaea promote Holliday Junction resolution by the introduction of symmetrically related nicks across the Junction, in reactions mediated by Holliday Junction resolvases. In 2008, after a search that lasted almost 20 years, a Holliday Junction resolvase was identified in humans. The protein, GEN1, was identified using MS following the brute-force fractionation of extracts prepared from human cells grown in tissue culture. GEN1 fits the paradigm developed from studies of prokaryotic Holliday Junction resolvases, in that it specifically recognizes Junctions and resolves them using a mechanism similar to that exhibited by the Escherichia coli RuvC protein.
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Identification of Holliday Junction resolvases from humans and yeast
Nature, 2008Co-Authors: Ulrich Rass, Miguel G. Blanco, Helen R. Flynn, J. Mark Skehel, Stephen C WestAbstract:Four-way DNA intermediates, also known as Holliday Junctions, are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. Here we identify nucleases from Saccharomyces cerevisiae and human cells that promote Holliday Junction resolution, in a manner analogous to that shown by the Escherichia coli Holliday Junction resolvase RuvC. The human Holliday Junction resolvase, GEN1, and its yeast orthologue, Yen1, were independently identified using two distinct experimental approaches: GEN1 was identified by mass spectrometry following extensive fractionation of HeLa cell-free extracts, whereas Yen1 was detected by screening a yeast gene fusion library for nucleases capable of Holliday Junction resolution. The eukaryotic Holliday Junction resolvases represent a new subclass of the Rad2/XPG family of nucleases. Recombinant GEN1 and Yen1 resolve Holliday Junctions by the introduction of symmetrically related cuts across the Junction point, to produce nicked duplex products in which the nicks can be readily ligated.
Kosuke Morikawa - One of the best experts on this subject based on the ideXlab platform.
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Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday Junction DNA complex
Biochemical and biophysical research communications, 2007Co-Authors: Kouta Mayanagi, Yoshie Fujiwara, Tomoko Miyata, Kosuke MorikawaAbstract:During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday Junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday Junction: complex I with the single RuvA tetramer on one side of the four way Junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday Junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday Junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB.
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dissection of the regional roles of the archaeal Holliday Junction resolvase hjc by structural and mutational analyses
Journal of Biological Chemistry, 2001Co-Authors: Tatsuya Nishino, Kayoko Komori, Yoshizumi Ishino, Kosuke MorikawaAbstract:Abstract Hjc is an archaeal DNA endonuclease, which resolves the Holliday Junction in the presence of divalent metals. Combined with mutational analyses, the x-ray structure of thePyrococcus furiosus Hjc crystal grown in the presence of ammonium sulfate revealed a positively charged interface, rich in conserved basic residues, and the catalytic center (Nishino, T., Komori, K., Tsuchiya, D., Ishino, Y., and Morikawa, K. (2001)Structure 9, 197–T204). This structural study also suggested that the N-terminal segment and some loops of Hjc play crucial roles in the cleavage of DNA. However, a structural view of the interaction between these regions and DNA remains elusive. To clarify the regional roles of Hjc in the recognition of the Holliday Junction, further structural and biochemical analyses were carried out. A new crystal form of Hjc was obtained from a polyethylene glycol solution in the absence of ammonium sulfate, and its structure has been determined at 2.16-A resolution. A comparison of the two crystal structures has revealed that the N-terminal segment undergoes a serious conformational change. The site-directed mutagenesis of the sulfate-binding site within the segment caused a dramatic decrease in the Junction binding, but the mutant was still capable of cleaving DNA with a 20-fold lower efficiency. The kinetic analysis of Hjc-Holliday Junction interaction indicated that mutations in the N-terminal segment greatly increased the dissociation rate constants of the Hjc-Holliday Junction complex, explaining the decreased stability of the complex. This segment is also responsible for the disruption of base pairs near the Junction center, through specific interactions with them. Taken together, these results imply that, in addition to the secondary effects of two basic loops, the flexible N-terminal segment plays predominant roles in the recognition of DNA conformation near the crossover and in correct positioning of the cleavage site to the catalytic center of the Hjc resolvase.
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crystal structure of the archaeal Holliday Junction resolvase hjc and implications for dna recognition
Structure, 2001Co-Authors: Tatsuya Nishino, Kayoko Komori, Yoshizumi Ishino, Daisuke Tsuchiya, Kosuke MorikawaAbstract:Abstract Background: Homologous recombination is a crucial mechanism in determining genetic diversity and repairing damaged chromosomes. Holliday Junction is the universal DNA intermediate whose interaction with proteins is one of the major events in the recombinational process. Hjc is an archaeal endonuclease, which specifically resolves the Junction DNA to produce two separate recombinant DNA duplexes. The atomic structure of Hjc should clarify the mechanisms of the specific recognition with Holliday Junction and the catalytic reaction. Results: The crystal structure of Hjc from the hyperthermophilic archaeon Pyrococcus furiosus has been determined at 2.0 A resolution. The active Hjc molecule forms a homodimer, where an extensive hydrophobic interface tightly assembles two subunits of a single compact domain. The folding of the Hjc subunit is clearly different from any other Holliday Junction resolvases thus far known. Instead, it resembles those of type II restriction endonucleases, including the configurations of the active site residues, which constitute the canonical catalytic motifs. The dimeric Hjc molecule displays an extensive basic surface on one side, which contains many conserved amino acids, including those in the active site. Conclusions: The architectural similarity of Hjc to restriction endonucleases allowed us to construct a putative model of the complex with Holliday Junction. This model accounts for how Hjc recognizes and resolves the Junction DNA in a specific manner. Mutational and biochemical analyses highlight the importance of some loops and the amino terminal region in interaction with DNA.
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mutational analysis of the pyrococcus furiosus Holliday Junction resolvase hjc revealed functionally important residues for dimer formation Junction dna binding and cleavage activities
Journal of Biological Chemistry, 2000Co-Authors: Kayoko Komori, Hideo Shinagawa, Kosuke Morikawa, Shinzi Sakae, Hiromi Daiyasu, Hiroyuki Toh, Yoshizumi IshinoAbstract:The Holliday Junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday Junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal Junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday Junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs. The products of these mutant genes were purified to homogeneity. With analysis of the activity of the mutant proteins to bind and cleave synthetic Holliday Junctions, one acidic residue, Glu-9, and two basic residues, Arg-10 and Arg-25, were found to play critical roles in enzyme action. This is in addition to the three conserved residues, Asp-33, Glu-46, and Lys-48, which are also conserved in the motif found in the type II restriction endonuclease family proteins. Two aromatic residues, Phe-68 and Phe-72, are important for the formation of the homodimer probably through hydrophobic interactions. The results of these studies have provided insights into the structure-function relationships of the archaeal Holliday Junction resolvase as well as the universality and diversity of the Holliday Junction cleavage reaction.
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Biochemical characterization of the hjc Holliday Junction resolvase of Pyrococcus furiosus.
Nucleic acids research, 2000Co-Authors: Kayoko Komori, Hideo Shinagawa, Kosuke Morikawa, Shinzi Sakae, Ryosuke Fujikane, Yoshizumi IshinoAbstract:The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday Junctions, the intermediates in homologous recombination. The amino acid sequence of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday Junction resolvases. In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday Junction resolvase, highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations. Hjc has specific binding activity to the Holliday-structured DNA, with an apparent dissociation constant (K:(d)) of 60 nM. The dimeric form of Hjc binds to the substrate DNA. The optimal reaction conditions were determined using a synthetic Holliday Junction as substrate. Hjc required a divalent cation for cleavage activity and Mg(2+) at 5-10 mM was optimal. Mn(2+) could substitute for Mg(2+), but it was much less efficient than Mg(2+) as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCl at approximately 200 mM. In addition to the high specific activity, Hjc was found to be extremely heat stable. In contrast to the case of SULFOLOBUS:, the Holliday Junction resolving activity detected in P. furiosus cell extract thus far is only derived from Hjc.
Yoshie Harada - One of the best experts on this subject based on the ideXlab platform.
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Synergistic effect of ATP for RuvA–RuvB–Holliday Junction DNA complex formation
Scientific reports, 2015Co-Authors: Takuma Iwasa, Yong-woon Han, Ryo Hiramatsu, Hiroaki Yokota, Kimiko Nakao, Ryuji Yokokawa, Teruo Ono, Yoshie HaradaAbstract:The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday Junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA-RuvB-Holliday Junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA-Holliday Junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA-Holliday Junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday Junction DNA complex formation.
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synergistic effect of atp for ruva ruvb Holliday Junction dna complex formation
Scientific Reports, 2015Co-Authors: Takuma Iwasa, Yong-woon Han, Ryo Hiramatsu, Hiroaki Yokota, Kimiko Nakao, Ryuji Yokokawa, Teruo Ono, Yoshie HaradaAbstract:The Escherichia coli RuvB hexameric ring motor proteins, together with RuvAs, promote branch migration of Holliday Junction DNA. Zero mode waveguides (ZMWs) constitute of nanosized holes and enable the visualization of a single fluorescent molecule under micromolar order of the molecules, which is applicable to characterize the formation of RuvA-RuvB-Holliday Junction DNA complex. In this study, we used ZMWs and counted the number of RuvBs binding to RuvA-Holliday Junction DNA complex. Our data demonstrated that different nucleotide analogs increased the amount of Cy5-RuvBs binding to RuvA-Holliday Junction DNA complex in the following order: no nucleotide, ADP, ATPγS, and mixture of ADP and ATPγS. These results suggest that not only ATP binding to RuvB but also ATP hydrolysis by RuvB facilitates a stable RuvA-RuvB-Holliday Junction DNA complex formation.
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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, Hiroaki Yokota, Hiroshi Iwasaki, Hideo Shinagawa, Masahito Hayashi, Takashi Hishida, 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.
Malcolm F. White - One of the best experts on this subject based on the ideXlab platform.
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Recombination Machinery: Holliday Junction-Resolving Enzymes
The Bacterial Chromosome, 2014Co-Authors: Malcolm F. WhiteAbstract:Holliday Junctions are resolved into recombinant duplex DNA species by a class of structure-specific endonucleases known as the Holliday Junction-resolving enzymes. The primary cellular resolving enzyme in bacteria is RuvC, which is the main focus of this chapter. The author also talks about the RusA protein, which may act as an alternative to RuvC in some bacterial species, and attempts to place RuvC in a wider context based on our knowledge of other Junction-resolving enzymes. The first cellular Holliday Junction-resolving enzyme identified was RuvC from Escherichia coli. Homologous recombination is ubiquitous among cellular life forms and many prokaryotic and eukaryotic viruses, and wherever Holliday Junctions are formed, Junction-resolving enzymes can be confidently expected. Resolving enzymes recognize the branched structure of the Holliday Junction and introduce paired phosphodiester bond cleavages in opposing strands to collapse the Junction, releasing nicked duplex DNA products. The study of homologous recombination and the Holliday Junction was for many years the realm of geneticists. Holliday Junction migration work has largely been driven by studies of the E. coli RuvABC resolvasome, emphasizing the continuing utility of bacteria as a model system to study some of the most interesting problems in biology.
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PCNA activates the Holliday Junction endonuclease Hjc.
Journal of molecular biology, 2006Co-Authors: Robert Dorazi, Joanne L. Parker, Malcolm F. WhiteAbstract:The resolving enzyme Hjc, which cleaves Holliday Junctions with a high degree of structural specificity, is conserved in all archaea. Like RuvC in Escherichia coli, Hjc functions in the related processes of homologous recombination and double-strand break repair. In bacteria, the RuvAB complex binds Holliday Junctions and catalyses ATP-dependent branch migration, but the equivalent proteins in archaea and eukarya are unknown. Here, we demonstrate that Hjc from Sulfolobus solfataricus forms a physical interaction with the sliding clamp PCNA via a C-terminal PCNA-interacting peptide (PIP) motif in Hjc. PCNA stimulates the Holliday Junction cleavage activity of Hjc in vitro, and deletion of the PIP motif abrogates this effect. This is the first report of a functional interaction between a sliding clamp and a Junction-resolving enzyme, and raises the possibility that PCNA could recruit a variety of different proteins to act on Holliday Junctions in vivo.
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structure of hjc a Holliday Junction resolvase from sulfolobus solfataricus
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Charles S Bond, Mamuka Kvaratskhelia, Malcolm F. White, Derek J Richard, William N HunterAbstract:The 2.15-A structure of Hjc, a Holliday Junction-resolving enzyme from the archaeon Sulfolobus solfataricus, reveals extensive structural homology with a superfamily of nucleases that includes type II restriction enzymes. Hjc is a dimer with a large DNA-binding surface consisting of numerous basic residues surrounding the metal-binding residues of the active sites. Residues critical for catalysis, identified on the basis of sequence comparisons and site-directed mutagenesis studies, are clustered to produce two active sites in the dimer, about 29 A apart, consistent with the requirement for the introduction of paired nicks in opposing strands of the four-way DNA Junction substrate. Hjc displays similarity to the restriction endonucleases in the way its specific DNA-cutting pattern is determined but uses a different arrangement of nuclease subunits. Further structural similarity to a broad group of metal/phosphate-binding proteins, including conservation of active-site location, is observed. A high degree of conservation of surface electrostatic character is observed between Hjc and T4-phage endonuclease VII despite a complete lack of structural homology. A model of the Hjc-Holliday Junction complex is proposed, based on the available functional and structural data.
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multiple Holliday Junction resolving enzyme activities in the crenarchaeota and euryarchaeota
FEBS Letters, 2001Co-Authors: Mamuka Kvaratskhelia, Benjamin N Wardleworth, Malcolm F. WhiteAbstract:Holliday Junction resolving enzymes are required by all life forms that catalyse homologous recombination, including all cellular organisms and many bacterial and eukaryotic viruses. Here we report the identification of three distinct Holliday Junction resolving enzyme activities present in two highly divergent archaeal species. Both Sulfolobus and Pyrococcus share the Hjc activity, and in addition possess unique secondary activities (Hje and Hjr). We propose by analogy with the two other domains of life that the latter enzymes are viral in origin, suggesting the widespread existence of archaeal viruses that rely on homologous recombination as part of their life cycle.
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A conserved nuclease domain in the archaeal Holliday Junction resolving enzyme Hjc.
The Journal of biological chemistry, 2000Co-Authors: Mamuka Kvaratskhelia, Benjamin N Wardleworth, David Norman, Malcolm F. WhiteAbstract:Abstract Holliday Junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday Junction resolving enzyme, Hjc, has recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday Junction into a 2-fold symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA 3′ to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this domain in an archaeal enzyme of known function as well as the first in a Holliday Junction resolving enzyme.
J B Rafferty - One of the best experts on this subject based on the ideXlab platform.
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Mutants of phage bIL67 RuvC with enhanced Holliday Junction binding selectivity and resolution symmetry.
Molecular microbiology, 2013Co-Authors: Victoria L. Green, J B Rafferty, S E Sedelnikova, Fiona Curtis, Gary J. SharplesAbstract:Viral and bacterial Holliday Junction resolvases differ in specificity with the former typically being more promiscuous, acting on a variety of branched DNA substrates, while the latter exclusively targets Holliday Junctions. We have determined the crystal structure of a RuvC resolvase from bacteriophage bIL67 to help identify features responsible for DNA branch discrimination. Comparisons between phage and bacterial RuvC structures revealed significant differences in the number and position of positively-charged residues in the outer sides of the Junction binding cleft. Substitutions were generated in phage RuvC residues implicated in branch recognition and six were found to confer defects in Holliday Junction and replication fork cleavage in vivo. Two mutants, R121A and R124A that flank the DNA binding site were purified and exhibited reduced in vitro binding to fork and linear duplex substrates relative to the wild-type, while retaining the ability to bind X Junctions. Crucially, these two variants cleaved Holliday Junctions with enhanced specificity and symmetry, a feature more akin to cellular RuvC resolvases. Thus, additional positive charges in the phage RuvC binding site apparently stabilize productive interactions with branched structures other than the canonical Holliday Junction, a feature advantageous for viral DNA processing but deleterious for their cellular counterparts.
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the structure of bacillus subtilis recu Holliday Junction resolvase and its role in substrate selection and sequence specific cleavage
Structure, 2005Co-Authors: Natalie Mcgregor, S E Sedelnikova, Sylvia Ayora, Begona Carrasco, Juan C Alonso, Paul Thaw, J B RaffertyAbstract:We have determined the structure of the enzyme RecU from Bacillus subtilis, that is the general Holliday Junction resolving enzyme in Gram-positive bacteria. The enzyme fold reveals a striking similarity to a class of resolvase enzymes found in archaeal sources and members of the type II restriction endonuclease family to which they are related. The structure confirms the presence of active sites formed around clusters of acidic residues that we have also shown to bind divalent cations. Mutagenesis data presented here support the key role of certain residues. The RecU structure suggests a basis for Holliday Junction selectivity and suggests how sequence-specific cleavage might be achieved. Models for a resolvase-DNA complex address how the enzyme might organize Junctions into an approximately 4-fold symmetric form.
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Crystallization of Escherichia coli RuvA complexed with a synthetic Holliday Junction.
Acta crystallographica. Section D Biological crystallography, 1999Co-Authors: D Hargreaves, J B Rafferty, S E Sedelnikova, R G Lloyd, D W RiceAbstract:During homologous recombination in Escherichia coli the RuvA, B and C proteins interact specifically with the Holliday Junction formed by the action of RecA to promote the strand-exchange reaction. RuvA, a homotetrameric protein of molecular weight 88 kDa, has been overexpressed in E. coli, purified and co-crystallized with a synthetic Holliday Junction substrate made from four 18-base deoxyoligonucleotides. Crystals were grown using the hanging-drop vapour-diffusion method with sodium acetate as the precipitant. The crystals diffract to a resolution of 6 A and belong to the monoclinic system, space group C2, with cell parameters a = 148, b = 148, c = 106 A and beta = 123 degrees. The X-ray analysis of these crystals should reveal the structure of the Holliday Junction and its mode of binding to RuvA, providing new insights into the molecular mechanism of genetic recombination.
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Crystallization of Escherichia coli RuvA complexed with a synthetic Holliday Junction.
Acta Crystallographica Section D Biological Crystallography, 1999Co-Authors: David J. Hargreaves, J B Rafferty, S E Sedelnikova, R G Lloyd, D W RiceAbstract:During homologous recombination in Escherichia coli the RuvA, B and C proteins interact specifically with the Holliday Junction formed by the action of RecA to promote the strand-exchange reaction. RuvA, a homotetrameric protein of molecular weight 88 kDa, has been overexpressed in E. coli, purified and co-crystallized with a synthetic Holliday Junction substrate made from four 18-base deoxyoligonucleotides. Crystals were grown using the hanging-drop vapour-diffusion method with sodium acetate as the precipitant. The crystals diffract to a resolution of 6 A and belong to the monoclinic system, space group C2, with cell parameters a = 148, b = 148, c = 106 A and β = 123°. The X-ray analysis of these crystals should reveal the structure of the Holliday Junction and its mode of binding to RuvA, providing new insights into the molecular mechanism of genetic recombination.
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crystal structure of e coli ruva with bound dna Holliday Junction at 6 a resolution
Nature Structural & Molecular Biology, 1998Co-Authors: David J. Hargreaves, S E Sedelnikova, R G Lloyd, D W Rice, Peter J Artymiuk, J B RaffertyAbstract:Here we present the crystal structure of the Escherichia coli protein RuvA bound to a key DNA intermediate in recombination, the Holliday Junction. The structure, solved by isomorphous replacement and density modification at 6 A resolution, reveals the molecular architecture at the heart of the branch migration and resolution reactions required to process Holliday intermediates into recombinant DNA molecules. It also reveals directly for the first time the structure of the Holliday Junction. A single RuvA tetramer is bound to one face of a Junction whose four DNA duplex arms are arranged in an open and essentially four-fold symmetric conformation. Protein-DNA contacts are mediated by two copies of a helix-hairpin-helix motif per RuvA subunit that contact the phosphate backbone in a very similar manner. The open structure of the Junction stabilized by RuvA binding exposes a DNA surface that could be bound by the RuvC endonuclease to promote resolution.