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S C West

Publications and source records attributed to S C West.

At least 37 records · Page 2Linked to original sources
PubMed2000

RuvAB-mediated branch migration does not involve extensive DNA opening within the RuvB hexamer.

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] [3] [4] [5] [6] and acts as a specificity factor that promotes the targeting of RuvB [7] [8], a hexameric ring protein that drives branch migration [9] [10] [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.

Adenosine Triphosphate↗
PubMed2000

Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest.

Individuals affected by the autosomal recessive disorder Werner's syndrome (WS) develop many of the symptoms characteristic of premature ageing. Primary fibroblasts cultured from WS patients exhibit karyotypic abnormalities and a reduced replicative life span. The WRN gene encodes a 3'-5' DNA helicase, and is a member of the RecQ family, which also includes the product of the Bloom's syndrome gene (BLM). In this work, we show that WRN promotes the ATP-dependent translocation of Holliday junctions, an activity that is also exhibited by BLM. In cells arrested in S-phase with hydroxyurea, WRN localizes to discrete nuclear foci that coincide with those formed by the single-stranded DNA binding protein replication protein A. These results are consistent with a model in which WRN prevents aberrant recombination events at sites of stalled replication forks by dissociating recombination intermediates.

Adenosine Triphosphatases↗
PubMed1999

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

Adenosine Triphosphatases↗
PubMed1999

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

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.

Adenosine Triphosphatases↗
PubMed1999

Human Dmc1 protein binds DNA as an octameric ring.

The bacterial RecA protein has been the most intensively studied enzyme in homologous genetic recombination. The core of RecA is structurally homologous to that of the F1-ATPase and helicases. Like the F1-ATPase and ring helicases, RecA forms a hexameric ring. The human Dmc1 (hDmc1) protein, a meiosis-specific recombinase, is homologous to RecA. We show that hDmc1 forms octameric rings. Unlike RecA and Rad51, however, hDmc1 protein does not form helical filaments. The hDmc1 ring binds DNA in the central channel, as do the ring helicases, which is likely to represent the active form of the protein. These observations indicate that the conservation of the RecA-like ring structure extends from bacteria to humans, and that some RecA homologs may form both rings and filaments, whereas others may function only as rings.

Bacteriophages↗
PubMed1999

Heteroduplex formation by human Rad51 protein: effects of DNA end-structure, hRP-A and hRad52.

Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.

DNA Repair↗
PubMed1999

Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of holliday junction resolution.

Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration.

Bacterial Proteins↗
PubMed1999

Binding of double-strand breaks in DNA by human Rad52 protein.

Double-strand breaks (DSBs) in DNA are caused by ionizing radiation. These chromosomal breaks can kill the cell unless repaired efficiently, and inefficient or inappropriate repair can lead to mutation, gene translocation and cancer. Two proteins that participate in the repair of DSBs are Rad52 and Ku: in lower eukaryotes such as yeast, DSBs are repaired by Rad52-dependent homologous recombination, whereas vertebrates repair DSBs primarily by Ku-dependent non-homologous end-joining. The contribution of homologous recombination to vertebrate DSB repair, however, is important. Biochemical studies indicate that Ku binds to DNA ends and facilitates end-joining. Here we show that human Rad52, like Ku, binds directly to DSBs, protects them from exonuclease attack and facilitates end-to-end interactions. A model for repair is proposed in which either Ku or Rad52 binds the DSB. Ku directs DSBs into the non-homologous end-joining repair pathway, whereas Rad52 initiates repair by homologous recombination. Ku and Rad52, therefore, direct entry into alternative pathways for the repair of DNA breaks.

Antigens, Nuclear↗
PubMed1999

Escherichia coli RuvBL268S: a mutant RuvB protein that exhibits wild-type activities in vitro but confers a UV-sensitive ruv phenotype in vivo.

The RuvABC proteins of Escherichia coli process recombination intermediates during genetic recombination and DNA repair. RuvA and RuvB promote branch migration of Holliday junctions, a process that extends heteroduplex DNA. Together with RuvC, they form a RuvABC complex capable of Holliday junction resolution. Branch migration by RuvAB is mediated by RuvB, a hexameric ring protein that acts as an ATP-driven molecular pump. To gain insight into the mechanism of branch migration, random mutations were introduced into the ruvB gene by PCR and a collection of mutant alleles were obtained. Mutation of leucine 268 to serine resulted in a severe UV-sensitive phenotype, characteristic of a ruv defect. Here, we report a biochemical analysis of the mutant protein RuvBL268S. Unexpectedly, the purified protein is fully active in vitro with regard to its ATPase, DNA binding and DNA unwinding activities. It also promotes efficient branch migration in combination with RuvA, and forms functional RuvABC-Holliday junction resolvase complexes. These results indicate that RuvB may perform some additional, and as yet undefined, function that is necessary for cell survival after UV-irradiation.

Adenosine Triphosphatases↗
PubMed1998

Visualisation of human rad52 protein and its complexes with hRad51 and DNA.

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.

Animals↗
PubMed1998

DNA end-joining catalyzed by human cell-free extracts.

Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PKcs, the three subunits of the DNA-activated protein kinase DNA-PK. Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this in vitro system provides an important advance in the study of the mechanism of DNA end-joining in human cells.

Androstadienes↗
PubMed1998

Coordinated actions of RuvABC in Holliday junction processing.

The RuvA, RuvB and RuvC proteins of Escherichia coli process Holliday junctions during genetic recombination and DNA repair. Biochemical studies have shown that RuvA and RuvB promote branch migration whereas RuvC resolves junctions by endonucleolytic cleavage. Here we show that RuvAB stimulate Holliday junction resolution by RuvC. Elevated RuvC activity was dependent upon RuvAB-mediated ATP-hydrolysis. These results show that the three Ruv proteins work in a coordinated manner to promote Holliday junction resolution, and account for the resolvase-defective phenotype exhibited by ruvA, ruvB or ruvC mutant strains.

Adenosine Triphosphate↗
PubMed1998

Sequence-specificity of Holliday junction resolution: identification of RuvC mutants defective in metal binding and target site recognition.

The RuvC protein of Escherichia coli resolves Holliday intermediates in recombination and DNA repair by a dual strand incision mechanism targeted to specific DNA sequences located symmetrically at the crossover. Two classes of amino acid substitutions are described that provide new insights into the sequence-specificity of the resolution reaction. The first includes D7N and G14S, which modify or eliminate metal binding and prevent catalysis. The second, defined by G114D, G114N, and A116T, interfere with the ability of RuvC to cleave at preferred sequences, but allow resolution at non-consensus target sites. All five mutant proteins bind junction DNA and impose an open conformation. D7N and G14S fail to induce hypersensitivity to hydroxyl radicals, a property of RuvC previously thought to reflect junction opening. A different mechanism is proposed whereby ferrous ions are co-ordinated in the complex to induce a high local concentration of radicals. The open structure imposed by wild-type RuvC in Mg2+ is similar to that observed previously using a junction with a different stacking preference. G114D and A116T impose slightly altered structures. This subtle change may be sufficient to explain the failure of these proteins to cleave the sequences normally preferred. Gly114 and Ala116 residues link two alpha-helices lining the wall of the catalytic cleft in each subunit of RuvC. We suggest that substitutions at these positions realign these helices and interfere with the ability to establish base-specific contacts at resolution hotspots.

Amino Acid Sequence↗
PubMed1998

Formation of RuvABC-Holliday junction complexes in vitro.

In Escherichia coli, the RuvA, RuvB and RuvC proteins are required for the late stages of homologous recombination and DNA repair. RuvA and RuvB form a complex that interacts with Holliday junctions--crossed DNA structures that are recombination intermediates--and promotes branch migration; RuvC is a junction-specific endonuclease that resolves Holliday junctions and completes the recombination process. Because genetic and biochemical experiments suggest that the processes of branch migration and resolution are linked, coimmunoprecipitation experiments were carried out to determine whether the three Ruv proteins interact to form a functional complex (RuvABC). Using a synthetic Holliday junction, a multisubunit complex containing the junction and RuvA, RuvB and RuvC was detected. In the absence of RuvB, RuvAC-junction complexes were observed. Complex formation was not facilitated by duplex DNA. The identification of a RuvABC-junction complex provides direct evidence that the RuvABC proteins interact at the Holliday junction.

Bacterial Proteins↗
PubMed1998

Functional interactions between the holliday junction resolvase and the branch migration motor of Escherichia coli.

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.

Bacterial Proteins↗
PubMed1998

Synergistic actions of Rad51 and Rad52 in recombination and DNA repair.

In the yeast Saccharomyces cerevisiae, mutations in the genes RAD51 or RAD52 result in severe defects in genetic recombination and the repair of double-strand DNA breaks. These genes, and others of the RAD52 epistasis group (RAD50, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2), were first identified by their sensitivity to X-rays. They were subsequently shown to be required for spontaneous and induced mitotic recombination, meiotic recombination, and mating-type switching. Human homologues of RAD50, RAD51, RAD52, RAD54 and MRE11 have been identified. Targeted disruption of the murine RAD51 gene results in an embryonic lethal phenotype, indicating that Rad51 protein is required during cell proliferation. Biochemical studies have shown that human RAD51 encodes a protein of relative molecular mass 36,966 (hRad51) that promotes ATP-dependent homologous pairing and DNA strand exchange. As a structural and functional homologue of the RecA protein from Escherichia coli, hRad51 is thought to play a central role in recombination. Yeast Rad51 has been shown to interact with Rad52 protein, as does the human homologue. Here we show that hRad52 stimulates homologous pairing by hRad51. The DNA-binding properties of hRad52 indicate that Rad52 is involved in an early stage of Rad51-mediated recombination.

Cloning, Molecular↗