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S C West

Publications and source records attributed to S C West.

At least 55 records · Page 3Linked to original sources
PubMed1998

Role of the human RAD51 protein in homologous recombination and double-stranded-break repair.

Eukaryotic cells possess several mechanisms for repairing double-stranded breaks in DNA. One mechanism involves genetic recombination with an intact sister duplex. The recent identification of the RAD51 protein, a eukaryotic homologue of Escherichia coli RecA, represents a landmark discovery in our understanding of the key reactions in this repair pathway. RAD51 is similar to RecA, both biochemically and structurally: it promotes homologous pairing and strand exchange within a regular nucleoprotein filament. The isolation of yeast and human RecA homologues shows that homologous recombination and recombinational repair have been conserved throughout evolution. The goal is now to identify other factors involved in recombinational repair and to define their roles in this essential process.

Animals↗
PubMed1997

Recombination initiation: easy as A, B, C, D... chi?

The octameric Chi (chi) sequence is a recombination hotspot in Escherichia coli. Recent studies suggest a singular mechanism by which chi regulates not only the nuclease activity of RecBCD enzyme, but also the ability of RecBCD to promote loading of the strand exchange protein, RecA, onto chi-containing DNA.

Binding Sites↗
PubMed1997

Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities.

Many DNA helicases utilise the energy derived from nucleoside triphosphate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB protein (a hexameric ring helicase), promotes the branch migration of Holliday junctions during genetic recombination and DNA repair. To analyse the relationship between ATP-dependent DNA helicase activity and branch migration, a site-directed mutation was introduced into the helicase II motif of RuvB. Over-expression of RuvBD113N in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical properties of RuvBD113N were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major alterations to the biochemical activities of RuvB, such that RuvBD113N was defective in DNA binding and ATP hydrolysis, while retaining the ability to form hexameric rings and interact with RuvA. RuvBD113N formed heterohexamers with wild-type RuvB, and could inhibit RuvB function by affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not all subunits of the ring need to be catalytically competent.

Adenosine Triphosphatases↗
PubMed1997

The human Rad51 protein: polarity of strand transfer and stimulation by hRP-A.

The human Rad51 protein is homologous to the RecA protein and catalyses homologous pairing and strand transfer reactions in vitro. Using single-stranded circular and homologous linear duplex DNA, we show that hRad51 forms stable joint molecules by transfer of the 5' end of the complementary strand of the linear duplex to the ssDNA. The polarity of strand transfer is therefore 3' to 5', defined relative to the ssDNA on which hRad51 initiates filament formation. This polarity is opposite to that observed with RecA. Homologous pairing and strand transfer require stoichiometric amounts of hRad51, corresponding to one hRad51 monomer per three nucleotides of ssDNA. Joint molecules are not observed when the protein is present in limiting or excessive amounts. The human ssDNA binding-protein, hRP-A, stimulates hRad51-mediated reactions. Its effect is consistent with a role in the removal of secondary structures from ssDNA, thereby facilitating the formation of continuous Rad51 filaments.

Adenosine Triphosphate↗
PubMed1997

Distribution of the Rad51 recombinase in human and mouse spermatocytes.

In vitro, the human Rad51 protein (hRad51) promotes homologous pairing and strand exchange reactions suggestive of a key role in genetic recombination. To analyse its role in this process, polyclonal antibodies raised against hRad51 were used to study the distribution of Rad51 in human and mouse spermatocytes during meiosis I. In human spermatocytes, hRad51 was found to form discrete nuclear foci from early zygotene to late pachytene. The foci always co-localized with lateral element proteins, components of the synaptonemal complex (SC). During zygotene, the largest foci were present in regions undergoing synapsis, suggesting that Rad51 is a component of early recombination nodules. Pachytene nuclei showed a greatly reduced level of Rad51 labelling, with the exceptions of any asynapsed autosomes and XY segments, which were intensely labelled. The distribution of Rad51 in mouse spermatocytes was similar to that found in human spermatocytes, except that in this case Rad51 was detectable at leptotene. From these results, we conclude that the Rad51 protein has a role in the interhomologue interactions that occur during meiotic recombination. These interactions are spatially and temporally associated with synapsis during meiotic prophase I.

Animals↗
PubMed1997

Effect of DNA topology on Holliday junction resolution by Escherichia coli RuvC and bacteriophage T7 endonuclease I.

Holliday junctions are key intermediates in homologous genetic recombination. Their resolution requires specialised nucleases that nick pairs of strands at the junction point, leading to the separation of mature recombinants. Resolution occurs in either of two orientations, according to which DNA strands are cut. We show that DNA topology can determine the efficiency and outcome of a recombination reaction. Using two Holliday junction resolvases, Escherichia coli RuvC protein and T7 endonuclease I, we observed that supercoiled figure-8 DNA molecules containing Holliday junctions were resolved with a specific orientation bias, and that this bias was reversed by the presence of a topological tether (catenation). In contrast, when all topological constraints were removed by restriction digestion, the recombination intermediates were resolved equally in the two orientations. These results show that topological constraints affecting Holliday junction structure influence the orientation of resolution by cellular resolvases.

Bacterial Proteins↗
PubMed1997

In vitro reconstitution of the late steps of genetic recombination in E. coli.

Purified proteins have been used to reconstitute an in vitro system for the medial-to-late stages of recombination in E. coli. In this system, RecA protein formed recombination intermediates that were processed by the actions of the RuvA, RuvB, and RuvC proteins. RuvAB was found to promote branch migration, to dissociate the RecA filament, and to modulate the orientation of cleavage of Holliday junction resolution by RuvC. Monoclonal antibodies directed against RuvA, RuvB, or RuvC inhibited resolution in the reconstituted system. Specific protein-protein interactions between the branch migration motor (RuvB) and the resolvase (RuvC) were also observed. These results provide evidence for coordinated action during the late stages of recombination, possibly involving the assembly of a RuvABC branch migration/resolution complex.

Antibodies, Monoclonal↗
PubMed1997

The RuvC protein dimer resolves Holliday junctions by a dual incision mechanism that involves base-specific contacts.

The Escherichia coli RuvC protein resolves DNA intermediates produced during genetic recombination. In vitro, RuvC binds specifically to Holliday junctions and resolves them by the introduction of nicks into two strands of like polarity. In contrast to junction recognition, which occurs without regard for DNA sequence, resolution occurs preferentially at sequences that exhibit the consensus 5'-(A/T)TT/(G/C)-3' (where / indicates the site of incision). Synthetic Holliday junctions containing modified cleavage sequences have been used to investigate the mechanism of cleavage. The results indicate that specific DNA sequences are required for the correct docking of DNA into the two active sites of the RuvC dimer. In addition, using chemically modified oligonucleotides to introduce a hydrolysis-resistant 3'-S-phosphorothiolate linkage at the cleavage site, it was found that, as long as the sequence requirements are fulfilled, the two incisions could be uncoupled from each other. These results indicate that RuvC protein resolves Holliday junctions by a mechanism similar to that exhibited by certain restriction enzymes.

Bacterial Proteins↗
PubMed1997

Structure and subunit composition of the RuvAB-Holliday junction complex.

The E. coli RuvA and RuvB proteins, which are involved in the late stages of recombination and the recombinational repair of damaged DNA, bind to Holliday junctions and promote branch migration. We have used electron microscopy and image analysis to examine RuvA and RuvB bound to model Holliday structures. The two hexameric rings of RuvB are oriented in a bipolar manner, so that the large end of each faces the junction. The results suggest a model for branch migration in which DNA is pumped out of the small end of each ring as ATP is hydrolyzed. The same structural polarity has been established for the bacteriophage T7 gp4 replicative helicase. Mass and image analysis of the RuvAB-junction complex suggests that two tetramers of RuvA form a symmetrical sandwich about the plane of the junction.

Bacterial Proteins↗
PubMed1997

Purification of human Rad51 protein by selective spermidine precipitation.

The human Rad51 protein is a structural homolog of Escherichia coli RecA. The exact role of human Rad51 within the cell is poorly understood but, like its bacterial and yeast homologs, hRad51 is believed to play a central role in homologous recombination. However, recent reports that transgenic mice lacking the RAD51 gene die early in development suggest an additional and essential function for mammalian Rad51 in cell proliferation or genome maintenance. In this paper we describe a simple and quick method for the purification of human Rad51 overproduced in E. coli. Dialysis of cell-free extracts against buffer containing low concentrations of spermidine result in the formation of hRad51 microcrystals as observed by light and electron microscopy. The crystals were easily redissolved in phosphate buffer and hRad51 was further purified to homogeneity using hydroxylapatite, affi-gel heparin and Q-sepharose chromatography. When purified by this method hRad51 is free of endo- and exonuclease activities and suitable for enzymological studies. Spermidine precipitation also provides a rapid method for the large scale purification of hRad51 suitable for physical analysis.

Chemical Precipitation↗
PubMed1997

Processing of recombination intermediates by the RuvABC proteins.

The RuvA, RuvB, and RuvC proteins in Escherichia coli play important roles in the late stages of homologous genetic recombination and the recombinational repair of damaged DNA. Two proteins, RuvA and RuvB, form a complex that promotes ATP-dependent branch migration of Holliday junctions, a process that is important for the formation of heteroduplex DNA. Individual roles for each protein have been defined, with RuvA acting as a specificity factor that targets RuvB, the branch migration motor to the junction. Structural studies indicate that two RuvA tetramers sandwich the junction and hold it in an unfolded square-planar configuration. Hexameric rings of RuvB face each other across the junction and promote a novel dual helicase action that "pumps" DNA through the RuvAB complex, using the free energy provided by ATP hydrolysis. The third protein, RuvC endonuclease, resolves the Holliday junction by introducing nicks into two DNA strands. Genetic and biochemical studies indicate that branch migration and resolution are coupled by direct interactions between the three proteins, possibly by the formation of a RuvABC complex.

Animals↗
PubMed1996

Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro.

The human testis Rad51 protein, a structural homolog of E. coli RecA, binds single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using circular ssDNA and linear dsDNA (3.0 kb in length), we demonstrate that hRad51 promotes homologous pairing and strand exchange reactions in vitro. Joint molecule formation was dependent upon ATP hydrolysis and DNA homology and was stimulated by the single-strand DNA-binding protein RP-A. In these reactions, the 5' terminus of the complementary strand of the linear duplex was efficiently transferred to the ssDNA. However, under standard conditions, extensive strand exchange was not observed. These results establish hRad51 as a functional homolog of RecA, but indicate that the human protein and its bacterial counterpart differ in their ability to promote extensive strand transfer. It is proposed that hRad51 mediates homology recognition and initiates strand exchange, but that extensive heteroduplex formation in higher organisms requires the actions of additional proteins.

Adenosine Triphosphate↗
PubMed1996

Bypass of DNA heterologies during RuvAB-mediated three- and four-strand branch migration.

During general genetic recombination and recombinational DNA repair, DNA damages and heterologies are often encountered which must be efficiently processed by the cellular recombination machinery. In RecA-mediated three-strand exchange reactions between single-stranded circular and linear duplex DNA, or four-strand exchange reactions between gapped circular and linear duplex DNA, heterologies can only be bypassed in vitro when they are short in length and are followed by homologous DNA downstream. Larger DNA inserts block RecA-mediated strand exchange, indicating that effective bypass requires other components of the recombination machinery. The RuvA and RuvB proteins of Escherichia coli form an important part of this machinery. In this work, we have analysed the ability of RuvA and RuvB to bypass large tracts of DNA heterology in both three- and four-strand exchange reactions, using recombination intermediates made by the E. coli RecA protein. Under optimal reaction conditions for RuvAB, up to 1000 bp of DNA heterology can by bypassed in three-strand reactions and 300 bp of DNA heterology can be bypassed in four-strand reactions. Whereas high concentrations of RuvB (in the absence of RuvA) can promote homologous branch migration, we find that RuvB alone is unable to catalyse heterologous bypass, indicating an essential role for both proteins in homologous recombination and recombinational DNA repair processes. Under certain conditions, the bypass of heterology is stimulated by the single-strand binding protein SSB.

Adenosine Triphosphate↗
PubMed1996

Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange.

The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA. Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration. Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C). To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence. We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange. In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.

Bacterial Proteins↗
PubMed1996

Role of RuvA in branch migration reactions catalyzed by the RuvA and RuvB proteins of Escherichia coli.

The RuvA and RuvB proteins of Escherichia coli promote ATP-dependent branch migration of Holliday junctions during homologous genetic recombination and DNA repair. In this process, RuvA acts as a specificity factor that targets RuvB, a hexameric ring motor protein, to the junction. Because elevated concentrations of RuvB can promote branch migration in the absence of RuvA, it has been suggested that RuvA acts as a molecular matchmaker. In the studies presented here, we compared the requirements for RuvAB- and RuvB-mediated branch migration reactions and found that reactions catalyzed by RuvB alone were highly sensitive to inhibition by NaCl, temperature, ADP, and ATPgammaS. Our observations indicate that the two reactions occur by distinct mechanisms and support the notion that RuvAB-mediated branch migration is physiologically more relevant than that catalyzed by RuvB. We also show that ongoing RuvAB-mediated branch migration reactions were blocked by the addition of polyclonal antibodies raised against RuvA. The role of RuvA is therefore unlikely to be restricted to RuvB targeting; instead, it is required continually during branch migration. Competition with excess synthetic Holliday junctions, sufficient to sequester released RuvA, failed to cause an immediate block and leads us to suggest that RuvAB promote branch migration by a processive mechanism.

Adenosine Diphosphate↗