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Biomedical subjects

S C West

Publications and source records attributed to S C West.

At least 91 records · Page 5Linked to original sources
PubMed1994

Targeted versus non-targeted DNA helicase activity of the RuvA and RuvB proteins of Escherichia coli.

The RuvA and RuvB proteins of Escherichia coli promote the branch migration of Holliday junctions in vitro. To understand the relationship between branch migration and the intrinsic 5'-->3' DNA helicase activity of RuvAB, the requirements and substrate specificity of the helicase reaction have been studied in more detail. We find that RuvAB-mediated DNA unwinding and branch migration reactions show similar requirements for Mg2+ and ATP and are inhibited to a similar extent by ADP and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)). The helicase activity, measured by the dissociation of a short fragment from circular single-stranded DNA, requires both RuvA and RuvB and is stimulated by subsaturating concentrations of single-strand binding protein (SSB). In contrast, saturating concentrations of SSB are inhibitory. Using substrates that contain a DNA junction, which permits the specific binding of RuvA, we find that the RuvA and RuvB proteins promote two types of helicase reactions: nonspecific reactions, which are sensitive to inhibition by stoichiometric amounts of SSB, and junction-targeted reactions, which are not inhibited by SSB. Using three-armed structures, we observe that junction-targeted reactions display a polarity and result in asymmetric product formation. Junction-specific binding and the subsequent initiation of DNA unwinding are likely to represent early steps in the process of branch migration.

Adenosine Triphosphatases↗
PubMed1994

Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins.

The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.

Bacterial Proteins↗
PubMed1994

XPG endonuclease makes the 3' incision in human DNA nucleotide excision repair.

Humans with a defect in the XPG protein suffer from xeroderma pigmentosum (XP) resulting from an inability to perform DNA nucleotide excision repair properly. Here we show that XPG makes a structure-specific endonucleolytic incision in a synthetic DNA substrate containing a duplex region and single-stranded arms. One strand of the duplex is cleaved at the border with single-stranded DNA. A cut with the same polarity is also made in a bubble structure, at the 3' side of the centrally unpaired region. Normal cell extracts introduce a nick 3' to a platinum-DNA lesion, but an XP-G cell extract is defective in making this incision. These data show that XPG has a direct role in making one of the incisions required to excise a damaged oligonucleotide, by cleaving 3' to DNA damage during nucleotide excision repair.

Base Sequence↗
PubMed1994

The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA.

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.

Algorithms↗
PubMed1994

Activation of RuvC Holliday junction resolvase in vitro.

The Escherichia coli RuvC protein is an endonuclease that resolves Holliday junctions. In vitro, the protein shows efficient structure-specific binding of Holliday junctions, yet the rate of junction resolution is remarkably low. We have mapped the sites of cleavage on a synthetic junction through which a crossover can branch migrate through 26 bp and find that > or = 90% of the junctions were cleaved at one site. This observation of sequence-specific cleavage suggests that inefficient resolution may be due to DNA binding events which occur away from the cleavage site and are therefore non-productive. Holliday junction resolution by RuvC protein can be stimulated by a number of factors including: (i) the presence of Mn2+ (rather than Mg2+) as the divalent metal cofactor, (ii) alkaline pH (< or = 10), and (iii) elevated temperature. These observations may indicate that other proteins are required for efficient RuvC-mediated resolution.

Bacterial Proteins↗
PubMed1994

Processing of Holliday junctions by the Escherichia coli RuvA, RuvB, RuvC and RecG proteins.

Recent work has led to significant advances in our understanding of the late steps of genetic recombination and the post-replicational repair of DNA. The RuvA and RuvB proteins have been shown to interact with recombination intermediates and catalyse the branch migration of Holliday junctions. Although both proteins are required for branch migration, each plays a defined role with RuvA acting as a specificity factor that directs RuvB (an ATPase) to the junction. The RuvB ATPase provides the motor for branch migration. The next step is catalysed by RuvC protein which recognises Holliday junctions and promotes their resolution by endonucleolytic cleavage. New data indicates an alternative pathway for Holliday junction processing. This pathway involves RecG, a branch migration protein which is functionally analogous to RuvAB, and a protein (activated by a rus mutation) which works with RecG to process intermediates independently of RuvA, RuvB and RuvC.

Adenosine Triphosphatases↗
PubMed1994

Cloning, overexpression, purification, and characterization of the Escherichia coli RuvC Holliday junction resolvase.

The ruvC gene has been cloned into the plasmid pT7-7 under the control of the T7 phi 10 promoter. Following induction with isopropyl-1-thio-beta-D-galactopyranoside, the 19-kDa RuvC protein was overexpressed to 20-30% of total cell protein. RuvC has been purified to homogeneity by a simple procedure involving precipitation from the crude lysate, followed by three chromatographic steps. The purified protein resolves synthetic Holliday junctions (60 nucleotides in length) by cleavage at the 3'-side of a phosphate group, to produce nicked duplex DNA. Under the same conditions no cleavage of linear duplex or single-stranded DNA was detected. However, low levels of cleavage were observed with supercoiled form I and single-stranded circular DNA substrates, consistent with the interaction of RuvC with secondary structures. Using synthetic Holliday junctions, we show that RuvC-mediated resolution requires Mg2+ (10 mM) and exhibits an alkaline pH optimum (pH 9.0). No energy cofactors are needed. When RuvC was analyzed by gel filtration and polyacrylamide gel electrophoresis, monomeric and dimeric forms of the protein were observed.

Bacterial Proteins↗
PubMed1994

Substrate specificity of the Escherichia coli RuvC protein. Resolution of three- and four-stranded recombination intermediates.

The specificity of the Escherichia coli RuvC Holliday junction resolvase has been investigated in vitro. RuvC protein cleaves synthetic DNA substrates that model three- or four-stranded recombination intermediates but fails to act upon Y junctions, G/A mismatches, heterologous loop structures, or two-stranded branched junctions. RuvC therefore differs from endonuclease VII of bacteriophage T4 which exhibits broad range specificity. Using related three- and four-stranded synthetic DNA junctions, we show that RuvC cleaves both junctions at the same DNA sequence and requires a region of homology at the junction point. The action of RuvC on three- and four-stranded recombination intermediates made by RecA was also investigated. We found that RuvC fails to resolve three-stranded intermediates in the presence of RecA, although four-stranded intermediates are resolved under the same conditions. However, both three- and four-stranded intermediates are substrates for the nuclease after removal of RecA. We interpret these differences in terms of the contiguity of the RecA nucleoprotein filament which may, under certain conditions, limit access to the Holliday junction resolvase.

Bacterial Proteins↗
PubMed1994

Resolution of recombination intermediates by a mammalian activity functionally analogous to Escherichia coli RuvC resolvase.

A mammalian endonuclease that resolves Holliday junctions has been partially purified from extracts of calf thymus and Chinese hamster ovary cells. The activity acts upon (i) synthetic Holliday junctions and (ii) recombination intermediates made by the Escherichia coli RecA protein and appears to be functionally analogous to the E. coli RuvC protein. Cleavage occurs by the introduction of symmetrically related nicks in strands of like polarity to produce nicked duplex DNA products. The nicks can be repaired by DNA ligase. The resolvase is specific for Holliday junctions and does not act upon Y junctions, G/A mismatches, or heterologous loops. The substrate specificity is therefore similar to that of E. coli RuvC protein and contrasts with the broad range specificity of other junction resolvases such as T4 endonuclease VII. The mammalian resolvase activity has been observed at normal levels in extracts prepared from a series of DNA repair-defective cells. These include the x-ray or UV-sensitive hamster lines xrs-5, xrs-6, and Chinese hamster ovary 43-3B (defective in ERCC-1), and murine cells that are severely immunodeficient and defective in both V(D)J rejoining and DNA repair.

Animals↗
PubMed1994

Recombination genes and proteins.

The recombination of DNA takes place by a multistep process involving numerous gene products. In the past year, studies using bacterial proteins have led to a number of significant advances in our understanding of the enzymes of recombination and of the reactions that they catalyze. Moreover, the identification of eukaryotic proteins that are structurally analogous to the principal bacterial recombination enzyme, RecA protein, suggests that the basic mechanisms of homologous pairing and strand exchange have been conserved through evolution from bacteria to man.

Base Sequence↗
PubMed1993

Resolution of Holliday junctions by RuvC resolvase: cleavage specificity and DNA distortion.

E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using small synthetic junctions, we show that junction recognition is structure-specific and occurs in the absence of metal cofactors. In the presence of Mg2+, Holliday junctions are resolved by the introduction of symmetrically related nicks at the 3' side of thymine residues. The nicked duplex products are repaired by the action of DNA ligase. Within the RuvC-Holliday junction complex, the DNA is distorted such that 2 of the 4 strands become hypersensitive to hydroxyl radical attack. The ionic requirements of binding, hydroxyl radical sensitivity, and strand cleavage indicate three distinct steps in the mechanism of RuvC-mediated Holliday junction resolution: structure-specific recognition, DNA distortion, and sequence-dependent cleavage.

Bacterial Proteins↗
PubMed1993

Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins. I. Comparison of RuvAB- and RuvB-mediated reactions.

The Escherichia coli RuvA and RuvB proteins mediate the branch migration of Holliday junctions in vitro. In the presence of stoichiometric amounts of RuvB (1 RuvB dimer/12 nucleotides), branch migration can occur without need for RuvA. However, RuvA is required when the RuvB concentration is reduced 4-fold or more. Under optimal conditions, we found the minimal protein requirement to be 1 RuvB dimer per 500-1100 nucleotides and 1 RuvA tetramer per 600-1200 nucleotides. To determine the roles of RuvA and RuvB in branch migration, we compared branch migration reactions mediated by RuvB only and by RuvA and RuvB. The time courses of the two reactions were similar, and both required ATP and Mg2+. However, RuvB-mediated branch migration occurred at lower ATP concentrations (> or = 200 microM) and higher Mg2+ concentrations (> or = 10 mM MgCl2) than the reaction mediated by RuvA and RuvB (> or = 1 mM ATP, > or = 5 mM MgCl2). The Mg2+ requirement for RuvB-mediated branch migration reflects the Mg2+ requirement of RuvB for DNA binding (Müller, B., Tsaneva, I.R., and West, S. C. (1993) J. Biol. Chem. 268, 17185-17189) and can be overcome by addition of RuvA. These results indicate that RuvA protein facilitates the interaction of RuvB with DNA.

Bacterial Proteins↗
PubMed1993

Branch migration of Holliday junctions promoted by the Escherichia coli RuvA and RuvB proteins. II. Interaction of RuvB with DNA.

Using recombination intermediates made by RecA protein, we have shown that the Escherichia coli RuvB protein can mediate the branch migration of Holliday junctions in vitro. The reaction is dependent on the presence of > or = 10 mM Mg2+ and stoichiometric amounts of RuvB. The presence of E. coli RuvA protein reduces the requirement for Mg2+ and also the stoichiometric requirement for RuvB (Müller, B., Tsaneva, I. R., and West, S. C. (1993) J. Biol. Chem. 268, 17179-17184). To determine the roles of the two proteins during branch migration, we have investigated the interaction of RuvB with DNA in the absence or presence of RuvA, by (i) gel retardation of protein-DNA complexes, (ii) stimulation of the RuvB ATPase, and (iii) protection of DNA from DNase I. The interaction of RuvB with duplex DNA was Mg(2+)-dependent and correlated with the Mg2+ requirement of the RuvB-mediated branch migration reaction. RuvB also interacted with ssDNA, but the affinity was significantly lower than for duplex DNA. In contrast to RuvB, the interaction of RuvA with duplex DNA occurred in the absence of Mg2+ and was inhibited by Mg2+ in a concentration-dependent manner. At 5 mM Mg2+, RuvA protein facilitated the interaction of RuvB with DNA, leading to the formation of a complex containing RuvA, RuvB, and duplex DNA.

Bacterial Proteins↗
PubMed1993

Formation of a RuvAB-Holliday junction complex in vitro.

The ruvA and ruvB genes of Escherichia coli encode a novel DNA helicase that interacts with Holliday junctions and promotes branch migration. In this work, we have investigated the protein-DNA complexes formed between RuvA, RuvB and Holliday junctions. As shown previously, RuvA protein binds a synthetic Holliday junction in vitro, to form a specific protein-DNA complex that can be detected by a band-shift assay. We now show that the combined presence of RuvA and RuvB results in a super-shift of this complex indicative of the formation of a RuvAB-Holliday junction complex. In the absence of RuvA, the RuvB protein fails to bind Holliday junctions. The RuvAB-Holliday junction complex was detected by the band-shift assay only under conditions that favoured its stability, e.g. complex formation in the presence of a nucleoside triphosphate that can not be hydrolysed by RuvB (adenosine 5'-[gamma-thio]triphosphate). In contrast, nucleoside triphosphates that can be hydrolysed (ATP, dATP, dCTP or TTP), lead to RuvAB-mediated branch migration of the junction. These results indicate that the formation of a (RuvAB-ATP)-Holliday junction complex represents the first step in the process of branch migration, and that branch migration is dependent upon ATP hydrolysis. In addition, we show that Holliday junction DNA stimulates the ATPase activity of RuvAB to a greater extent than either single-stranded or linear duplex DNA.

Adenosine Triphosphatases↗
PubMed1993

RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro.

The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity. The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.

Amino Acid Sequence↗