Biomedical subjects

R J Bennett

Publications and source records attributed to R J Bennett.

At least 19 recordsLinked to original sources

PubMed2001

Association of yeast DNA topoisomerase III and Sgs1 DNA helicase: studies of fusion proteins.

The Sgs1 protein of the budding yeast Saccharomyces cerevisiae is a member of the RecQ DNA helicase family that includes the human Bloom, Werner, and Rothmund-Thompson syndrome proteins. The N-terminal region outside the central DNA helicase core of Sgs1, particularly the part containing the first 100 amino acid residues of the 1,447-residue protein, is known to be functionally important and has been implicated in Sgs1-DNA topoisomerase III (Top3) interaction. We show in this work that the functionality of a truncated Sgs1 lacking its N-terminal 106 residues can be restored by replacing the truncated region with Top3. Fusion of Top3 to a mutant Sgs1 with a Val-29 to Glu substitution, which interferes with Sgs1-Top3 interaction, similarly restores the functionality of the mutant Sgs1(V29E) protein. The Top3-Sgs1(Delta1-106) and Top3-Sgs1(V29E) fusion proteins behave like wild-type Sgs1 in complementing several aspects of the sgs1 phenotype, including the hypersensitivity of sgs1 cells to methyl methanesulfonate and hydroxyurea. Complementation by the fusion proteins required both the topoisomerase activity of Top3 and the helicase activity of the Sgs1 polypeptide. These results suggest that the sole function of the N-terminal 106 amino acid residues of Sgs1 is for Top3 binding, and that the coordinated actions of Sgs1 and Top3 are important in cellular processes such as the processing of DNA after exposure of cells to DNA-damaging agents.

Adenosine Triphosphatases↗

PubMed2000

Interaction between yeast sgs1 helicase and DNA topoisomerase III.

The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases that includes the human Bloom's syndrome and Werner's syndrome proteins. In this work, we report studies on the interaction between Sgs1 and DNA topoisomerase III in vitro and in vivo. Affinity chromatography experiments with various fragments of Sgs1, a 1447-amino acid polypeptide, suggested that its N-terminal one-fifth was sufficient for interaction with DNA topoisomerase III. Gel electrophoretic mobility shift assays also indicated that a fragment Sgs1(1-283), containing residues 1-283, inhibited the binding of DNA topoisomerase III to single-stranded DNA. A shorter protein fragment containing residues 1-107 also showed partial inhibition in these assays. Studies of a sgs1 top1 double mutant lacking both Sgs1 and DNA topoisomerase I showed that the slow growth phenotype of this double mutant is suppressed by expressing full-length Sgs1, but not Sgs1 without the N-terminal 107 amino acid residues. In sgs1 top3 cells devoid of DNA topoisomerase III, however, expression of full-length Sgs1 or Sgs1 lacking the N-terminal 107 amino acid residues has the same effect of reducing the growth rate of the double mutant. These in vitro and in vivo data indicate that Sgs1 and DNA topoisomerase III physically interact and that this interaction is physiologically significant.

Base Sequence↗

PubMed2000

Inhibition of unwinding of G-quadruplex structures by Sgs1 helicase in the presence of N,N'-bis[2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide, a G-quadruplex-interactive ligand.

N,N'-Bis[2-(1-piperidino)ethyl]-3,4,9,10-perylenetetracarboxylic diimide (PIPER), a perylene derivative, is a very potent and selective G-quadruplex DNA-interactive agent. It has been shown to inhibit DNA polymerase and telomerase by stacking externally to the G-tetrads in the G-quadruplex structures. Recently, we have demonstrated that this small molecule greatly accelerates the assembly of G-quadruplex structures in a cell-free system. In this report, we present data demonstrating that PIPER prevents the unwinding of G-quadruplex structures by yeast Sgs1 helicase. Sgs1 belongs to the RecQ DNA helicase family whose members include other G-quadruplex DNA unwinding helicases, such as human Bloom's syndrome and human Werner's syndrome helicases. PIPER specifically prevents the unwinding of G-quadruplex DNA but not duplex DNA by Sgs1. Competition experiments indicate that this inhibitory activity is due to the interaction of PIPER with G-quadruplex structures rather than the helicase itself. These results combined with previous studies suggest a possible mechanism of action for these G-quadruplex-interactive agents inside cells: they might induce G-quadruplex formation in G-rich regions on genomic DNA, stabilize these structures, and prevent them from being cleared by enzymes such as helicases. The G-quadruplex structures may, in turn, disrupt some critical cellular events such as DNA replication, transcription regulation, and telomere maintenance.

Anthracenes↗

PubMed2000

Development of a measure of workplace deviance.

The purpose of this research was to develop broad, theoretically derived measure(s) of deviant behavior in the workplace. Two scales were developed: a 12-item scale of organizational deviance (deviant behaviors directly harmful to the organization) and a 7-item scale of interpersonal deviance (deviant behaviors directly harmful to other individuals within the organization). These scales were found to have internal reliabilities of .81 and .78, respectively. Confirmatory factor analysis verified that a 2-factor structure had acceptable fit. Preliminary evidence of construct validity is also provided. The implications of this instrument for future empirical research on workplace deviance are discussed.

Adult↗

PubMed1999

Binding specificity determines polarity of DNA unwinding by the Sgs1 protein of S. cerevisiae.

Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ DNA helicase family which also includes the products of the human Bloom's syndrome and Werner's syndrome genes. We have studied the substrate specificity of a recombinant Sgs1 helicase (amino acid residues 400-1268 of the Sgs1 protein). Sgs1 shows a strong preference for binding branched DNA substrates, including duplex structures with a 3' single-stranded overhang and DNA junctions with multiple branches. Duplex DNA with a 5' rather than a 3' single-stranded tail is not recognized or unwound by Sgs1. DNase I and hydroxyl radical footprinting of the Sgs1-DNA complex shows that the protein binds specifically to the junction of a double-stranded DNA and its 3' overhang. Binding and unwinding of duplex DNA with a 3' overhang are much reduced if the backbone polarity of the 3' overhang is reversed in the junction region, but are unaffected if polarity reversal occurs four nucleotides away from the junction. These results indicate that the 3' to 5' polarity of unwinding by the recombinant Sgs1 protein is a direct consequence of the binding of the helicase to the single-stranded/double-stranded DNA junction and its recognition of the polarity of the single-stranded DNA at the junction. The recombinant Sgs1 also unwinds four-way junctions (synthetic Holliday junctions), a result that may be significant in terms of its role in suppressing DNA recombination in vivo.

Base Sequence↗

PubMed1999

The Saccharomyces cerevisiae Sgs1 helicase efficiently unwinds G-G paired DNAs.

The Saccharomyces cerevisiae Sgs1p helicase localizes to the nucleolus and is required to maintain the integrity of the rDNA repeats. Sgs1p is a member of the RecQ DNA helicase family, which also includes Schizo-saccharomyces pombe Rqh1, and the human BLM and WRN genes. These genes encode proteins which are essential to maintenance of genomic integrity and which share a highly conserved helicase domain. Here we show that recombinant Sgs1p helicase efficiently unwinds guanine-guanine (G-G) paired DNA. Unwinding of G-G paired DNA is ATP- and Mg2+-dependent and requires a short 3' single-stranded tail. Strikingly, Sgs1p unwinds G-G paired substrates more efficiently than duplex DNAs, as measured either in direct assays or by competition experiments. Sgs1p efficiently unwinds G-G paired telomeric sequences, suggesting that one function of Sgs1p may be to prevent telomere-telomere interactions which can lead to chromosome non-disjunction. The rDNA is G-rich and has considerable potential for G-G pairing. Diminished ability to unwind G-G paired regions may also explain the deleterious effect of mutation of Sgs1 on rDNA stability, and the accelerated aging characteristic of yeast strains that lack Sgs1 as well as humans deficient in the related WRN helicase.

Base Pairing↗

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

Purification and characterization of the Sgs1 DNA helicase activity of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae Sgs1 protein is a member of a family of DNA helicases that include the Escherichia coli RecQ protein and the products of human Bloom's syndrome and Werner's syndrome genes. To study the enzymatic characteristics of the protein, a recombinant Sgs1 fragment (amino acids 400-1268 of the 1447-amino acid full-length protein) was overexpressed in yeast and purified to near homogeneity. The purified protein exhibits an ATPase activity in the presence of single- or double-stranded DNA. In the presence of ATP or dATP, unwinding of duplex DNA or a DNA-RNA heteroduplex by the recombinant Sgs1 fragment was readily observed. Similar to the E. coli RecQ helicase, displacement of the DNA strand occurs in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. The efficiency of unwinding was found to correlate inversely with the length of the duplex region and was enhanced by the presence of E. coli single-stranded DNA-binding protein. In addition, the recombinant Sgs1 fragment was found to bind more tightly to a forked DNA substrate than to either single- or double-stranded DNA.

Adenosine Triphosphatases↗

PubMed1997

Ascorbic acid absorption in patients with systemic sclerosis.

OBJECTIVE: To investigate whether reduced circulating levels of ascorbic acid in patients with systemic sclerosis (SSc) are a result of malabsorption. METHODS: Eight patients with SSc, but with no evidence of bacterial overgrowth, and 8 healthy controls were recruited. On the first day of study, each subject was given orally an aliquot of [14C] ascorbic acid, which was then "flushed out" by oral intake of unlabeled ascorbic acid for the following 7 days. Plasma samples were collected at specified intervals and urine was collected continuously over the 8 day study period. [14C] content of plasma and urine were measured by scintillation counting. For each subject, a plasma [14C] decay curve was drawn. Each subject's ascorbic acid absorption was assessed using the area under the curve (AUC) and the apparent renal clearance (CLr[app]). Ascorbic acid intake was assessed using dietary history and food composition tables. RESULTS: There were no differences in the dietary intake of vitamin C (p = 0.16) and body mass indices (p = 0.91) between patients and controls. The plasma [14C] AUC and CLr(app) were similar between patients and controls [AUC patient mean (standard deviation, SD) = 37.1 (6.8), AUC control mean (SD) = 38.6 (9.9), p = 0.74; CLr(app) patient mean (SD) = 0.57 (0.24), CLr(app) control mean (SD) = 0.47 (0.27), p = 0.45]. CONCLUSION: There was no evidence of impaired absorption of ascorbic acid in patients with SSc without bacterial overgrowth compared to healthy controls.

Absorption↗

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↗

PubMed1995

Structural analysis of the RuvC-Holliday junction complex reveals an unfolded junction.

The RuvC protein of Escherichia coli is an endonuclease that specifically recognises and cleaves Holliday junctions during genetic recombination. The structure of the RuvC-Holliday junctions complex has been investigated by DNAse I footprinting and by gel electrophoretic analysis. We find that RuvC binds to the Holliday junction to form a complex that exhibits 2-fold symmetry, and in which the three-dimensional structure of the Holliday junction is altered to an unfolded form. This structure is observed in the absence or presence of divalent metal ions and differs from either the unfolded square or the folded stacked X-structures that have been observed with protein-free Holliday junctions. KMnO4 was used to probe the junction DNA upon binding by RuvC, and indicates that base-pairing at the crossover is disrupted within the RuvC-Holliday junction.

Bacterial Proteins↗

PubMed1995

RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands.

The RuvC protein of Escherichia coli resolves Holliday junctions during genetic recombination and the postreplicational repair of DNA damage. Using synthetic Holliday junctions that are constrained to adopt defined isomeric configurations, we show that resolution occurs by symmetric cleavage of the continuous (noncrossing) pair of DNA strands. This result contrasts with that observed with phage T4 endonuclease VII, which cleaves the pair of crossing strands. In the presence of RuvC, the pair of continuous strands (i.e., the target strands for cleavage) exhibit a hypersensitivity to hydroxyl radicals. These results indicate that the continuous strands are distorted within the RuvC/Holliday junction complex and that RuvC-mediated resolution events require protein-directed structural changes to the four-way junction.

Bacterial Proteins↗

PubMed1995

Structure of a multisubunit complex that promotes DNA branch migration.

The RuvA and RuvB proteins of Escherichia coli, which are induced in response to DNA damage, are important in the formation of heteroduplex DNA during genetic recombination and related recombinational repair processes. In vitro studies show that RuvA binds Holiday junctions and acts as a specificity factor that targets the RuvB ATPase, a hexameric ring protein, to the junction. Together, RuvA and RuvB promote branch migration, an ATP-dependent reaction that increases the length of the heteroduplex DNA. Electron microscopic visualization of RuvAB now provides a new insight into the mechanism of this process. We observe the formation of a tripartite protein complex in which RuvA binds the crossover and is sandwiched between two hexameric rings of RuvB. The Holliday junction within this complex adopts a square-planar structure. We propose a molecular model for branch migration, a unique feature of which is the role played by the two oppositely oriented RuvB ring motors.

Adenosine Triphosphate↗

PubMed1994

Genetic recombination in E. coli: RuvC protein cleaves Holliday junctions at resolution hotspots in vitro.

The E. coli RuvC protein resolves Holliday junctions during genetic recombination and postreplication repair. Using recombination intermediates made by RecA protein, we have identified specific "hotspots" for RuvC resolution. Characterization of these sites reveals a common tetranucleotide sequence, with the consensus 5'-A/TTT decreases G/C-3'. The correct orientation of the resolution site is required for cleavage. These observations suggest that the strand bias of this sequence will affect the outcome of recombinational crosses by directing resolution to either "patch" or "splice" recombinant products. Mutation of the consensus site in synthetic Holliday junctions abolishes or significantly reduces the efficiency of cleavage, although binding is unaffected, demonstrating that junction recognition and incision are biochemically separable events. We propose that efficient RuvC resolution requires the translocation of Holliday junctions to specific cleavage sites, thus providing a biochemical basis for the similar genetic defects observed in ruvA, ruvB, and ruvC mutants.

Bacterial Proteins↗

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

Abnormal intragastric distribution of food during gastric emptying in functional dyspepsia patients.

Although delayed gastric emptying is found in some patients with functional dyspepsia, there seems to be little relation between rate of emptying and symptoms. This study examined the hypothesis that food maldistribution rather than gastric stasis may equate to symptoms in such patients and used scintigraphic techniques to quantify the partition of gastric contents between proximal and distal stomach during gastric emptying. Eleven patients with functional dyspepsia characterised by chronic severe postprandial bloating without organic abnormality, and 12 healthy volunteers, ingested a standard meal labelled with technetium-99M (99mTc). Serial images of the gastric area in anterior and posterior projections were taken for 90 minutes, regions of interest for proximal, distal, and total stomach were defined, and activity time curves were derived from the geometric means of anterior and posterior counts. Total emptying in patients (median: 46 minutes; range: 30-76) was not significantly different from controls (45 minutes; 28-58) and only three showed delayed gastric emptying. In controls, food remained predominantly in the proximal half of the stomach after ingestion and then redistributed to the distal half. In the patients, however, initial activity in the proximal half after ingestion (48%; 40-65) was significantly lower (p < 0.05) than in controls (60%; 39-73) and distributed more fully to the distal half of the stomach with a peak distal activity (56%; 34-58), which was consistently higher than in controls (36%; 33-42) (p < 0.05). It is concluded that this subgroup of functional dyspepsia patients show abnormal intragastric distribution of food, independent of gastric emptying rate.

Adult↗

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↗

PubMed1989

Ascorbic acid absorption in Crohn's disease. Studies using L-[carboxyl-14C]ascorbic acid.

Total body pool and intestinal absorption of ascorbic acid were studied in 12 patients undergoing operation for Crohn's disease (six with fistulae and six without) and in six control patients undergoing operation for reasons other than Crohn's disease. L-[carboxyl-14C]Ascorbic acid, 0.19-0.40 megabecquerels (MBq), was given orally. After a period of equilibration, the labeled ascorbic acid was flushed out of the patient's body tissues using large doses of unlabeled ascorbic acid. Intestinal absorption of ascorbic acid, assessed from the total cumulative urinary 14C recovery, was found to be similar in patients with fistulizing Crohn's disease (73.9 +/- 8.45%), those without fistulas (72.8 +/- 11.53%), and in controls (80.3 +/- 8.11%). Total body pools of ascorbic acid, calculated using the plasma 14C decay curves, were similar in patients with Crohn's disease with fistulas (17.1 +/- 5.91 mg/kg), patients without fistulas (9.6 +/- 3.58 mg/kg), and in controls (13.3 +/- 4.28 mg/kg). The results indicate that ascorbic acid absorption is normal in patients with both fistulizing and nonfistulizing Crohn's disease. The results suggest that routine supplements of vitamin C are not necessary unless oral ascorbic acid intake is low.

Adult↗