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S C West

Publications and source records attributed to S C West.

142 records · Page 8Linked to original sources
PubMed1981

Genetic recombination: recA protein promotes homologous pairing between duplex DNA molecules without strand unwinding.

The recA protein of Escherichia coli promotes pairing in vitro between covalent circular duplex DNA and homologous circular duplex DNA containing a single stranded region. We have used a filter binding assay to investigate the frequency of homologous pairing between gapped and intact duplex DNA when unwinding of the free 3' and 5' ends of the gapped molecules was blocked. In order to obtain DNA without free ends, the gapped DNA was treated with trimethylpsoralen and 360 nm light so as to introduce about 6 crosslinks per DNA molecule and the double stranded regions on either side of the gaps were then digested up to the first crosslinks with exonuclease III and lambda exonuclease. This treatment did not diminish the frequency of homologous pairing, an observation which is difficult to reconcile with models for recombination requiring strand unwinding before pairing.

Bacterial Proteins↗
PubMed1981

Homologous pairing can occur before DNA strand separation in general genetic recombination.

In the presence of ATP and Mg2+, purified Escherichia coli recA protein promotes the formation of joint molecules between closed circular duplex DNA and homologous circular single-stranded DNA carrying a short annealed fragment. The presence of this fragment is essential for pairing between molecules. In similar conditions recA protein is unable to act as a helicase and does not cause strand separation of the fragment from the single-stranded circle. Thus, homologous pairing between DNA molecules can take place without prior unwinding of a free end.

Bacterial Proteins↗
PubMed1981

Heteroduplex formation by recA protein: polarity of strand exchanges.

Purified recA protein promotes strand exchanges between linear duplex DNA and homologous circular single-stranded phage phi X174 DNA that carries a short hybridized fragment [West, S. C., Cassuto, E. & Howard-Flanders, P. (1981) Proc. Natl. Acad. Sci. USA 78, 2100-2104]. In this paper we investigate the mechanism of this strand exchange reaction. We show that recA protein initiates strand exchanges by pairing the free end of the duplex fragment with the single-stranded DNA. In addition, we find that strand exchanges are polar, stable heteroduplex molecules being formed by the directional transfer transfer of the (-) strands starting at 3' termini.

Bacterial Proteins↗
PubMed1981

recA protein promotes homologous-pairing and strand-exchange reactions between duplex DNA molecules.

Purified recA protein, product of the recA+ gene, promotes homologous pairing between intact covalent circular duplex DNA and circular single-stranded DNA carrying a short hybridized fragment [West, S. C., Cassuto, E. & Howard-Flanders, P. (1981) Nature (London) 290, 29-33.]. In this paper we investigate the interaction of duplex fragments with circular single-stranded DNA carrying the hybridized fragment and find that recA protein promotes an efficient strand-exchange reaction between interacting DNA molecules. The exchange is dependent upon linear duplex DNA fragments that are homologous to, but extend beyond, the short fragment present on the hybridized DNA substrate. The reactions require stoichiometric amounts of recA protein and the presence of ATP.

Bacterial Proteins↗
PubMed1980

Recognition of duplex DNA containing single-stranded regions by recA protein.

Genetic recombination in Escherichia coli requires recA protein, the product of the recA+ gene. In this paper we show that purified recA protein, which binds strongly to denatured DNA, cooperatively recognizes DNA containing short single-stranded regions. The interaction of varying amounts of recA protein with DNA molecules was investigated by measuring its DNA-dependent ATPase activity. In 3mM Mg2+, the ATPase activity was stimulated by excess single-stranded DNA and was minimal with either intact circular or blunt-ended linear duplexes. Single-strand gaps of about 30 nucleotides were sufficient to increase the ATPase activity to a level almost as great as that observed with single-stranded DNA. Sedimentation studies at neutral pH showed cooperative binding of recA protein to single-stranded DNA or to duplex DNA containing single-stranded regions. In the presence of ATP, an intermediate rate of sedimentation was observed; in contrast, adenosine 5'-gamma-thiotriphosphate (ATP[S]) caused the formation of fast-sedimenting DNA-protein complexes. Gapped plasmid DNA plus recA protein and ATP[S] formed large aggregates containing thousands of molecules. Complex formation and stimulation of the ATPase activity of recA protein with duplex DNA containing single-stranded regions indicates that recA protein may change the conformation of the normally duplex molecules to a conformation prepared for homologous pairing.

Adenosine Triphosphatases↗
PubMed1980

Initiation of genetic recombination: homologous pairing between duplex DNA molecules promoted by recA protein.

recA protein has been shown to promote hydrogen bonding between single-stranded DNA fragments and duplex DNA molecules homologous to them. However, genetic and biochemical evidence indicates that genetic exchanges generally take place between duplex molecules. We therefore chose to study the interactions promoted by recA protein between intact duplex DNA molecules and molecules containing gaps that are believed to increase the frequency of genetic exchanges. In the present paper, we show that incubation of intact and gap-containing plasmid DNA in the presence of recA protein leads to homologous pairing between duplex molecules which can be detected by centrifugation analysis and electron microscopy. The reaction is completely dependent on an active recA gene product, on genetic homology between the DNA species involved, and on the presence of ATP; under certain conditions, its efficiency can be increased considerably by the presence of the single-stranded DNA binding protein of Escherichia coli.

Adenosine Triphosphatases↗
PubMed1980

A proposed neurophysiological basis of alexithymia.

The possibility that alexithymia respresents a symptom of subtle brain dysfunction is discussed. Several lines of research have shown that emotional thought, behavior and, in particular, the use of affect-laden speech are influenced by impaired function of the right hemisphere or a disconnection between the two hemispheres. A patient with agenesis of the corpus callosum is presented demonstrating severe alexithymia.

Adult↗
PubMed1977

Induction of protein synthesis in Escherichia coli following UV- or gamma-irradiation, mitomycin C treatment or tif Expression.

The rate of synthesis of total cellular proteins has been studied by pulse labelling cells at various periods after irradiation with UV or gamma-rays, after treatment with mitomycin C (MMC) or after expression of the temperature sensitive mutation tif. Subsequent gel electrophoresis and autoradiography reveals changes in the rate of synthesis of several proteins. The most striking change is in a protein of molecular weight 40,000, protein X, which has been previously most extensively studied in cells treated with nalidixic acid (Gudas, 1976). Synthesis of large quantities of protein X is induced by UV, gamma-rays, MMC treatment or tif expression in rec+ but not recA cells. A feature of recA cells is that they break down their DNA excessively after irradiation or MMC treatment. However, if protein synthesis following irradiation is prohibited by chloramphenicol, post-irradiation degradation becomes excessive in recA+ cells. This inverse relationship between DNA degradation and new protein synthesis consistent with the hypothesis that an induced protein such as X is responsible for controlling DNA degradation following irradiation. Protein X is not induced in a lexB mutant following MMC treatment. In this respect the lexB mutant behaves like lexA and recA mutants in that the ability to induce protein X can be correlated with excessive DNA degradation. Studies on the induction of proteins in inf, tif and tif sfi mutants fail to reveal any correlation between induction of protein X and either the induction of prophage lambda or septation.

Bacterial Proteins↗
PubMed1975

recA+-dependent inactivation of the lambda repressor in Escherichia coli lysogens by gamma-radiation and by tif expression.

When gamma lysogens of E. coli are induced by gamma-radiation the gamma repressor, as measured by its specific binding to gamma DNA, is rapidly inactivated by a recA+-dependent process which does not require new protein synthesis. This rapid inactivation is similar to inactivation of repressor by expression of the temperature sensitive E. coli mutation tif. In contrast, induction by UV irradiation or mitomycin C treatment requires new protein synthesis and there is a lag before the repressor is inactivated (Tomizawa and Ogawa, 1967; Shinagawa and Itoh, 1973).

Coliphages↗
PubMed1975

Single dose metyrapone test: 11 beta-hydroxylase inhibition by metyrapone and reduced metyrapone assayed by radioimmunoassay.

To assess the effects of metyrapone and reduced metyrapone on 11 beta-hydroxylase inhibition, the plasma levels of cortisol, 11-deoxycortisol, and the inhibitors were measured by radioimmunoassays in 34 normal subjects 8 h after they received a single oral dose of metyrapone at midnight. The ratio of 11-deoxycortisol to cortisol, as an index of 11 beta-hydroxylase inhibition, was compared to plasma levels of metyrapone and reduced metyrapone. One subject received an infusion of metyrapone ditartrate in order to study the sequential conversion of metyrapone to reduced metyrapone. A new radioimmunoassay was developed for measurement of plasma concentrations of metyrapone and reduced metyrapone. Following intravenous administration of metyrapone, it is rapidly converted to an active metabolite, reduced metyrapone. At 8 h after a dose was given, the average reduced metyrapone level was 1.5 times higher than the average metyrapone level. Following oral administration of the drug, we found a high correlation when plasma levels of metyrapone were compared to reduced metyrapone and when the ratio of 11-deoxycortisol to cortisol was related to metyrapone or to total metyrapone levels. In conclusion, the conversion of metyrapone to reduced metyrapone is such that by 8 hours after a single oral dose, more than one-half of the inhibitory effect on 11 beta-hydroxylase appears to be produced by reduced metyrapone. The inhibitory action of metyrapone and reduced metyrapone on the enzyme system is reflected by their concentration in plasma.

Humans↗
PubMedDate not supplied

Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins.

The recombination of DNA molecules has been reconstituted in vitro using two purified enzymes from Escherichia coli. RecA protein catalyses homologous pairing and strand exchange reactions to form intermediate DNA structures that are acted upon by RuvC. The newly identified RuvC protein resolves the intermediates by specific endonucleolytic cleavage to produce recombinant DNA molecules.

Bacterial Proteins↗