PubMed Health⌕ Search

Biomedical subjects

S K Bryan

Publications and source records attributed to S K Bryan.

14 recordsLinked to original sources
PubMed1995

Inactivation of the 5'-3' exonuclease of Thermus aquaticus DNA polymerase.

The gene for Thermus aquaticus (Taq) DNA polymerase enzyme (Taq Pol I) was mutagenized and sixty-two candidate clones were screened for enzyme activity. Two of the clones expressed enzymes (*Taq-3 and *Taq-5) that showed very reduced 5'-3' exonuclease activity and normal DNA polymerase activity. These two enzymes showed heat resitance and storage stability similar to Taq Pol I and had similar effectiveness in PCR. Processivity of the polymerases was compared by measuring the extension of an end-labeled primer annealed to a single stranded DNA, as well as by a PCR method. The processivity of *Taq-3 and *Taq-5 was similar to Taq Pol I (50-80 nucleotides) and more processive than a Taq Pol I deficient in the 5'-3' exonuclease due to absence of the first 290 amino acids (Stoffel fragment). The results indicate two amino acids which are required for normal 5'-3' exonuclease activity in Taq Pol I (Arg-25 and Arg-74).

Cloning, Molecular↗
PubMed1995

Discriminative stimulus effects of cocaine: antagonism by dopamine D1 receptor blockade in the amygdala.

Mesolimbic dopamine (DA) D1 and D2 receptors appear to be involved in mediating the discriminative stimulus effects of cocaine. The purpose of the present study was to investigate the role of the amygdala DA D1 receptors, in modulating the stimulus effects of cocaine. Thus, rats were trained to discriminate cocaine (10 mg/kg, IP) from saline using a two-lever, water-reinforced FR 20 drug discrimination task. In substitution tests, systemic (IP) administration of cocaine (0.625-20 mg/kg) produced a dose-related increase in cocaine-lever responding. Intracranial bilateral injections of cocaine (20-200 micrograms, total dose) into the central amygdala engendered, at best, a partial substitution (< 60% drug-lever responding) for the systemic cocaine cue. Central amygdala microinjections of artificial cerebrospinal fluid (ACSF; 1 microliter/side) or SCH 23390 (0.5-2 microgram, total dose) resulted in primarily saline-appropriate responding. In antagonism tests, bilateral injections of the DA D1 receptor antagonist SCH 23390 (0.5-2 microgram, total dose) into the central amygdala produced a dose-related blockade of a systemic dose of cocaine (5 mg/kg) that engendered > 85% cocaine-lever responding when given alone. Additionally, bilateral injection of a fixed dose of SCH 23390 (2 micrograms) into the central amygdala resulted in a rightward shift in the cocaine dose-response curve (2.5-20 mg/kg). Although administration of cocaine into the central amygdala does not mimic the systemic cocaine cue, the present results demonstrate that DA D1 receptors located within the central amygdala appear to have a modulatory role upon the discriminative stimulus properties of cocaine.

Amygdala↗
PubMed1991

Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits.

DNA polymerase III of Escherichia coli requires multiple auxiliary factors to enable it to serve as a replicative complex. We demonstrate that auxiliary components of the DNA polymerase III holoenzyme, the gamma delta complex and beta subunit, markedly stimulate DNA polymerase II on long single-stranded templates. DNA polymerase II activity is enhanced by single-stranded DNA binding protein, but the stimulation by gamma delta and beta can be observed either in the absence or presence of single-stranded DNA binding protein. In contrast with DNA polymerase III, the requirement of DNA polymerase II for gamma delta cannot be bypassed by large excesses of the beta subunit at low ionic strength in the absence of the single-stranded DNA binding protein. The product of the DNA polymerase II-gamma delta-beta reaction on a uniquely primed single-stranded circle is of full template length; the reconstituted enzyme apparently is incapable of strand displacement synthesis. The possible biological implications of these observations are discussed.

DNA Polymerase II↗
PubMed1990

Holoenzyme DNA polymerase III fixes mutations.

DNA polymerase III is required for mutagenesis after damage to the chromosome. This effect is not modulated by the presence or absence of DNA polymerase II activity in the cell. In cells containing a temperature-sensitive dnaE mutation, the alpha-subunit of DNA polymerase III is inactivated at the restrictive temperature, resulting in lethality. Cells containing the pcbA1 mutation can continue replication if DNA polymerase I activity is present. When such cells are shifted from the permissive to the restrictive temperature, mutagenesis decreases rapidly after 10 min. These results are compatible with conversion of the replicative apparatus from one containing a functional DNA polymerase III synthetic subunit to one containing DNA polymerase I. We also find that DNA polymerase I dependent replication is markedly sensitive to coumermycin A1. We conclude that DNA polymerase III holoenzyme with the alpha-subunit is required for fixing mutations in the genome.

Aminocoumarins↗
PubMed1989

Cloning the polB gene of Escherichia coli and identification of its product.

Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDa protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.

Antigen-Antibody Complex↗
PubMed1989

Suppression of dnaE nonsense mutations by pcbA1.

DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli. The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene. We have reported that E. coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present. Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations. We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C). Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype. We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C.

Blotting, Western↗
PubMed1987

DNA polymerase III of Escherichia coli is required for UV and ethyl methanesulfonate mutagenesis.

Strains of Escherichia coli possessing the pcbA1 mutation, a functional DNA polymerase I, and a temperature-sensitive mutation in DNA polymerase III can survive at the restrictive temperature (43 degrees C) for DNA polymerase III. The mutation rate of the bacterial genome of such strains after exposure to either UV light or ethyl methanesulfonate was measured by its rifampicin resistance or amino acid requirements. In addition, Weigle mutagenesis of preirradiated lambda phage was also measured. In all cases, no increase in mutagenesis was noted at the restrictive temperature for DNA polymerase III. Introduction of a cloned DNA polymerase III gene returned the mutation rate of the bacterial genome as well as the Weigle mutagenesis to normal at 43 degrees C. Using a recA-lacZ fusion, the SOS response after UV irradiation was measured and found to be normal at the restrictive and permissive temperature for DNA polymerase III, as was induction of lambda prophage. Recombination was also normal at either temperature. Our studies demonstrate that a functional DNA polymerase III is strictly required for mutagenesis at a step other than SOS induction.

DNA Polymerase III↗
PubMed1987

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.

Alleles↗
PubMed1984

Map location of the pcbA mutation and physiology of the mutant.

Many temperature-resistant revertants of a polA1 polB polCts (HS432) strain are PolI+ (by either suppression of the polA1 amber allele or intragenic reversion) but remain polCts (contain a temperature-sensitive DNA polymerase III). It appears that DNA replication in such temperature-resistant revertants depends on an extragenic mutation, pcbA, already present in the parent strain and not linked to any of the DNA polymerase loci. This allele allows DNA replication dependent on DNA polymerase I and bypasses a temperature-sensitive DNA polymerase III (polC bypass), so that reversion to PolI+ makes the strain temperature resistant. This pathway of DNA replication also supports phage and plasmid DNA replication. At restrictive temperature, these mutants display a normal response to UV irradiation but show increased sensitivity to the alkylating agent methyl methanesulfonate. We have located pcbA linked to dnaA.

Bacterial Proteins↗
PubMed1981

Alternate pathways of DNA replication: DNA polymerase I-dependent replication.

We have previously shown that some Escherichia coli [derivatives of strain HS432 (polA1, polB100, polC1026)] can replicate DNA at a restrictive temperature in the presence of a polCts mutation and that such revertants contain apparent DNA polymerase I activity. We demonstrate here that this strain of E. coli becomes temperature-resistant upon the introduction of a normal gene for DNA polymerase I or suppression of the polA1 nonsense mutation. Such temperature-resistant phenocopies become temperature-sensitive upon introduction of a temperature-sensitive DNA polymerase I gene. Our results confirm that DNA replication is DNA polymerase I-dependent in the temperature-resistant revertants, indicating that an alternative pathway of replication exists in E. coli. HS432 contains a transducible locus (which we term pcbA) that can support an alternate pathway in other E. coli strains, so the effect of suppression of polCts is a general one.

DNA Polymerase I↗
PubMed1979

Replication at restrictive temperatures in Escherichia coli containing a polCts mutation.

Escherichia coli cells with a polCts mutation contain a temperature-sensitive DNA polymerase II and fall to replicate DNA at the restrictive temperature (43 degreees C). Mutants deficient in polymerizing activity of the other two recognized DNA polymerases in E. coli can replicate DNA. We have isolated temperature-resistant revertants from a strain containing polA-, polB-, and polCts mutations. These revertants grow at 43 degrees C, but analysis of partially purified DNA polymerase III from several such revertants shows a temperature-sensitive DNA polymerase III activity. Genetic analysis by P1 transduction confirms that such revertants can contain a polCts mutation and also a polA- mutation. We find that such revertants behave phenotypically as PolI+ cells (DNA polymerase I-containing), and extracts of such cells show a DNA polymerase I-like activity. Revertants of polA-, dnaAts and polA-, dnaBts strains do not show such a DNA polymerase activity.

DNA Polymerase I↗