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N C Brown

Publications and source records attributed to N C Brown.

At least 19 recordsLinked to original sources
PubMed1995

Characterization and overexpression of the gene encoding Staphylococcus aureus DNA polymerase III.

The polC gene specifying DNA polymerase III (PolIII) of Staphylococcus aureus (Sa), was cloned with a novel strategy and found to contain a 4305-bp open reading frame (ORF) encoding a polypeptide of approx. 162 kDa. The 1435-codon ORF was engineered into an Escherichia coli (Ec) expression plasmid under the control of the lac promoter and its repressor. Derepression of Ec transformants carrying the recombinant (re-) vector generated high-level synthesis of active re-Sa PolIII. The re-PolIII was purified to > 98% homogeneity and was shown by N-terminal amino acid sequence analysis to be the bona fide product of the Sa polC ORF. The physical and catalytic properties of re-Sa PolIII and its responsiveness to inhibitors of the HPUra type were generally similar to those of Bacillus subtilis (Bs) PolIII. Comparative analysis of the primary structures of Sa PolIII, Bs PolIII and Mycoplasma pulmonis PolIII indicated strong conservation of essential catalytic domains and a novel zinc-finger motif. Comparison of the primary structures of Ec PolIII and these three Gram+ enzymes revealed a region of novel homology and reinforced the likelihood of a specific evolutionary relationship between PolIII of Gram+ and Gram- eubacteria. The polC gene mapped between omega 1074 [Tn551] and recA/ngr on the Sa NCTC 8325 genome.

Base Sequence↗
PubMed1995

The 3'-5' exonuclease site of DNA polymerase III from gram-positive bacteria: definition of a novel motif structure.

The primary structure of the 3'-5' exonuclease (Exo) site of the Gram+ bacterial DNA polymerase III (Pol III) was examined by site-directed mutagenesis of Bacillus subtilis Pol III (BsPol III). It was found to differ significantly from the conventional three-motif substructure established for the Exo site of DNA polymerase I of Escherichia coli (EcPol I) and the majority of other DNA polymerase-exonucleases. Motifs I and II were conventionally organized and anchored functionally by the predicted carboxylate residues. However, the conventional downstream motif, motif III, was replaced by motif III epsilon, a novel 55-amino-acid (aa) segment incorporating three essential aa (His565, Asp533 and Asp570) which are strictly conserved in three Gram+ Pol III and in the Ec Exo epsilon (epsilon). Despite its unique substructure, the Gram+ Pol III-specific Exo site was conventionally independent of Pol, the site of 2'-deoxyribonucleoside 5-triphosphate (dNTP) binding and polymerization. The entire Exo site, including motif III epsilon, could be deleted without profoundly affecting the enzyme's capacity to polymerize dNTPs. Conversely, Pol and all other sequences downstream of the Exo site could be deleted with little apparent effect on Exo activity. Whether the three essential aa within the unique motif III epsilon substructure participate in the conventional two-metal-ion mechanism elucidated for the model Exo site of EcPol I, remains to be established.

Amino Acid Sequence↗
PubMed1994

DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC.

Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorously investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.

Amino Acid Sequence↗
PubMed1992

Properties of the nuclear P1 protein, a mammalian homologue of the yeast Mcm3 replication protein.

Polyclonal antibodies were raised against a multiprotein 'holoenzyme' form of calf thymus DNA polymerase alpha-primase and used to probe a human cDNA-protein expression library constructed in the lambda gt11 vector. The probe identified a series of cDNA clones derived from a 3.2 kb mRNA which encodes a novel 105 kDa polypeptide, the P1 protein. In intact cells, the P1 protein was specifically associated with the nucleus, and in cell extracts, it was associated with complex forms of DNA polymerase alpha-primase. The synthesis of human P1-specific mRNA was stimulated upon addition of fresh serum to growth-arrested cells, and RNA blot analyses with the human P1-cDNA probe indicated that P1 is encoded by a strictly conserved mammalian gene. The amino acid sequence deduced from a 240-codon open reading frame resident in the largest human P1-cDNA (0.84 kb) displayed greater than 96% identity with that deduced from the equivalent segment of a 795-codon open reading frame of a larger mouse P1-cDNA (2.8 kb). Throughout its length, the primary structure of mammalian P1 displayed strong homology with that of Mcm3, a 125 kDa yeast protein thought to be involved in the initiation of DNA replication (Gibson et al. 1990. Mol. Cell. Biol. 10: 5707-5720). The P1-Mcm3 homology, the strong conservation of P1 among mammals, its nuclear localization, and its association with the replication-specific DNA polymerase alpha strongly suggest an important role of the P1 protein in the replication of mammalian DNA.

Amino Acid Sequence↗
PubMed1992

Localization of the exonuclease and polymerase domains of Bacillus subtilis DNA polymerase III.

Structural gene mutants were cloned and exploited to identify the major catalytic domains of Bacillus subtilis DNA polymerase III (BsPolIII), a 162.4-kDa [1437 amino acids (aa)] polymerase: 3'-5' exonuclease (Exo) required for replicative DNA synthesis. Analysis of the sequence, mutagenicity, and catalytic behavior of natural and site-directed point mutants of BsPolIII unequivocally located the domain involved in exonuclease catalysis within a 155-aa residue segment displaying homology with the Exo domain of Escherichia coli DNA polymerase I. Sequence analysis of four structural gene mutations which specifically alter then enzyme's reactivity to the inhibitory dGTP analog, 6-(p-hydroxyphenylhydrazino)uracil, and the inhibitory arabinonucleotide, araCTP, defined a domain (Pol) involved in dNTP binding. The Pol domain was in the C-terminal fourth of the enzyme within a 98-aa segment spanning aa 1175-1273. The primary structure of the domain was unique, displaying no obvious conservation in any other DNA polymerase, including the distantly related PolIIIs of the Gram- organisms, E. coli and Salmonella typhimurium.

Amino Acid Sequence↗
PubMed1992

Overproduction and purification of Bacillus subtilis DNA polymerase III.

The objectives of this work were to engineer the cloned polC gene encoding Bacillus subtilis DNA polymerase III for controlled overexpression in Escherichia coli and to devise a facile purification scheme permitting the large-scale production of pure recombinant polymerase. The translational signals of polC were restructured by expression cassette PCR (MacFerrin et al., 1990, Proc. Natl. Acad. Sci. USA 87, 1937-1941), and the modified gene was inserted into the expression plasmid, pKC30 (Rosenberg et al., 1983, in "Methods in Enzymology," Vol. 101, pp. 123-138, Academic Press, San Diego), under the strict control of the coliphage lambda pL promoter and its repressor, cI. When the system was derepressed at 32 degrees C, soluble DNA polymerase III accumulated at levels approximating 2% of total cellular protein. The recombinant protein was purified to greater than 99% purity by utilizing a tandem combination of Cibacron blue agarose, phenyl-Sepharose, and MonoQ FPLC chromatography. The properties of the purified recombinant protein were indistinguishable from those of native B. subtilis DNA polymerase III.

Bacillus subtilis↗
PubMed1991

The molecular mechanism of inhibition of alpha-type DNA polymerases by N2-(butylphenyl)dGTP and 2-(butylanilino)dATP: variation in susceptibility to polymerization.

Calf thymus DNA polymerase alpha (pol alpha) and bacteriophage T4 DNA polymerase (pol T4) were exploited as model enzymes to investigate the molecular mechanism of inhibitory action of N2-(p-n-butylphenyl)dGTP (BuPdGTP) and 2-(p-n-butyl-anilino)dATP (BuAdATP) on the BuPdNTP-susceptible alpha polymerase family. Kinetic analysis of inhibition of pol alpha with mixtures of complementary and noncomplementary template:primers indicated that both nucleotides induced the formation of a polymerase: inhibitor:primer-template complex. Primer extension experiments using the guanine form as the model analog indicated that pol alpha cannot utilize these nucleotides to extend primer termini. In contrast, pol T4 polymerized BuPdGTP, indicating that resistance to polymerization is not a common feature of the inhibitor mechanism among the broad membership of the alpha polymerase family.

Adenosine Triphosphate↗
PubMed1991

Bacillus subtilis DNA polymerase III: complete sequence, overexpression, and characterization of the polC gene.

Genomic DNA encompassing polC, the structural gene specifying Bacillus subtilis DNA polymerase III (PolIII), was sequenced and found to contain a 4311-bp open reading frame (ORF) encoding a 162.4-kDa polypeptide of 1437 amino acids (aa). The ORF was engineered into an Escherichia coli expression plasmid under the control of the coliphage lambda repressor. Derepression of E. coli transformants carrying the recombinant vector resulted in the high-level synthesis of a recombinant DNA polymerase indistinguishable from native PolIII. N-terminal aa sequence analysis of the recombinant polymerase unequivocally identified the 4311-bp ORF as that of polC. Comparative aa sequence analysis indicated significant homology of the B. subtilis enzyme with the catalytic alpha subunit of the E. coli PolIII and, with the exception of an exonuclease domain, little homology with other DNA polymerases. The respective sequences of the mutant polC alleles, dnaF and ts-6, were identified, and the expression of specifically truncated forms of polC was exploited to assess the dependence of polymerase activity on the structure of the enzyme's C terminus.

Alleles↗
PubMed1991

Use of sex-linked minisatellite fragments to investigate genetic differentiation and migration of North American populations of the peregrine falcon (Falco peregrinus).

The M13 repeat detects different levels of genetic variation in falcons. First, this minisatellite probe reveals typically highly variant restriction fragments that show no apparent unequal distribution between the sexes. Secondly, the M13 repeat detects sets of fragments that are only present in DNAs from female falcons. The level of polymorphism displayed by the sex-linked fragments is greatly reduced relative to most autosomal minisatellites. In addition, the size of these fragments (in kilobase pairs) is species-specific among Mauritius kestrels (Falco punctatus) and peregrines (Falco peregrinus). Variation observed at one o of the sex-linked fragments in peregrines has proven to be useful in distinguishing a subset of the tundrius subspecies of this endangered raptor. This correlation has enabled a genetic test to be used to examine the representation of tundrius peregrines during mass migration.

Animals↗
PubMed1990

Development of novel inhibitor probes of DNA polymerase III based on dGTP analogs of the HPUra type: base, nucleoside and nucleotide derivatives of N2-(3,4-dichlorobenzyl)guanine.

6-(p-Hydroxyphenylhydrazino)uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific class III DNA polymerase (pol III) of Gr+ bacteria. Although formally a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. We describe the successful conversion of the H2-HPUra inhibitor prototype to a bona fide purine, using N2-(benzyl)guanine (BG) as the basis. Structure-activity relationships of BGs carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl)guanine (DCBG) as a nucleus equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2'-deoxyribonucleoside 5'-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of B. subtilis DNA pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization.

Base Sequence↗
PubMed1990

(Difluoromethylene)phosphates of guanine nucleosides as probes of DNA polymerases and G proteins.

5'-Polyphosphates of N2-(p-n-butylphenyl)-2'-deoxyguanosine and -guanosine which contain a difluoromethylene group in place of a phosphoanhydride oxygen have been synthesized. 5'-[beta,gamma-(Difluoromethylene)triphosphates], including that of 2'-deoxyguanosine, were prepared by reaction of the corresponding 5'-phosphates, activated by 1,1'-carbonyldiimidazole, with difluoromethanediphosphonate. The 5'-[(difluoromethylene)diphosphate] of N2-(p-n-butylphenyl)guanosine was prepared by treatment of a protected 5'-tosyl nucleoside with difluoromethanediphosphonate, followed by deprotection. Condensation of this nucleotide, activated with 1,1'-carbonyldiimidazole, with orthophosphate gave N2-(p-n-butylphenyl)guanosine 5'-[(alpha,beta-difluoromethylene)triphosphate]. Products were characterized by 31P and 19F NMR spectroscopy. The phosphonates were tested for their ability to displace [3H]GDP from the GTP binding proteins cellular (EC) and oncogenic (Leu-61) Ha-ras p21, and for their ability to inhibit DNA polymerase alpha from Chinese hamster ovary cells. The p21s bound weakly to a triphosphonate when the CF2 group was in the beta,gamma position, but not when it was in the alpha,beta position, and they did not bind to the corresponding (difluoromethylene)diphosphate. In contrast, the CF2 group had no effect on inhibition of DNA polymerase alpha by N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphospate]. 2'-Deoxyguanosine 5'-[(beta,gamma-difluoromethylene)triphosphate] was found to be a bona fide substrate for several DNA polymerases and had a lower apparent Km than dGTP with Bacillus subtilis DNA polymerase III.

Bacillus subtilis↗
PubMed1990

Deoxyribonucleotide analogs as inhibitors and substrates of DNA polymerases.

Inhibitory and substrate properties of analogs of deoxyribonucleoside triphosphates toward DNA polymerases are reviewed. A general introduction is followed by a description of DNA polymerases and the reaction that they catalyze, and sites at which substrate analogs may inhibit them. Effects of modifications in the major family of compounds, nucleotide derivatives, at the base, sugar and triphosphate portions of the molecule, are summarized with respect to retention of substrate properties and generation of inhibitory properties. Structure-activity relationships and the basis of selectivity in the second family of compounds, deoxyribonucleotide mimics, are also presented. Conclusions are drawn regarding the structural basis of inhibitor selectivity and mechanism, relationship between in vitro and in vivo effects of inhibitors, and the promise of inhibitors as probes for study of active sites of DNA polymerases.

Animals↗
PubMed1989

The cloned polC gene of Bacillus subtilis: characterization of the azp12 mutation and controlled in vitro synthesis of active DNA polymerase III.

Wild type (wt) Bacillus subtilis polC and polCazp12, a mutant derivative specifying a form of DNA polymerase III resistant to hydroxyphenylazopyrimidines, were cloned as genomic fragments approximating the length required to encode the entire polymerase. The cloned DNA fragments were subjected to restriction and partial sequence analysis to locate the 5' end of the polC-specific coding sequence and the azp12 mutation, which was identified as a T----G transversion specifying replacement of serine with alanine. The cloned wt and azp12-coding sequences were recloned in an Escherichia coli expression vector with their respective 5' ends under the control of the bacteriophage lambda PL promoter and cIts857-encoded repressor. In response to induction, the wt- and azp12-specific recombinant plasmids expressed active DNA polymerases indistinguishable from the native enzymes derived from the respective B. subtilis hosts.

Amino Acid Sequence↗
PubMed1989

Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta.

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.

Animals↗
PubMed1988

Calcium-dependent calmodulin-binding proteins associated with mammalian DNA polymerase alpha.

Complex, multiprotein forms of bovine (calf thymus), hamster (Chinese hamster ovary cell), and human (HeLa) cell DNA polymerase alpha (Pol alpha) were analyzed for their content of calmodulin-binding proteins. The approach used an established autoradiographic technique employing 125I-labeled calmodulin to probe proteins in denaturing SDS-polyacrylamide gel electropherograms. All three Pol alpha enzymes were associated with discrete, Ca2+-dependent calmodulin-binding proteins. Conventionally purified calf thymus Pol alpha holoenzyme contained three prominent, trifluoperazine-sensitive species with apparent molecular masses of approx. 120, 80 and 48 kDa. The 120 and 48 kDa species remained associated with the polymerase.primase core of the calf enzyme during immunopurification with monoclonal antibodies directed specifically against the polymerase subunit. The patterns of the calmodulin-binding proteins displayed by conventionally purified preparations of hamster and human Pol alpha enzymes were similar to each other and distinctly different from the pattern of comparable preparations of calf thymus Pol alpha. Immunopurified preparations of the human and hamster Pol alphas retained significant calmodulin-binding activity of apparent molecular masses of approx. 55, 80 and 150-200 kDa.

Animals↗