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N C Brown

Publications and source records attributed to N C Brown.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1988

Isolation and molecular characterization of a highly polymorphic centromeric tandem repeat in the family Falconidae.

An abundant tandem repeat has been cloned from genomic DNA of the merlin (Falco columbarius). The cloned sequence is 174 bp in length, and maps by in situ hybridization to the centromeric regions of several of the large chromosomes within the merlin karyotype. Complementary sequences have been identified within a variety of falcon species; these sequences are either absent or in very low copy number in the family Accipitridae. The cloned merlin repeat reveals highly polymorphic restriction patterns in the peregrine falcon (Falco peregrinus). These polymorphisms, which have been shown to be stably inherited within a family of captive peregrines, can be used to differentiate the Greenland and Argentina populations of this endangered raptor species.

Animals↗
PubMed1987

Mammalian DNA polymerase alpha: a replication-competent holoenzyme form from calf thymus.

Calf thymus DNA polymerase alpha, like the replication-specific DNA polymerase III holoenzyme of Escherichia coli, can be isolated as a distinct complex. A specific multiprotein form of the polymerase alpha, a form designated replication-competent (RC) holoenzyme, consists of a complex of a polymerase-primase core and at least six other polypeptides. The RC holoenzyme can efficiently replicate several naturally occurring templates, including the genomic DNA of the porcine circovirus (PCV). The DNA of this virion consists of a single-stranded circle with a defined replication origin, and its replication requires the cellular DNA replication machinery. It might therefore provide an invaluable opportunity to investigate chromosomal replication mechanisms, analogous to the way that studies on E. coli bacteriophage DNA replication elucidated host DNA replication mechanisms. Calf RC holoenzyme alpha selectively initiates PCV DNA replication in vitro at a site that possibly represents a consensus sequence of cellular DNA replication origins. The cell-free PCV replication system will be exploited for the in vitro dissection and reconstitution of the RC holoenzyme and the functional analysis of its component polypeptides.

Animals↗
PubMed1987

Synthesis, cell growth inhibition, and antitumor screening of 2-(p-n-butylanilino)purines and their nucleoside analogues.

Derivatives of N2-(p-n-butylphenyl)guanine (BuPG) and 2-(p-n-butylanilino)adenine (BuAA) were synthesized and tested as inhibitors of mammalian DNA polymerase alpha, cell growth, and macromolecule synthesis. 2-(p-n-Butylanilino)-6-chloropurine (BuACl) served as a useful intermediate to prepare a series of 6-substituted analogues. BuACl, as its sodium salt, reacted with 2-deoxy-3,5-di-p-toluoyl-beta-D-ribofuranosyl chloride in acetonitrile to give 64% of the corresponding 9-beta nucleoside (blocked BuAdCl) and only 14% of the 7-beta isomer. Deblocking and substitution of chlorine in BuAdCl generated a series of 2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl)purine derivatives. Reaction of the sodium salt of BuACl with (2-acetoxyethoxy)methyl bromide also afforded, after deblocking and substitution of the 6-chloro group, a series of 2-(p-n-butylanilino)-9-[(2-hydroxyethoxy)methyl]purines. The bases synthesized were inhibitors of DNA polymerase alpha isolated from Chinese hamster ovary cells, the most potent compounds being 6-methoxy and 6-methylthio derivatives of 2-(p-n-butylanilino)purine. When tested for their ability to inhibit [3H]thymidine incorporation into DNA in HeLa cell cultures and the growth of exponentially growing HeLa cells, 9-(2-deoxy-beta-D-ribofuranosyl) derivatives had greater potency than their base counterparts, but "adenine" analogues, such as 2-(p-n-butylanilino)-2'-deoxyadenosine (BuAdA, IC50 = 1 microM), were considerably more potent than N2-(p-n-butylphenyl)-2'-deoxyguanosine (BuPdG, IC50 = 25 microM). Derivatives bearing the 9-[(2-hydroxyethoxy)methyl] group were nearly as potent inhibitors of [3H]thymidine incorporation in these experiments as the corresponding deoxyribonucleosides. Base and deoxynucleoside derivatives also inhibited cellular RNA synthesis, and several compounds, at high concentrations, inhibited protein synthesis. BuPG, BuAA, and four deoxyribonucleoside derivatives of 2-(p-n-butylanilino)purines were tested against P-388 lymphocytic leukemia in mice. None of the compounds increased the survival time of test animals, but two of them, BuAdA and its 6-desamino derivative BuAdP, were lethal at the highest concentration used (400 mg/kg).

Aniline Compounds↗
PubMed1986

Protein properties of the subunits of ribonucleotide reductase and the specificity of the allosteric site(s).

Ribonucleotide reductase catalyzes the critical reaction in which the deoxyribonucleotides required for DNA replication are synthesized de novo. This enzyme consists of two non-identical protein subunits, both of which are required for enzymatic activity. These subunits consist of a non-heme iron and an effector-binding subunit. These subunits are not coordinately regulated as the cells pass from G1 to the S phase of the cell cycle. Studies carried out with the holoenzyme and the isolated subunits indicate that the effector-binding subunit is more susceptible to chymotrypsin and the sulfhydryl reagents, pCMB and NEM, than is the non-heme iron subunit. The non-heme iron subunit is more susceptible to trypsin than is the effector-binding subunit. The presence of ATP or dATP protects the effector-binding subunit from proteolysis by either trypsin or chymotrypsin. The loss of activity in the holoenzyme, as a result of proteolysis, parallels the loss of the particular subunit. These results demonstrate that the protein properties of the subunits are significantly different to account for the differential turnover. The binding of nucleotides to the effector-binding site(s), which in turn regulates ribonucleotide reductase activity, is very specific. Formycin 5'-triphosphate and etheno-ATP could not replace ATP in the CDP reductase reaction. 2',3'-DideoxyATP was 5-fold less active than dATP as a negative effector; etheno-dATP was not inhibitory. AraGTP and BuPdGTP could not replace dGTP as a positive effector of ADP reduction. BuPdGTP, but not araGTP, served as an inhibitor of CDP reduction. 2',3'-DideoxyTTP was much less active as either an activator of GDP reduction or an inhibitor of ADP reduction. These studies indicate that the binding to the allosteric sites is highly specific and suggest that the structural requirements for the binding of activators are different from the structural requirements for the binding of inhibitors.

Adenine Nucleotides↗
PubMed1986

Inhibitors of Bacillus subtilis DNA polymerase III. Influence of modifications in the pyrimidine ring of anilino- and (benzylamino)pyrimidines.

Substituent effects governing inhibition of DNA polymerase III from Bacillus subtilis were examined in several series of N6-substituted 6-aminopyrimidines. The presence of alkyl groups as large as n-butyl in the 3-position of 6-(5-indanylamino)uracil had no effect on inhibitor-enzyme binding. Substituents in the 4-position of a series of 2-amino-6-(benzylamino)pyrimidines had complex effects: alkoxy and phenoxy derivatives were less active than the parent 4-oxo (isocytosine) compound, but alkylphenoxy and halophenoxy derivatives were more active than the 4-phenoxy compound itself, suggesting that hydrophobic binding can occur between 4-substitutents and the enzyme surface and that space between the pyrimidine ring and pol III may represent the active site of the enzyme. Replacement of 5-H by methyl and ethyl groups drastically decreased inhibitory activity of 6-(benzylamino)- and 6-p-toluidinouracils, but 5-bromo and 5-iodo analogues were equipotent with the parent compounds. These results indicate that the phenyl rings of these compounds must exist in conformations in which they are perpendicular to the pyrimidine ring plane and that charge-transfer stabilization of such "active conformations" may compensate for steric barriers from 5-halo groups in the inhibitor-enzyme complex.

Amines↗
PubMed1986

Cloning and characterization of the polC region of Bacillus subtilis.

The polC gene of Bacillus subtilis is defined by five temperature-sensitive mutations and the 6-(p-hydroxyphenylazo)-uracil (HPUra) resistance mutation azp-12. Biochemical evidence suggests that polC codes for the 160-kilodalton DNA polymerase III. A recombinant plasmid, p154t, was isolated and found to contain the azp-12 marker and one end of the polC gene (N. C. Brown and M. H. Barnes, J. Cell. Biochem. 78 [Suppl.]: 116, 1983). The azp-12 marker was localized to a 1-kilobase DNA segment which was used as a probe to isolate recombinant lambda phages containing polC region sequences. A complete polC gene was constructed by in vitro ligation of DNA segments derived from two of the recombinant phages. The resulting plasmid, pRO10, directed the synthesis of four proteins of 160, 76, 39, and 32 kilodaltons in Escherichia coli maxicells. Recombination-deficient (recE) B. subtilis PSL1 containing pRO10 produced an HPUra-resistant polymerase III activity which was lost when the strain was cured of pRO10. In vivo, the HPUra resistance of the plasmid-encoded polymerase III appeared to be recessive to the resident HPUra-sensitive polymerase III enzyme.

Bacillus subtilis↗
PubMed1986

Rational design of substrate analogues targeted to selectively inhibit replication-specific DNA polymerases.

The authors' approach to the design of DNA polymerase-specific inhibitors, an approach based on the mechanism of action of 6-(p-hydroxyphenylazo)uracil, has been to disguise nucleic acid bases to mimic the purine substrates dGTP and dATP. Specifically, the strategy has been to synthesize bases with substituents that endow them with the capacity: to seek and react with unique features of the active site of a polymerase; and to form H bonds with complementary template pyrimidines apposing the active site. This strategy has yielded a series of novel, enzyme-specific dATP and dGTP analogues which are non-polymerizable and which inhibit their target polymerase by sequestering it to a complementary pyrimidine residue in primer:template. The work has involved primarily two replication-specific polymerases, B. subtilis DNA polymerase III (pol III) and mammalian DNA polymerase alpha (pol alpha). The initial design exploited the pyrimidine nucleus and produced inhibitors with Ki values in the micromolar range. Principles established with the pyrimidine derivatives have led to the development of bona fide purine nucleotide analogues which act as DNA polymerase inhibitors of high selectivity and unprecedented potency. For example, BuPdGTP, the 2'-deoxyribonucleoside 5'-triphosphate of N2-(p-n-butylphenyl)guanine (BuPG), lacks discernible activity against mammalian polymerases beta and gamma, whereas it inhibits mammalian pol alpha with a Ki of less than 10 nanomolar. Currently, the authors are exploiting BuPdGTP, BuPdGDP, and similar butylanilino derivatives of dATP to probe the active site of pol alpha and to develop other N2-substituted analogues which can bind selectively to the substrate sites of other important polymerases and nucleotide binding proteins.

Animals↗
PubMed1985

Elucidation of the mechanism of selective inhibition of mammalian DNA polymerase alpha by 2-butylanilinopurines: development and characterization of 2-(p-n-butylanilino)adenine and its deoxyribonucleotides.

2-(p-n-Butylanilino)adenine (BuAA), an homolog of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n-butylphenyl)guanine (BuPG), was transformed to its 2'-deoxyribonucleoside, BuAdA, and the corresponding 2'-deoxyribonucleoside 5'-phosphates, BuAdAMP, BuAdADP, and BuAdATP. All five forms of BuAA are highly selective inhibitors of mammalian pol alpha, and the action of each is subject to specific competitive antagonism by dATP. BuAdADP, and BuAdATP, like the corresponding forms of BuPG, are very potent pol alpha inhibitors, displaying apparent Ki's of less than 3 nanomolar on natural activated templates. BuAdATP, like BuPdGTP, also inhibits pol alpha-catalysed reactions directed by non-complementary, thymine-deficient templates, and it does so via a mechanism subject to specific antagonism by its natural homolog, dATP. The results of the BuAdATP-homopolymer experiments complement those of analogous experiments with BuPdGTP and the dCTP-specific pol alpha inhibitor, aphidicolin, and strengthen the suggestion that mammalian pol alpha contains dNDP and dNTP binding sites which can recognize specific bases without direction by templates.

Adenine↗
PubMed1985

Inhibition of RNA-directed DNA polymerase from avian myeloblastosis virus by a 5-benzyl-6-aminouracil.

5-(p-Chlorobenzyl)-6-aminouracil (5-ClAU) inhibited RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus. Inhibition was expressed only in the presence of a polyribonucleotide template such as mRNA or poly(rA), and kinetic analysis suggested that the action of 5-ClAU is competitive with template:primer. 5-ClAU did not inhibit HeLa DNA polymerase gamma, an enzyme that efficiently copies polyribonucleotide templates. A mechanism is proposed in which a 5-ClAU:template complex interferes with enzyme function, based partly on NMR studies indicating that 5-ClAU can form a hydrogen-bonded complex with deoxyadenosine in solution.

Animals↗
PubMed1985

Purification and characterization of DNA polymerase alpha of Chinese hamster ovary cells.

The major pol alpha activity of CHO cells was purified 2 800-fold to near homogeneity and was characterized with respect to its physical and catalytic properties. The purified enzyme, upon analysis in denaturing 'activity' gels, displayed a major, 120 kilodalton, catalytically active core and two minor, catalytically inactive components of 180 and 135 kilodaltons. The native form of the enzyme behaved in velocity sedimentation and gel permeation experiments as an asymmetric protein of an apparent Mr. of 515 kilodaltons. The purified enzyme displayed catalytic behavior and inhibitor sensitivity typical of that displayed by other mammalian pol alphas. Specifically, the enzyme: was sensitive to n-ethylmaleimide and the pol alpha-specific inhibitors, BuPdGTP and aphidicolin; was subject to neutralization by specific monoclonal antibodies raised against human pol alpha; was devoid of detectable 3' to 5' exonuclease activity, and displayed a ribonucleotide-dependent DNA primase activity.

Animals↗