Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The conformations and binding site environments of Mg2+TTP and Mg2+dATP bound to Escherichia coli DNA polymerase I and its large (Klenow) fragment have been investigated by proton NMR. The effect of the large fragment of Pol I on the NMR line widths of the protons of Mg2+TTP detected one binding site for this substrate with a dissociation constant of 300 +/- 100 microM and established simple competitive binding of deoxynucleoside triphosphates at this site in accord with previous equilibrium dialysis experiments with whole Pol I [Englund, P. T., Huberman, J.A., Jovin, T.M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038]. Primary negative nuclear Overhauser effects were used to calculate interproton distances on enzyme-bound Mg2+dATP and Mg2+TTP. These distances established that each substrate was bound with an anti-glycosidic torsional angle (chi) of 50 +/- 10 degrees for Mg2+dATP and 40 +/- 10 degrees for Mg2+TTP. The sugar pucker of both substrates was predominantly O1'-endo, with a C5'-C4'-C3'-O3' exocyclic torsional angle (delta) of 95 +/- 10 degrees for Mg2+dATP and 100 +/- 10 degrees for Mg2+TTP. The consistency of these conformations with those previously proposed, on the basis of distances from Mn2+ at the active site [Sloan, D. L., Loeb, L. A., Mildvan, A.S., & Feldman, R.J. (1975) J. Biol. Chem. 250, 8913], indicates a unique conformation for each bound nucleotide. The chi and delta values of the bound substrates are appropriate for nucleotide units of B DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nuclear Overhauser effect studies of the conformations and binding site environments of deoxynucleoside triphosphate substrates bound to DNA polymerase I and its large fragment. 390 5

(Sp)-2'-Deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate has been synthesized by desulfurization of (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1,1-18O2]diphosphate) with N-bromosuccinimide in [17O]water, followed by phosphorylation with phosphoenolpyruvate-pyruvate kinase. A careful characterization of the product using high-resolution 31P NMR revealed that the desulfurization reaction proceeded with approximately 88% direct in-line attack at the alpha-phosphorus and 12% participation by the beta-phosphate to form a cyclic alpha,beta-diphosphate. The latter intermediate underwent hydrolysis by a predominant nucleophilic attack on the beta-phosphate. This complexity of the desulfurization reaction, however, does not affect the stereochemical integrity of the product but rather causes a minor dilution with nonchiral species. The usefulness of the (Sp)-2'-deoxyadenosine 5'-O-[1-17O,1-18O,1,2-18O]triphosphate in determining the stereochemical course of deoxyribonucleotidyl-transfer enzymes is demonstrated by using it to delineate the stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. Upon incubation of this oxygen-chiral substrate with Klenow fragment of DNA polymerase I in the presence of poly[d(A-T)] and Mg2+, a quantitative conversion into 2'-deoxyadenosine 5'-O-[16O,17O,18O]monophosphate was observed. The stereochemistry of this product was determined to be Rp. Since the overall template-primer-dependent conversion of a deoxynucleoside triphosphate into the deoxynucleoside monophosphate involves incorporation into the polymer followed by excision by the 3'----5'-exonuclease activity and since the stereochemical course of the incorporation reaction is known to be inversion, it can be concluded that the stereochemical course of the 3'----5'-exonuclease is also inversion.
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PMID:Stereochemical course of the 3'----5'-exonuclease activity of DNA polymerase I. 609 2

T4 DNA polymerase converts (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O2]triphosphate) to 2'-deoxyadenosine 5'-O-[18O]-phosphorothioate in the presence of poly(d(A-T).poly(d(A-T)) template-primer. Control experiments involving either omitting the poly(d(A-T)).poly(d(A-T) template-primer or employing the (Rp)-2'-deoxyadenosine 5'-O-(1-thiotriphosphate) diastereomer showed no reaction. It is assumed, therefore, that this conversion as in the P--O case involves incorporation of the thionucleotide into the poly(d(A-T)) followed by hydrolysis resulting from the 3' goes to 5'-exonuclease activity. The 2'-deoxyadenosine 5'-O-[18O] phosphorothioate was converted to (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O]triphosphate), with no change in the configuration at P alpha by using the coupled adenylate kinase-pyruvate kinase enzyme system. A 31P NMR spectrum of the product showed that the 18O was entirely in the nonbridging position, indicating an overall retention in the net turnover process (i.e. incorporation followed by excision). Since the incorporation process involves an inversion of configuration around the phosphorus (Romaniuk, P. J., and Eckstein, F. (1982) J. Biol. Chem. 257, 7684-7688), it must be inferred that the 3' goes to 5'-exonuclease activity of T4 polymerase proceeds with inversion of configuration at the phosphorus atom, most simply via a direct displacement mechanism. This finding represents the first example of phosphodiester hydrolysis catalyzed by an exonuclease that does not involve a covalent phosphoryl-enzyme intermediate (Knowles, J. R. (1980) Annu. Rev. Biochem. 49, 877-919).
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PMID:Template-prime-dependent turnover of (Sp)-dATP alpha S by T4 DNA polymerase. The stereochemistry of the associated 3' goes to 5'-exonuclease. 628 51

N2-(p-n-Butylphenyl)-2'-deoxyguanosine (BuPdG) and its 5'-triphosphate (BuPdGTP), expected to be inhibitors of eukaryotic DNA polymerase alpha, have been synthesized. BuPdG was synthesized by two methods and characterized by 1H NMR and by chemical relation to guanosine. Direct synthesis involving silylated N2-(p-n-butylphenyl)guanine (BuPG) and 1-chloro-3,5-di-p-toluoyl-2-deoxyribofuranose in the presence of trimethylsilyl trifluoromethanesulfonate gave one alpha and two beta isomers of deoxyribonucleoside as determined by 1H NMR. However, NMR and UV spectra were equivocal in distinguishing between 7 and 9 isomers. The identity of the desired 9-beta-BuPdG was ultimately proved by its independent synthesis from the corresponding ribonucleoside. 1H NMR spectra of the O'-acetylated ribonucleosides of BuPG showed characteristic patterns of O'-acetylated guanosines, and their identity was proved by relating the products of the reaction of isomeric O'-acetylated 2-bromoinosines with p-n-butylaniline and with ammonia: the 2-bromoinosine which gave guanosine also gave the suspected 9-beta-ribonucleoside, BuPGr, and that which gave N7-beta-ribofuranosylguanine also gave the 7-beta isomer of BuPGr. BuPGr was transformed in a multistep procedure to give BuPdG, identical with the major beta isomer obtained by direct deoxynucleoside synthesis. The 5'-monophosphate of BuPdG was obtained by treatment of the nucleoside with phosphoryl chloride in trimethyl phosphate; the monophosphate reacted as the phosphoimidazolyl derivative with pyrophosphate to yield the 5'-triphosphate, BuPdGTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and characterization of N2-(p-n-butylphenyl)-2'-deoxyguanosine and its 5'-triphosphate and their inhibition of HeLa DNA polymerase alpha. 669 66

An inhibitor of alkaline phosphodiesterase was isolated from a soil Streptomyces. The agent was identified with 2-crotonyloxymethyl-4,5,6-trihydroxycylohex-2-enone (COTC) by UV, IR, 1H HMR and 13C NMR spectrometry. The mechanism of tumor-inhibitory action of COTC was studied with murine lymphoblastma L5178Y cells. COTC blocked alkaline phosphodiesterase; IC 50 was 60 micrograms/ml by the method employed. The growth of L5178Y cells was inhibited by COTC; IC50 was 4.4 micrograms/ml. DNA biosynthesis was preferentially prevented by COTC over RNA and protein syntheses; IC50 of DNA synthesis was 7 or approximately 25 micrograms/ml. COTC significantly inhibited DNA polymerase alpha even in the presence of dithiothreitol. The mitosis was markedly blocked by COTC; complete inhibition was observed at a drug concentration of 20 microgram/ml. Adriamycin-, aclarubicin- and bleomycin-resisant cell subline showed collateral sensitivity to COTC. COTC and aclarubicin exhibited synergistic activity on aclarubicin-resistant cells, but not on the parental cells. COTC increased uptake of [3H]adriamycin or blocked the drug efflux in the resistance cells, but not in the parental cells. The effects of COTC on macromolecular syntheses, mitosis and membrane functions may be attributed to the interaction with the sulfhydryl group of various enzymes. Although COTC is multifunctional drug, the inhibition of DNA polymerase alpha and a certain mitotic process seems to be related to the lethal action.
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PMID:Mechanism of action of 2-crotonyloxymethyl-4,5,6-trihydroxycyclohex-2-enone, a SH inhibitory antitumor antibiotic, and its effect on drug-resistant neoplastic cells. 714 23

A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.
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PMID:NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides. 772 21

We have shown that deoxycytidine-5'-triphosphate modified by O-(4-aminobutyl)hydroxylamine in the pyrimidine ring, is effectively incorporated into DNA synthesizing in vitro, replacing deoxythymidine-5'-triphosphate or deoxycytidine-5'-triphosphate and inducing A-->G and G-->A transitions, respectively. UV spectroscopy and NMR spectroscopy have shown that the modified cytidine-5'-triphosphate is identical to N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate. When the modified deoxycytidine-5'-triphosphate was inserted into DNA in vitro by DNA polymerase I of E. coli Klenow fragment, retardation sites correlating with poly-A sites (when the modified triphosphate replaced deoxythymidine-5'-triphosphate) or with poly-G sites (when it replaced deoxycytidine-5'-triphosphate) were revealed. Our data show high mutagenic effect of the modified deoxycytidine-5'-triphosphate inserted into DNA, allowing us to recommend this compound for localized static mutagenesis.
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PMID:[The mutagenic activity of N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate]. 778 36

Irradiation of d(GTATTATG) with 254 nm light gave rise to four major photoproducts, two of which were readily identified by NMR as the cis-syn cyclobutane dimer and the (6-4) photoproduct of the central TT site. Analysis of the NMR data for the other two photoproducts indicated that they were not any of the other known photoproducts of a TT site and might be TA* photoproducts [Bose, S. N., et al. (1983) Science 220, 723-725]. In support of this possibility, the fluorescence spectra of the products of acid hydrolysis of the two photoproducts were very similar to that reported for the hydrolysis product of the TA* photoproduct of TpdA. Only one of the two TA*-containing octamers could be ligated at both ends to form a 49-mer oligonucleotide in the presence of a complementary oligonucleotide scaffold, suggesting that the TA* photoproduct had formed between T5 and A6. The position of the TA* photoproduct was confirmed by mapping the arrest sites for 3'-->5' exonucleolytic degradation of the 49-mer by T4 DNA polymerase and for primer extension opposite the 49-mer by exonuclease deficient Klenow fragment (KF) and Sequenase Version 2.0. The TA* product could also be bypassed by both polymerases, but it was less of a block to KF. Treatment with 1 M aqueous piperidine at 100 degrees C led to a maximum of about 34% cleavage of the DNA at the site of the TA* product.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Preparation and characterization of a deoxyoligonucleotide 49-mer containing a site-specific thymidylyl-(3',5')-deoxyadenosine photoproduct. 782 86

The mechanisms by which DNA polymerases achieve their remarkable fidelity, including base selection and proofreading, are briefly reviewed. Nine proofreading models from the current literature are evaluated in the light of steady-state and transient kinetic studies of E. coli DNA polymerase I, the best-studied DNA polymerase. One model is demonstrated to predict quantitatively the response of DNA polymerase I to three mutagenic probes of proofreading: exogenous pyrophosphate, deoxynucleoside monophosphates, and the next correct deoxynucleoside triphosphate substrate, as well as the response to combinations of these probes. The theoretical analysis allows elimination of many possible proofreading mechanisms based on the kinetic data. A structural hypothesis links the kinetic analysis with crystallographic, NMR and genetic studies. It would appear that DNA polymerase I proofreads each potential error twice, at the same time undergoing two conformational changes within a catalytic cycle. Multi-stage proofreading is more efficient, and may be utilized in other biological systems as well. In fact, recent evidence suggests that fidelity of transfer RNA charging may be ensured by a similar mechanism.
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PMID:Multi-stage proofreading in DNA replication. 802 69

DNA polymerase beta consists of an N-terminal single-stranded DNA binding domain and a C-terminal catalytic domain separable by mild proteolysis [Kumar et al. (1990) J. Biol. Chem. 265, 2124-2131]. The N-terminal domain participates in template and gapped DNA recognition and contributes significantly to catalysis. The secondary structure and tertiary contacts within the cloned N-terminal domain (residues 2-87) of mammalian DNA polymerase beta have been determined using multidimensional NMR. Assignments of backbone 1H, 15N, and 13C resonances and side chain 1H and 13C resonances have been obtained from double- and triple-resonance 3D NMR experiments. The 13C-edited TOCSY experiment has allowed nearly complete assignments of 1H and 13C resonances within side chains. The 13C-edited NOESY experiment has been used for determination of medium- and long-range NOEs and a determination of tertiary contacts. The N-terminal domain is found to consist of four helices, helix-1 (15-26), helix-2 (36-47), helix-3 (56-61), and helix-4 (69-78), which on the basis of long-range NOEs are tightly packed of form a hydrophobic core. The remainder of the domain consists of two turns (48-51 and 62-65), an omega-type loop (27-35), and extended structure. The aromatic side chains of Y36, Y39, Y49, and F76 display tertiary contacts indicative of at least partial hydrophobic packing. The S30 and H34 residues which cross-link to single-stranded DNA [Prasad et al. (1993) J. Biol. Chem. 268, 15906-15911] are contained within the K27-K35 loop.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Assignments of 1H, 15N, and 13C resonances for the backbone and side chains of the N-terminal domain of DNA polymerase beta. Determination of the secondary structure and tertiary contacts. 806 28


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