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)

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma- diphosphate and 2'-deoxythymidine-5'-(alpha-methylphosphonyl)-beta, gamma- diphosphate have been synthesized. Both compounds are incorporated into DNA chains during catalysis by reverse transcriptases of human immunodeficiency (HIV) and avian myeloblastosis (AMV) viruses, DNA polymerase beta from rat liver, terminal deoxynucleotidyl transferase from calf thymus and (at a very low rate) is by E. coli DNA polymerase I, Klenow fragment. The first compound is a termination substrate while the second is capable of multiple incorporation into the DNA chains. For instance, reverse transcriptase catalysis resulted in the appearance of 8 residues of second compound. DNA polymerases alpha and epsilon from human placenta incorporated none of the above compounds into DNA chains, although an inhibition of DNA synthesis by both compounds was observed with all enzymes mentioned. The 3'----5'-exonuclease activity of DNA polymerase I, Klenow fragment, hydrolyzed DNA fragments containing phosphonomethyl internucleoside groups, while such DNA fragments were resistant to the E. coli exonuclease III.
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PMID:Formation of phosphonester bonds catalyzed by DNA polymerase. 137 65

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta, gamma-diphosphate (I) and 2'-deoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma-diphosphate (II) were synthesised. Reverse transcriptases of HIV and avian myeloblastosis virus, rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase and E. coli DNA polymerase I KF incorporated both compounds into the growing DNA chain, KF being the least effective. Compound I revealed termination substrate properties, but II was repeatedly incorporated into the DNA chain, for example, by HIV reverse transcriptase - up to 8 residues. Human placenta DNA polymerases alpha and epsilon incorporated neither I nor II into the DNA chain, although DNA synthesis, catalyzed by all the investigated enzymes, was inhibited in the presence of I or II and compound II was a more effective inhibitor then I. The DNA fragments containing alpha-phosphonomethyl groups were hydrolyzed by 3'----5' exonuclease of DNA polymerase I and not hydrolyzed by ExoIII from E. coli.
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PMID:[Formation of phosphonoester bonds, catalyzed by DNA polymerases]. 172 22

The antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine (iNDG) is phosphorylated by HSV1 thymidine kinase, and its phosphorylated products inhibit DNA polymerase activity. iNDG exists in two enantiomeric forms, each with a primary and a secondary hydroxyl; thus, a number of possibilities for preferential phosphorylation exist, which were explored in this study. HSV1 thymidine kinase phosphorylates the primary hydroxyl of both the R and the S isomers of iNDG. This was established by comparison with analogues in which either the primary or the secondary hydroxyl was replaced by fluorine or hydrogen and also by a study of the NMR spectrum of the monophosphate. GMP kinase phosphorylates the R and the S monophosphates to the respective diphosphates. Further phosphorylation, however, is much more efficient with the S than with the R isomer. Furthermore, (S)-iNDG triphosphate is a more potent inhibitor of HSV1 DNA polymerase than (R)-iNDG triphosphate. These differences in the biochemical specificities of the two isomers account for the observed higher antiviral potency of (S)-iNDG as compared to that of (R)-iNDG.
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PMID:Enzymatic phosphorylation of the antiherpetic agent 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine. 300 16

2'-Fluoro-5-methyl-beta-L-arabinofuranosyl uracil (L-FMAU) was discovered to have potent antiviral activity against hepatitis B virus (HBV). L-FMAU was more potent than its D-enantiomer and produced dose-dependent inhibition of the viral DNA replication in 2.2.15 cells (human HepG2 cells with the HBV genome), with a 50% inhibitory concentration of 0.1 microM. There was no inhibitory effect on HBV transcription or protein synthesis. In the 2.2.15 cell system, L-FMAU did not show any toxicity up to 200 microM, whereas the D-enantiomer was toxic, with a 50% inhibitory concentration of 50 microM. Repeated treatments of HepG2 cells with L-FMAU at a 1 microM concentration for 9 days did not result in any decrease in the total mitochondrial DNA content, suggesting that a mode of toxicity similar to that produced by 2',3'-dideoxycytidine is unlikely. Also at concentrations as high as 200 microM, L-FMAU did not adversely affect mitochondrial function as determined by lactic acid production by L-FMAU-treated hepatoma cells. L-FMAU was metabolized in the cells to its mono-, di-, and triphosphates, A dose-dependent inhibition of HBV DNA synthesis by L-FMAU triphosphate was observed in the DNA polymerase assays with isolated HBV particles, suggesting that the mode of action of this compound could involve viral polymerase. However, L-FMAU was not incorporated into the cellular DNA. Considering the potent inhibition of the viral DNA synthesis and the nontoxicity of L-FMAU towards the host DNA synthetic machinery, this compound should be further explored for development as asn anti-HBV drug.
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PMID:Inhibition of hepatitis B virus by a novel L-nucleoside, 2'-fluoro-5-methyl-beta-L-arabinofuranosyl uracil. 883 84

Fragmentation is a major factor limiting mass range and resolution in the analysis of DNA by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protonation of the nucleobase leads to base loss and backbone cleavage by a mechanism similar to the depurination reactions employed in the chemical degradation method of DNA sequencing. In a previous study [Tang,W., Zhu,L. and Smith,L.M. (1997) Anal. Chem ., 69, 302-312], the stabilizing effect of substituting the 24 hydrogen with an electronegative group such as hydroxyl or fluorine was investigated. These 24 substitutions stabilized the N-glycosidic linkage, blocking base loss and subsequent backbone cleavage. For such chemical modifications to be of practical significance, it would be useful to be able to employ the corresponding 24-modified nucleoside triphosphates in the polymerase-directed synthesis of DNA. This would provide an avenue to the preparation of 24-modified PCR fragments and dideoxy sequencing ladders stabilized for MALDI analysis. In this paper methods are described for the polymerase-directed synthesis of 24-fluoro modified DNA, using commercially available 24-fluoronucleoside triphosphates. The ability of a number of DNA and RNA polymerases to incorporate the 24-fluoro analogs was tested. Four thermostable DNA polymerases [Pfu (exo-), Vent (exo-), Deep Vent (exo-) and UlTma] were found that were able to incorporate 24-fluoronucleotides with reasonable efficiency. In order to perform Sanger sequencing reactions, the enzymes' ability to incorporate dideoxy terminators in conjunction with the 24-fluoronucleotides was evaluated. UlTma DNA polymerase was found to be the best of the enzymes tested for this purpose. MALDI analysis of enzymatically produced 24-fluoro modified DNA using the matrix 2,5-dihydroxy benzoic acid showed no base loss or backbone fragmentation, in contrast to the extensive fragmentation evident with unmodified DNA of the same sequence.
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PMID:2'-Fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry. 935 69

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.
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PMID:Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation. 1690 31

Adduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the "B-DNA" major groove, S is "stacked" into the helix with base-displacement, and W is "wedged" into the minor groove. Here, we conducted (19)F NMR, CD, T(m), and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B or S-conformers while the FAF-G:A mismatch ones preferred the B and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and T(m) results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller & Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA-protein interaction.
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PMID:Examination of the long-range effects of aminofluorene-induced conformational heterogeneity and its relevance to the mechanism of translesional DNA synthesis. 1721 58

The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3' OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.
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PMID:DNA polymerase beta fidelity: halomethylene-modified leaving groups in pre-steady-state kinetic analysis reveal differences at the chemical transition state. 1816 50

Deoxyinosine (dI) and deoxyxanthosine (dX) are both formed in DNA at appreciable levels in vivo by deamination of deoxyadenosine (dA) and deoxyguanosine (dG), respectively, and can miscode. Structure-activity relationships for dA pairing have been examined extensively using analogs but relatively few studies have probed the roles of the individual hydrogen-bonding atoms of dG in DNA replication. The replicative bacteriophage T7 DNA polymerase/exonuclease and the translesion DNA polymerase Sulfolobus solfataricus pol IV were used as models to discern the mechanisms of miscoding by DNA polymerases. Removal of the 2-amino group from the template dG (i.e., dI) had little impact on the catalytic efficiency of either polymerase, as judged by either steady-state or pre-steady-state kinetic analysis, although the misincorporation frequency was increased by an order of magnitude. dX was highly miscoding with both polymerases, and incorporation of several bases was observed. The addition of an electronegative fluorine atom at the 2-position of dI lowered the oligonucleotide T(m) and strongly inhibited incorporation of dCTP. The addition of bromine or oxygen (dX) at C2 lowered the T(m) further, strongly inhibited both polymerases, and increased the frequency of misincorporation. Linear activity models show the effects of oxygen (dX) and the halogens at C2 on both DNA polymerases as mainly due to a combination of both steric and electrostatic factors, producing a clash with the paired cytosine O2 atom, as opposed to either bulk or perturbation of purine ring electron density alone.
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PMID:Steric and electrostatic effects at the C2 atom substituent influence replication and miscoding of the DNA deamination product deoxyxanthosine and analogs by DNA polymerases. 1960 42

Beta,gamma-fluoromethylene analogues of nucleotides are considered to be useful mimics of the natural substrates, but direct structural evidence defining their active site interactions has not been available, including the influence of the new chiral center introduced at the CHF carbon, as in beta,gamma-fluoromethylene-dGTP, which forms an active site complex with DNA polymerase beta, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. We report X-ray crystallographic results for a series of beta,gamma-CXY dGTP analogues, where X,Y = H, F, Cl, Br, and/or CH(3). For all three R/S monofluorinated analogues examined (CHF, 3/4; CCH(3)F, 13/14; CClF 15/16), a single CXF-diastereomer (3, 13, 16) is observed in the active site complex, with the CXF fluorine atom at a approximately 3 A (bonding) distance to a guanidinium N of Arg183. In contrast, for the CHCl, CHBr, and CHCH(3) analogues, both diasteromers (6/7, 8/9, 10/11) populate the dGTP site in the enzyme complex about equally. The structures of the bound dichloro (5) and dimethyl (12) analogue complexes indicate little to no steric effect on the placement of the bound nucleotide backbone. The results suggest that introduction of a single fluorine atom at the beta,gamma-bridging carbon atom of these dNTP analogues enables a new, stereospecific interaction within the preorganized active site complex that is unique to fluorine. The results also provide the first diverse structural data set permitting an assessment of how closely this class of dNTP analogues mimics the conformation of the parent nucleotide within the active site complex.
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PMID:Halogenated beta,gamma-methylene- and ethylidene-dGTP-DNA ternary complexes with DNA polymerase beta: structural evidence for stereospecific binding of the fluoromethylene analogues. 2046 17


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