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

---

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

During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, O.C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25 degrees C and to temperatures as high as 100 degrees C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.
...
PMID:Polyphosphate present in DNA preparations from filamentous fungal species of Colletotrichum inhibits restriction endonucleases and other enzymes. 838 89

2'-Deoxythymidine 5'-triphosphate and 2'-deoxyadenosine 5'-triphosphate analogs containing a methylene group between the alpha phosphorus and 5' oxygen were synthesized. The substrate properties of these compounds toward some mammalian DNA polymerases and retroviral reverse transcriptases were evaluated using a system containing phage M13mp10 DNA, a synthetic oligonucleotide, and the enzyme. The compounds containing a hydroxyl at the 3' position were incorporated into the DNA chain by DNA polymerase alpha and terminal deoxynucleotidyl transferase, but were not recognized by retroviral reverse transcriptases and mammalian DNA polymerases epsilon and beta. The selectivity of the compounds synthesized was capitalized on during simultaneous isolation of DNA polymerases alpha and epsilon from human placenta. A methylene group was also introduced into the acyclovir molecule. It was shown that this modification inactivates furanose-related nucleotide analogs, but has a minor effect on the substrate properties of acyclic nucleotide analogs.
...
PMID:[New nucleotide inhibitors of human DNA polymerase alpha]. 855 70

A high-resolution structure of a 16-nucleotide bacteriophage T4 RNA hairpin, 5'-GCCU[AAUAACUC]GGGC (loop bases in square brackets), has been determined in solution by proton, phosphorus, and carbon (natural abundance) NMR spectroscopy. This RNA hairpin is known to play a crucial role in the translational repression of bacteriophage T4 DNA polymerase. Ultraviolet absorbance melting curves indicate that the structure formed is unimolecular. The NMR spectra indicate that a single conformation consistent with a hairpin structure is formed. Strong imino-imino NOEs confirm the formation of the G.U base pair at the stem-loop junction. There is no evidence that A5 is protonated (at pH 6.0) and involved in an A+.C pair. However, the NMR data indicate that the stem is extended beyond the G.U pair and that A-form stacking continues for three nucleotides on the 5' side and one nucleotide on the 3' side. Structure calculations using restraints obtained from NMR data give a precisely defined structure with an average root mean square deviation (RMSD) of approximately 1.2 A for the entire molecule. The assignment of all the protons and most of the 31P resonances in the loop yielded a large number of distance and torsion angle restraints for these nucleotides. These helped obtain a well-defined loop with an average RMSD of 1.1 A for the loop nucleotides of 11 converged structures.
...
PMID:NMR structure of a bacteriophage T4 RNA hairpin involved in translational repression. 867 67

The solution structure of a 16-nucleotide RNA hairpin, 5'-GCCUAG[CAAC]CUGGGC (loop bases in square brackets), has been determined by proton, phosphorus, and carbon (natural abundance) nuclear magnetic resonance (NMR) spectroscopy. This RNA tetraloop hairpin varies in four loop nucleotides from the wild-type T4 RNA hairpin (with eight loop nucleotides) involved in the translational repression of bacteriophage T4 DNA polymerase. Despite the differences in their sequence and proposed secondary structures, these two hairpins bind T4 DNA polymerase with equal affinity. The NMR spectra of the mutant hairpin indicate that its stem is extended in comparison to that of the wild-type hairpin by the formation of two additional Watson-Crick base pairs. The NMR data provide a precisely defined structure for the mutant hairpin with an average root mean square deviation of approximately 0.7 A for all 16 residues in the molecule. The structure of the mutant loop is very similar to that determined previously for the wild-type hairpin. The three loop bases that are conserved between the mutant and wild-type hairpins point out in solution with the groups capable of hydrogen bond formation exposed to the solution. This is exactly what was seen for the wild-type hairpin. Also, unusual, long-range NOEs, loop hydrogen bonds, and even the position at which the loop bends are common features between the two loops. This explains how two different hairpins, by adopting similar three-dimensional structures, have the same affinity for the DNA polymerase.
...
PMID:A quadruple mutant T4 RNA hairpin with the same structure as the wild-type translational repressor. 867 68

(R)-9-(2-Phosphonylmethoxypropyl)adenine (PMPA) is an acyclic nucleoside phosphonate that has been shown to be effective in the treatment of AIDS although it has a shorter separation between the adenine and phosphorus than dideoxy-AMP and dAMP. By using pre-steady state kinetic methods, we examined the incorporation of the diphosphate of PMPA, 2',3'-dideoxyadenosine 5'-triphosphate (ddATP), and dATP catalyzed by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, an exonuclease-deficient T7 DNA polymerase (T7 exo-), and wild-type rat DNA polymerase beta in order to evaluate the selectivity of PMPA as an antiviral inhibitor. With a DNA/DNA or DNA/RNA 22/43-mer duplex, the diphosphate of PMPA (PMPApp) is as effective as ddATP in reactions catalyzed by HIV-1 reverse transcriptase in that both analogs have similar substrate specificity constants (kp/Kd) which are only 5-fold lower than dATP. In contrast, PMPApp is a much weaker inhibitor of the reaction catalyzed by T7 exo- (with the DNA/DNA 22/43-mer duplex) in that PMPApp has a 5 x 10(-4)-fold lower kp/Kd than ddATP and dATP. The lower kp/Kd of PMPApp is due to a 1000-2000-fold lower incorporation rate (kp) and a 35-45-fold lower binding constant (Kd). Similarly, PMPApp is 800-fold less inhibitory toward polymerase beta with the DNA/DNA 22/43-mer duplex, whereas in studies with a single nucleotide gapped DNA (22-20/43-mer) PMPApp is 13-fold less inhibitory than ddATP. Although parallel studies will need to be performed using appropriate human polymerases, these results begin to define the mechanistic basis for the reported lower toxicity of PMPA in the treatment of AIDS.
...
PMID:Selective inhibition of HIV-1 reverse transcriptase by an antiviral inhibitor, (R)-9-(2-Phosphonylmethoxypropyl)adenine. 976 48

The structure of Mycobacterium tuberculosis dUTP nucleotidohydrolase (dUTPase) has been determined at 1.3 Angstrom resolution in complex with magnesium ion and the non-hydrolyzable substrate analog, alpha,beta-imido dUTP. dUTPase is an enzyme essential for depleting potentially toxic concentrations of dUTP in the cell. Given the importance of its biological role, it has been proposed that inhibiting M.tuberculosis dUTPase might be an effective means to treat tuberculosis infection in humans. The crystal structure presented here offers some insight into the potential for designing a specific inhibitor of the M.tuberculosis dUTPase enzyme. The structure also offers new insights into the mechanism of dUTP hydrolysis by providing an accurate representation of the enzyme-substrate complex in which both the metal ion and dUTP analog are included. The structure suggests that inclusion of a magnesium ion is important for stabilizing the position of the alpha-phosphorus for an in-line nucleophilic attack. In the absence of magnesium, the alpha-phosphate of dUTP can have either of the two positions which differ by 4.5 Angstrom. A transiently ordered C-terminal loop further assists catalysis by shielding the general base, Asp83, from solvent thus elevating its pK(a) so that it might in turn activate a tightly bound water molecule for nucleophilic attack. The metal ion coordinates alpha, beta, and gamma phosphate groups with tridentate geometry identical with that observed in the crystal structure of DNA polymerase beta complexed with magnesium and dNTP analog, revealing some common features in catalytic mechanism.
...
PMID:Crystal structure of the Mycobacterium tuberculosis dUTPase: insights into the catalytic mechanism. 1527 40

A reference system for DNA replication fidelity was studied by free energy perturbation (FEP) and linear interaction energy (LIE) methods. The studied system included a hydrated duplex DNA with the 5'-CG dangling end of the templating strand, and dCTP4-.Mg2+ or dTTP4-.Mg2+ inserted opposite the dangling G to form a correct (i.e., Watson-Crick) or incorrect (i.e., wobble) base pair, respectively. The average distance between the 3'-terminal oxygen of the primer strand and the alpha-phosphorus of dNTP was found to be 0.2 A shorter for the correct base pair than for the incorrect base pair. Binding of the incorrect dNTP was found to be disfavored by 0.4 kcal/mol relative to the correct dNTP. We estimated that improved binding and more near-attack configurations sampled by the correct base pair should translate in aqueous solution and in the absence of DNA polymerase into a six times faster rate for the incorporation of the correct dNTP into DNA. The accuracy of the calculated binding free energy difference was verified by examining the relative free energy for melting duplex DNA containing GC and GT terminal base pairs flanked by a 5' dangling C. The calculated LIE and FEP free energies of 1.7 and 1.1 kcal/mol, respectively, compared favorably with the experimental estimate of 1.4 kcal/mol obtained using the nearest neighbor parameters. To decompose the calculated free energies into additive electrostatic and van der Waals contributions and to provide a set of rigorous theoretical data for the parametrization of the LIE method, we suggested a variant of the FEP approach, for which we coined a binding-relevant free energy (BRFE) acronym. BRFE approach is characterized by its unique perturbation pathway and by its exclusion of the intramolecular energy of a rigid part of the ligand from the total potential energy.
...
PMID:Free energy simulations of uncatalyzed DNA replication fidelity: structure and stability of T.G and dTTP.G terminal DNA mismatches flanked by a single dangling nucleotide. 1672 67

The structures and energy of phosphate dimethyl ester and vanadate dimethyl ester have been calculated using B3LYP/TZVP density functional quantum chemical methods and polarized continuum (PCM) and Langevin dipoles solvation models. These calculations were carried out to obtain fundamental information on the ability of vanadate esters to function as transition state analogues for the nucleotidyl transfer reaction catalyzed by DNA polymerases. Base-catalyzed methanolysis of the phosphate and vanadate dimethyl esters were the model reactions examined in this study. The structures of the phosphate and vanadate dimethyl esters and pentavalent intermediates in aqueous solution were optimized and evaluated at the PCM/B3LYP/TZVP level. The three-dimensional free energy surfaces for the studied reactions were determined at the PCM/B3LYP/TZVP//B3LYP/TZVP level. Comparison with experimental structural data obtained from the Cambridge Structural Database and with the observed kinetics of phosphate diester hydrolysis demonstrated that the level of theory chosen for these studies was appropriate. The results showed that structurally and electrostatically the vanadate dimethylester and a five-coordinate nearly trigonal bipyramidal intermediate were reasonable analogues for the parent phosphorus systems. Despite these similarities in structure, the energetics of the two systems were different, and the transition states of the two model reactions were found on different areas of the potential energy surface. When the binding energy of a transition state-DNA polymerase complex was extrapolated to a transition state analogue-DNA polymerase complex, the formation of a simple dianionic pentavalent vanadate ester adduct in the enzyme active site was not found to be sufficiently favorable. This finding suggests that additional stabilization of this adduct is needed before this type of transition state analogue will be likely to yield stable adducts with this class of enzymes. New possible candidates for such complexes are suggested.
...
PMID:Transition state analogues for nucleotidyl transfer reactions: Structure and stability of pentavalent vanadate and phosphate ester dianions. 1686 14

To evaluate the effects of aging on DNA damage, spontaneous and chemical-induced DNA damage and its repair were examined using comet assays at pH 9, 12.1 and 13, and an 8-OH-dG assay in the liver and kidney of young (9-week-old) and aged (20-month-old) rats. Additionally, blood chemistry was examined to investigate any correlation between vital functions and age-dependent DNA damage. DNA migration at pH 13 and 8-OH-dG levels increased in the liver and/or kidney of aged rats, but DNA migration did not increase at pH 9 or 12.1; that is, alkali-labile sites and 8-OH-dG were concomitantly accumulated in aged rats. These results suggest that 8-OH-dG production caused by reactive oxygen species exceeded glycosylation and that the glycosylation activity is far more than the AP endonucleation in aged rats. Methyl methanesulfonate (MMS, 80 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr after treatment in young and aged rats. The DNA damage in aged rats was less and decreased more slowly compared with young rats. The pictures of MMS-induced DNA migrations at pH 12.1 and 13 were very similar to each other. These results suggest that the adduct glycosylation and repair of the single-strand breaks (SSBs) of aged rats are less than those of young rats, although AP endonucleation is sufficient to remove the AP sites. N-nitrosodiethylamine (160 mg/kg, i.p.) increased DNA migration at pH 12.1 and 13 in the liver and kidney at 3 and 24 hr in young rats and at pH 12.1 and 13 in the kidney at 24 hr in aged rats. These results showed that SSBs were predominantly detected as chemical-induced DNA damage and DNA repairs such as N-glycosylase, DNA polymerase and DNA ligase, and that the metabolic activation declined in aged rats. Aspartate aminotransferase, alanine aminotransferase, total bilirubin, total cholesterol, total protein, globulin, creatinine and chloride age-dependently increased and alkaline phosphates, albumin/globulin ratio, inorganic phosphorus and potassium age-dependently decreased, and these changes were correlated with the DNA migration at pH 13 and/or 8-OH-dG. These results suggest that the activity of DNA repair and metabolic activation enzymes declines in aged rats and that the accumulation of spontaneous DNA damage may affect vital functions.
...
PMID:DNA damage measured by comet assay and 8-OH-dG formation related to blood chemical analyses in aged rats. 1778 42

The structural and dynamical changes occurring before nucleotide addition were studied using molecular dynamics (MD) simulations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexes containing one or two Mg2+ ions in the presence of dNTP. Our models revealed that the formation of a catalytically competent DNA polymerase complex required subtle rearrangements at the catalytic site A, which occurred only when an Mg2+ ion was bound. This model has been validated using pre-steady-state kinetics to show that free Mg2+ is necessary to obtain a catalytically competent polymerase. Kinetic studies carried out with Be2+ as a cofactor permitted the functional discrimination between metal sites A and B. At low concentrations, Be2+ increased the catalytic efficiency of the polymerase, while at higher concentrations, it competed with Mg2+ for binding to site A, and inhibited DNA polymerization. In agreement with experimental data, MD simulations revealed that the catalytic attack distance between the 3-OH of the primer and the phosphorus in complexes containing Be2+ instead of Mg2+ at site A was above 4.5 A. Our findings provide a detailed description of the mechanism of DNA polymerization and should be helpful to understand the molecular basis of DNA replication fidelity.
...
PMID:A Mg2+-induced conformational switch rendering a competent DNA polymerase catalytic complex. 1796 36


<< Previous 1 2 3 Next >>

---