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)

Progress of DNA- and/or RNA-directed DNA polymerization reactions can be measured continuously using circular dichroism (CD) or ultraviolet (UV) spectroscopy. In the presence of the Klenow fragment of Escherichia coli DNA polymerase I, a CD change of -0.27 +/- 0.06 millidegree at 248 nm and a UV change of -2.7 +/- 0.3 milliabsorbance units at 275 nm occur upon incorporation of 120 pmol of dTMP in a reaction volume of 120 microliters (1 microM dTMP incorporation) into a synthetic template-primer, p(dA)40-60.p(dT)20. The transcription of poly(A).p(dT)12-18 by reverse transcriptases can also be monitored using these methods. Kinetic parameters for the polymerization reaction catalyzed by the Klenow fragment were determined from initial velocity measurements using CD or UV assays and were in close agreement with those measured by the standard single point radiochemical filtration assay. The generality of optical techniques for the measurement of DNA polymerase activity was shown by the use of a partially self-complementary hairpin-shaped oligonucleotide substrate for the Klenow fragment. Addition of a single nucleotide residue under steady-state conditions to this 35-mer at a concentration of 1.5-3 microM gave an easily measurable absorbance decrease at 275 nm, and the absorbance changes upon sequential addition of nucleotide units were additive.
...
PMID:Continuous microspectrophotometric measurement of DNA polymerase activity: application to the Klenow fragment of Escherichia coli DNA polymerase I and human immunodeficiency virus type 1 reverse transcriptase. 170 25

A phosphorothioate homocytidine 10-mer containing a cholesteryl moiety covalently linked to the 5'-end (Chol-SdC10) inhibited syncytium formation in susceptible T cells induced by HIV-1 and HIV-2. The syncytium inhibition effect was minimal with unmodified cytidine homopolymer of the same net charge. Chol-SdC10 was shown to protect CEM cells against infection by cell-free HIV-1 particles without any apparent toxicity to the growth of CD4+ T cells. The DNA polymerase activity of the purified reverse transcriptase (RT) of HIV-1 was markedly inhibited by Chol-SdC10 but the effect on the RNase H activity of RT was minimal. Analysis of the kinetics of reverse transcriptase inhibition mediated by the drug revealed that the inhibition at a higher concentration was competitive with respect to template primer binding and noncompetitive at lower concentrations. Chol-SdC10 also partially blocked the binding of gp120 to CD4 in a solid-phase ELISA. These results confirm that the anti-HIV activity of phosphorothioate cytidine homopolymers increases markedly by covalent modification with the cholesteryl moiety at the 5'-end and demonstrates that the cytoprotective effect is manifested at multiple steps in the virus life cycle. These steps include inhibition of retroviral replication activity as well as the binding and fusion of HIV with CD4+ T cells.
...
PMID:Mode of action of 5'-linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncytia formation and infection by HIV-1 and HIV-2 in vitro. 170 17

The conformation of a complex of a 41 mer/31 mer DNA fragment and the Klenow fragment of DNA polymerase I of Escherichia coli was studied by scanning tunnelling microscopy (STM). The results shows that near two turns of double helix of this DNA fragment was outside of enzyme while another part containing more than one turn of helix and 10 nucleotides single strand was combined with enzyme. The dimension and shape of DNA polymerase I (KF) in complex were different from that of free enzyme. The conformation of DNA-DNA polymerase I (KF) complex and the application of STM in studying structure of complex of DNA polymerase with DNA were discussed.
...
PMID:Conformation of DNA-DNA polymerase I complex observed by scanning tunneling microscopy. 174 60

The elementary steps of DNA polymerization catalyzed by T7 DNA polymerase have been resolved by transient-state analysis of single nucleotide incorporation, leading to the complete pathway: [formula: see text] where E, D, N, and P represent T7 DNA polymerase, DNA primer/template, deoxynucleoside triphosphate, and inorganic pyrophosphate, respectively. A DNA primer/template consisting of a synthetic 25/36-mer has been used as a substrate for correct nucleotide incorporation of dTTP in all the experiments. The rate constants and equilibrium constants of each step have been established by direct measurement of individual reactions and fit by computer simulation of the data to obtain a single set of rate constants accounting for all the data. Analysis of the single-turnover kinetics provided measurements of equilibrium dissociation constants for 25/36-mer, dTTP, and PPi equal to 18 nM (koff/kon), 18 microM (k-1/k1), and 2 mM (k5/k-5), respectively. The rate-limiting step during single-nucleotide incorporation has been identified as a conformational change, E.Dn.N----E'.Dn.N, which occurs at a rate of 300 s-1 (k2) upon binding of the correct dNTP. Accordingly, tighter binding of the transition states for the reaction resulting from the conformational change facilitates the phosphodiester bond formation. The chemical step itself was excluded as the rate-limiting step because of the small phosphothioate elemental effect. An observed rate constant of 70 s-1 for dTTP (alpha S) incorporation suggest that the chemical step (k3) occurs at a fast rate, greater than or equal to 9000 s-1. Following chemistry, the resulting ternary complex, E'.Dn+1.P, undergoes a second conformational change at a rate of 1200 s-1 (k4), leading to release of PPi and translocation of the DNA to continue subsequent cycles of polymerization. The rate constants of the reverse steps, 100 s-1 (k-2), greater than or equal to 18,000 s-1 (k-3) and 18 s-1 (k-4), were derived as fits to the data based upon simulation of single-turnover kinetics of pyrophosphorolysis including measurements of pyrophosphate exchange and the overall equilibrium constant of 1.0 x 10(4) for elongation of E.25/36-mer and analysis of the kinetics of the pulse-chase experiment. These studies provide the first complete and self-consistent thermodynamic descriptions of DNA polymerase and establish the basis for quantitative assessment of the reactions contributing to its extraordinary fidelity.(ABSTRACT TRUNCATED AT 400 WORDS)
...
PMID:Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. 184 98

We report here a simple and efficient method for site-directed mutagenesis using polymerase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restriction gene, we made two point mutations. While one created a new SalI site prior to the SD sequence, the other replaced Glu144 with Lys. A 1.5 kb SalI-PstI fragment isolated from pER101 was used as the template. Two 25 mer oligonucleotide primers containing the desired mutations were synthesized and used to direct PCR amplification with Taq DNA polymerase. About 0.5 microgram of the 0.49 kb fragment was obtained from 0.05 microgram of the 1.5 kb fragment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically, 99% of the product contained the desired mutations. The product was cloned into pUC19 using SalI and PstI, two of the transformed colonies were randomly chosen for sequence analysis, and both of them were shown to contain the desired mutations. Finally, the amplified fragment was cloned into pER304 to place the EcoRI (Lys144) gene directly under the control of the lambda PL promoter.
...
PMID:An efficient site-directed mutagenesis using polymerase chain reaction. 188 38

6-Nitroso-1,2-benzopyrone, an oxidation product of 6-amino-1,2-benzopyrone, binds to the DNA-recognizing domain of the ADP-ribose transferase protein and preferentially destabilizes Zn2+ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of 65Zn2+ from the 65Zn(2+)-isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc-deficient enzyme still binds to a DNA template, consisting of a 17-mer DNA primer annealed to M13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I. Auto-poly-ADP-ribosylated ADP-ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer-template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP-ribose transferase in intact cells can induce significant alteration in DNA structure and replication.
...
PMID:Destabilization of Zn2+ coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function. 191 72

The replication of adenovirus DNA requires, in addition to several host factors, three virus-encoded proteins: a DNA binding protein, the precursor of the terminal protein (pTP), and a DNA polymerase (Ad pol). Ad pol and pTP form a tight complex that is necessary for the initiation step in DNA replication. To perform mutation scanning of the adenovirus type 5 pTP and Ad pol a series of in-frame linker insertions of a 12-mer oligonucleotide d(CCCATCGATGGG) were introduced into cloned viral DNA fragments containing coding sequences of these proteins. The insertions are located at recognition sites for several blunt end-cutting restriction endonucleases. Forty different sites were mutagenized and the mutated genes were transferred to a plasmid that contains the left 42% of the adenovirus genome. They were rebuilt into the viral genome by means of in vivo recombination between plasmid DNA and digested adenovirus DNA-TP complex. The resulting viral genomes were tested for viability and rescued virus was analyzed for the presence of the inserted linker oligonucleotide. This procedure resulted in recovery of a number of viable virus mutants with insertions in the pTP or Ad pol genes, all of which are phenotypically silent. The other mutations did not allow virus production. The positions of these apparent lethal codon insertion mutations were useful to identify regions of functional importance in both proteins. It can be concluded that the precursor-specific region of pTP plays an important role in virus multiplication.
...
PMID:Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase. 198 53

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-). 199 Nov 25

Terminal deoxynucleotidyl transferase (terminal transferase) was specifically modified in the DNA binding site by a photoactive DNA substrate (hetero-40-mer duplex containing eight 5-azido-dUMP residues at one 3' end). Under optimal photolabeling conditions, 27-40% of the DNA was covalently cross-linked to terminal transferase. The specificity of the DNA and protein interaction was demonstrated by protection of photolabeling at the DNA binding domain with natural DNA substrates. In order to recover high yields of modified peptides from limited amounts of starting material, protein modified with 32P-labeled photoactive DNA and digested with trypsin was extracted 4 times with phenol followed by gel filtration chromatography. All peptides not cross-linked to DNA were extracted into the phenol phase while the photolyzed DNA and the covalently cross-linked peptides remained in the aqueous phase. The 32P-containing peptide-DNA fraction was subjected to amino acid sequence analysis. Two sequences, Asp221-Lys231 (peptide B8) and Cys234-Lys249 (peptide B10), present in similar yield, were identified. Structure predictions placed the two peptides in an alpha-helical array of 39 A which would accommodate a DNA helix span of 11 nucleotides. These peptides share sequence similarity with a region in DNA polymerase beta that has been implicated in the binding of DNA template.
...
PMID:Interactions of photoactive DNAs with terminal deoxynucleotidyl transferase: identification of peptides in the DNA binding domain. 200 41

Synthesis of a 25-mer oligonucleotide template containing O4-methylthymine (m4T) at a unique site is reported. The sequence used is analogous to that studied previously to determine the mutation frequency of O6-methylguanine in vitro and in vivo. The templates containing m4T or unmodified T were used in a primer-extension gel assay to determine kinetic parameters for incorporation by DNA polymerases of dGTP and dATP opposite either m4T or T. Both Escherichia coli DNA polymerase I (Klenow fragment, Kf) and Drosophila melanogaster polymerase alpha-primase complex (pol alpha) were used. On the basis of the Vmax/Km ratios, the pairing of m4T.G was preferred over that of both m4T.A and T.G by more than 10-fold. The two polymerases gave almost identical values for the frequency of formation of all pairs investigated including m4T.G pairs, suggesting that the 3'----5' exonuclease activity of the Klenow fragment does not efficiently edit such pairs. Extension beyond m4T.G was demonstrated with both Klenow and pol alpha. In similar kinetic experiments, bacteriophage T4 DNA polymerase, which has a very high 3'----5' exonuclease activity, allows stable incorporation of G opposite m4T in contrast to G opposite T. This kinetic approach allows quantitation of the mutagenic potential in the absence of alkylation repair and additionally provides qualitative data on mutagenesis that are in accord with our previous in vivo studies showing that replication of m4T causes T----C transitions.
...
PMID:Comparative efficiency of forming m4T.G versus m4T.A base pairs at a unique site by use of Escherichia coli DNA polymerase I (Klenow fragment) and Drosophila melanogaster polymerase alpha-primase complex. 211 81


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---