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)

The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.
...
PMID:Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase. 769 3

Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host.
...
PMID:Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay. 841 59

We have determined the DNA sequence of the mitochondrial plasmid from Neurospora intermedia strain Fiji N6-6. The plasmid contains a 1278-codon open reading frame that is 49% identical to the open reading frame of the mitochondrial plasmid from the LaBelle strain of N. intermedia, which is known to encode a DNA-dependent DNA polymerase. The results of polymerase assays and photolabeling studies, the high degree of identity with the LaBelle plasmid polymerase, and the observation that the Fiji polymerase activity in a reaction utilizing endogenous template is not affected by removal of RNA suggest that the Fiji plasmid also encodes a DNA-dependent DNA polymerase. Comparison of regions of amino acids that are highly conserved in the two plasmid polymerases to family B polymerases reveals good correlates for the three major polymerase motifs and suggests that previously identified motifs characteristic of reverse transcriptase found in the LaBelle sequence are not significant. The polymerases encoded by the Fiji and LaBelle plasmids are unusual in that the amino acid sequence Asp-Thr-Asp, which forms the core of the third motif in family B polymerases, is not present in either Fiji or LaBelle. A version of the motif containing Thr-Thr-Asp exists in both sequences.
...
PMID:Two Neurospora mitochondrial plasmids encode DNA polymerases containing motifs characteristic of family B DNA polymerases but lack the sequence Asp-Thr-Asp. 848 47

In the presence of Mg2+ ions, polynucleotide phosphorylase (PNPase, EC 2.7.7.8) is known to synthesize RNA-like polymers using ribonucleoside-5'-diphosphate (NDP) substrates but to be unable to utilize deoxyribonucleoside substrates. Our experiments show that when MgCl2 is replaced by FeCl3, PNPase becomes able to synthesize deoxyheteropolymers using deoxyribonucleoside-5'-diphosphates (dNDPs). The deoxyheteropolymer formed from the four dNDPs is degraded by pancreatic DNase, but not by RNase, and is readily used as a template by DNA-dependent DNA polymerase. Synthesis of this DNA-like polymer is accomplished de novo without the help of any primer or preexisting template. What is more, dA/dG and dC/dT ratios of polymers synthesized by different bacterial PNPases closely match ratios found in DNA of the bacterial species the enzyme came from.
...
PMID:De Novo Synthesis of DNA-Like Molecules by Polynucleotide Phosphorylase In Vitro 866 1

The DNA-dependent DNA polymerase (DDDP) and RNA-dependent DNA polymerase (RDDP) activities of hepadnavirus polymerases are both essential for viral replication. Human hepatitis B virus (HBV) polymerase has been successfully expressed in Escherichia coli as a fusion protein in frame with maltose-binding protein. The present study was undertaken to characterize these two activities and introduce an in vitro assay system. In situ activity gel assays show that the polymerase has both types of activities. One hundred thirty-four kilodaltons of active full-length product was proteolytically cleaved into approximately 73 kDa of active fragment by proteinase K preincubation. Mutation of conserved YMDD motif also confirms that the activities were due to the recombinant polymerase and that this motif is essential to polymerase activity. Two activities of the polymerase show their optima under conditions of 1 mM (DDDP) or 0.25 mM (RDDP) of MnCl2, 400 mM KCl, 37 degrees C (DDDP) or 24 degrees C (RDDP), and pH 7.0-7.7. Substitution of Mg2+ for Mn2+ results in reduction of processivity, which may explain why Mn2+ supports nucleotide incorporation to a higher level than Mg2+. The polymerase is resistant to aphidicolin. Actinomycin D acts selectively on DDDP activity, whereas phosphonoformic acid inhibits both activities. The in vitro HBV polymerase assay system demonstrated herein will be useful for screening potential HBV polymerase inhibitor for the development of anti-HBV drugs.
...
PMID:The catalytic properties of human hepatitis B virus polymerase. 867 Feb 70

In the search for effective antiviral agents, we have found 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate (RD4-2025) to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. The 50% effective concentration of RD4-2025 for HIV-1-induced cytopathic effect in MT-4 cells was 37 nM, yet no antiviral activity was observed against HIV-2. In HIV-1 reverse transcriptase (RT) assays, RD4-2025 inhibited both RNA-dependent and DNA-dependent DNA polymerase activities of a recombinant HIV-1 RT with 50% inhibitory concentrations of 0.11 and 3.5 microM, respectively. However, the compound did not affect the activity of human DNA polymerase alpha. Kinetic studies revealed that the inhibition was noncompetitive with respect to dGTP as the substrate and poly(C)/(dG) 12-18 as the template/primer. These results were in accordance with those of nonnucleoside RT inhibitors (NNRTIs), such as R89439 (an alpha-anilinophenylacetamide derivative) and nevirapine, indicating that RD4-2025 also belongs to the family of NNRTIs.
...
PMID:Inhibitory effect of 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate on replication of human immunodeficiency virus type 1 and the mechanism of action. 879 26

The replication of the hepatitis B viral DNA genome proceeds through a pregenomic RNA intermediate. This pregenomic RNA subsequently serves as the template for the formation of the viral DNA by the reverse transcriptase activity of the viral P gene product. The P gene product is believed to be a multifunctional enzyme with DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, and RNase H activities. Detailed biochemical studies of this protein have not been performed because of the inability to obtain sufficient amounts of the enzyme from the virus and by the inability to produce the enzyme in heterologous expression systems. The RNase H activity is essential for viral replication and is believed to be responsible for the degradation of the RNA pregenomic intermediate as well as for generating the short RNA primer that is required for DNA second strand synthesis. We have assembled an expression vector which directs the synthesis of a protein that corresponds to the putative RNase H domain of the P gene product and having a carboxyl-terminal polyhistidine tag to facilitate purification. The protein has been expressed in Escherichia coli and purified to yield 1-2 mg of protein/liter of culture. This protein has RNase H activity as defined by its ability to degrade the RNA component of RNA-DNA hybrids but not the DNA component. The RNase H has a basic optimum pH, is active only in the presence of reducing agents, and is dependent on the presence of divalent cations, with magnesium being preferred over manganese.
...
PMID:Expression, purification, and characterization of an active RNase H domain of the hepatitis B viral polymerase. 895 90

HIV-1 reverse transcriptase (RT) is multifunctional, with RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP), and ribonuclease H (RNase H) activities. N-(4-tert-Butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) inhibited both the polymerase and the RNase H activities of HIV-1 RT in vitro. IC50 values for inhibition of RDDP were 0.8-3.4 microM, depending on the template/primer (T/P) used in the assay. The IC50 for DDDP inhibition was about 12 microM, while that for inhibition of RNase H was 3.5 microM. EC50 for inhibition of HIV-1 replication in cord blood mononuclear cells was 1.5 microM. BBNH inhibition of RNase H in vitro was time-dependent, whereas inhibition of RT polymerase activities was immediate. BBNH was a linear mixed-type inhibitor of RT RDDP activity with respect to both T/P and to dNTP, whereas BBNH inhibition of RT RNase H activity was linear competitive. Protection experiments using an azidonevirapine photolabel showed that BBNH binds to the non-nucleoside RT inhibitor (NNRTI) binding pocket. Importantly, the compound inhibited recombinant RT containing mutations associated with high-level resistance to other NNRTI. While BBNH did not inhibit the DNA polymerase activities of other retroviral reverse transcriptases and DNA polymerases, the compound inhibited Escherichia coli RNase HI and the RNase H activity of murine leukemia virus RT. BBNH also inhibited HIV-1 RT RNase H in the presence of high concentrations of other non-nucleoside inhibitors with higher affinities for the NNRTI binding pocket, and of RT in which the NNRTI binding pocket had been irreversibly blocked by the azidonevirapine photolabel. We conclude that BBNH may therefore bind to two sites on HIV-1 RT. One site is the polymerase non-nucleoside inhibitor binding site and the second may be located in the RNase H domain. BBNH is therefore a promising lead compound for the development of multisite inhibitors of HIV-1 RT.
...
PMID:Inhibition of the ribonuclease H and DNA polymerase activities of HIV-1 reverse transcriptase by N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone. 911 94

Ongoing and extensive screening for potentially selective and potent inhibitors of RNA-dependent DNA polymerases (reverse transcriptases) is important for the discovery of therapeutic agents active against retroviruses. Traditional systems for assaying the activity of reverse transcriptase enzymes have relied upon the use of radioactive nucleotides for monitoring complementary DNA (cDNA) synthesis. Moreover, such assays have also typically been programmed by the addition of synthetic template-primers. The recent trend, however, is towards the use of more natural nucleic acid template-primer-directed systems, which provides for a more realistic estimation of the activity of potential therapeutic anti-reverse transcriptase agents. A novel agarose gel-electrophoretic method originally developed to evaluate DNA polymerase activity during enzyme purification was adapted for screening purposes. The system was modified so that one can detect the synthesis of non-radioactively labeled cDNA synthesized in both DNA and RNA template-directed reverse transcription reactions. Such assays are programmed with either M13mp9 single-stranded DNA or rabbit globin mRNA as appropriate templates. Following incubation of reaction mixtures, agarose gels are used to determine the activity of the DNA-dependent DNA polymerase and RNA-dependent DNA polymerase activities of Maloney murine leukemia virus. This is done through a detection of the amount of double-stranded nucleic acid molecules (either DNA:DNA or RNA:DNA hybrids) generated by the enzyme in the presence and absence of various known enzyme inhibitors. The assay systems that have been developed will be useful in initial, general, rapid, inexpensive and safer screening programs for potential inhibitors of both the DNA- and RNA-dependent DNA-polymerase functions of reverse transcriptase enzymes. The system was tested and evaluated with potent inhibitors of reverse transcription, such as aurintricarboxylic acid and with several plant flavonoids known to be moderately potent inhibitors of reverse transcriptases. The details concerning the RNA-dependent DNA polymerizing system and some of our results are presented.
...
PMID:A non-radioactive assay system for screening for inhibitors of RNA-dependent reverse transcriptase activity; an analysis using aurintricarboxylic acid and plant flavonoids. 917 31

We have constructed a plasmid that induces in bacteria the synthesis of an enzymically active reverse transcriptase (RT) of mouse mammary tumour virus (MMTV), a retrovirus with a typical B-type morphology. The highest catalytic activity was detected only when 27 residues from the C-terminus of the protease were included in the N-terminus of the recombinant RT, after an extra deoxyadenosine was added between the pro and pol genes to overcome the -1 frameshift event (which occurs naturally in virus-infected cells). The recombinant protein with a six-histidine tag was purified to homogeneity by a two-column purification procedure, Ni2+ nitriloacetic acid/agarose followed by carboxymethyl-Sepharose chromatography. Unlike most RTs, the purified MMTV RT is enzymically active as a monomer even after binding a DNA substrate. Like all RTs studied, the recombinant MMTV RT possesses RNA-dependent and DNA-dependent DNA polymerase activities as well as RNase H activity, all of which show a preference for Mg2+ over Mn2+ ions. Other features of these enzymic activities, such as extension of DNA primers, processivity of DNA synthesis, pH dependence, steady-state kinetic constants, effects of Na+ or K+ ions and sensitivity to a thiol-specific reagent and to a zinc chelator, have been evaluated. The catalytic properties of MMTV RT were compared with those of the well-studied RT of HIV-1, the causative agent of AIDS. Interestingly, MMTV RT exhibits a high sensitivity to nucleoside triphosphate analogues (which are known to be potent inhibitors of HIV RTs and are being used as the major anti-AIDS drugs), as high as that of HIV-1 and HIV-2 RTs. Furthermore the recombinant MMTV RT shows a processivity of DNA synthesis higher than that of HIV-1 RT.
...
PMID:Reverse transcriptase of mouse mammary tumour virus: expression in bacteria, purification and biochemical characterization. 944 85


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

---