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)

An RNA-directed DNA polymerase was purified from mouse spleen infected with leukaemia virus activated during N-methyl-N-nitroso urea- (MNU-) induced leukaemogenesis. The enzyme was isolated from the microsomal fraction and purified by successive chromatography of Sephadex G-200 and phosphocellulose. Estimation of molecular weight from the sedimentation rate of the purified enzyme in a sucrose gradient gave a value of 70,000. The enzyme had a pH optimum of 7.4, a KC1 optimum of 50 mmol/l, an Mn2+ optimum of 0.2 mmol/l, and a temperature optimum of 25 degrees C, when (rA)n . (dT) 10 was used as the template-primer. It preferred (rA)n . (dT)10 as the template-primer and transcribed (rC)n . (dG) 12 and (OMeC)n . (dG)12. A comparison of the properties of this DNA polymerase with the enzyme purified from murine type C retroviruses showed that the MNU-activated virus enzyme was both biochemically and biophysically indistinguishable from murine leukaemia virus DNA polymerases.
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PMID:Characterization of an RNA-directed DNA polymerase from mouse spleen infected with leukaemia virus activated during N-methyl-N-nitroso urea-induced leukaemogenesis. 617 78

The synthesis of single-stranded globin cDNA by the RNA-directed DNA polymerase activity of reverse transcriptase in the presence of oligothymidylate primers was investigated in order to determine the limitations to higher yields. The results indicated that the associated ribonuclease H activity, an integral part of reverse transcriptase, plays a large role in the synthesis of the first strand of cDNA and that the interplay of the two enzyme activities for any specific set of conditions determines the yield of single-stranded products. In both the presence and the absence of polymerization, the associated ribonuclease H catalyzed the deadenylation of mRNA, producing molecules that were somewhat shorter, highly homogeneous in size, and fully translatable into globin protein. They were also entirely lacking in the ability to serve as templates for cDNA synthesis. The reaction was completely dependent on oligothymidylate and completely independent of deoxyribonucleoside triphosphates. The initial rate of deadenylation was one-fourth the initial rate of initiation of polymerization when saturating levels of deoxyribonucleoside triphosphates were used in the polymerase reaction. In the presence of ribonuclease H activity, the DNA polymerase catalyzed the synthesis of an array of cDNAs including some that were full length. The initiation of polymerization was rate limiting: once synthesis had begun, it required 1-1.5 min to transcribe globin mRNA. However, most primers that were elongated were aborted prematurely. Maximum synthesis of full-length cDNA required stoichiometric levels of enzyme and high triphosphate levels, but regardless of conditions, the sum of completed cDNA and deadenylated mRNA accounted for only 50% of the input mRNA. The data fit a model in which synthesis of full-length cDNA molecules depends on the arrangement of primers and transcription initiation complexes on the poly(A) "tail" of mRNA.
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PMID:Reverse transcriptase and its associated ribonuclease H: interplay of two enzyme activities controls the yield of single-stranded complementary deoxyribonucleic acid. 619 May 7

A RNA-directed DNA polymerase was partially purified from a human homologous, mixed mesodermal sarcoma by DEAE-cellulose chromatography after sucrose density centrifugation. The enzyme transcribed poly(rA) most effectively but also transcribed poly(rI), poly(dA) and poly(rG) and to a lesser extent, poly(rmC). It was unable to transcribe poly(rU). The product with poly(rA) as template contained large material (greater than 28S) in addition to some proper size product demonstrating a slippage reaction. This pattern of transcription, while similar to avian myeloblastosis virus DNA polymerase, reveals qualitative differences making direct extrapolation from studies with animal oncornaviruses to human cancer difficult. In this paper, the detection and purification of RNA-directed DNA polymerase from a patient with an uncommon uterine sarcoma is reported along with the template specificities of the enzyme.
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PMID:Template specificities of a RNA-directed DNA polymerase from a human homologous mixed mesodermal sarcoma. 619 Dec 64

A novel enzyme inhibitor against RNA-directed DNA polymerase of avian myeloblastosis virus was produced by an isolate of a new streptomycete for which the name Streptomyces retrostaticus is proposed. This enzyme inhibitor, which was named retrostatin, did not inhibit DNA-directed DNA polymerase of Escherichia coli and DNA-directed RNA polymerase of Ehrlich ascites tumor cells. Retrostatin was produced by the microorganism together with streptonigrin. These two substances were extracted from the culture broth with ethyl acetate at acidic pH. Retrostatin is an acidic pH indicator and the free acid was recovered as a red powder. Retrostatin had weak antibiotic activities against Gram-positive bacteria and yeasts.
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PMID:Retrostatin, a new specific enzyme inhibitor against avian myeloblastosis virus reverse transcriptase. 619 91

The expression of a mouse mammary tumor virus is inducible by hormones, and the virus contains a hormone-responsive element. Viral particles and RNA-directed DNA polymerase (RDDP, EC 2.7.7.7; reverse transcriptase) are both detectable in human breast tumors but the frequency and significance of these findings are unknown. Breast tumor biopsy specimens (from either the primary site or a metastasis), frozen in liquid nitrogen at the time of surgery, were routinely obtained to determine estrogen receptor (ERP) concentration. A sample of the tissue was pulverized, homogenized and centrifuged at low speed to remove nuclei and mitochondria. The supernate was then centrifuged at 225,000 g to obtain the cytosol fraction for estrogen and progestin receptor (PgR) assays. Partially purified membranes for the RDDP assays were prepared from the high-speed pellet by discontinuous sucrose density gradient centrifugation. The RDDP assay involved measuring primer-dependent poly(dT) synthesis in the presence of poly(A) as template and oligo-(dT)12-18 as primer. To date, we have studied biopsy specimens from 46 patients with breast cancer. 27 (59%) had ERP and 23 (50%) were RDDP-positive. There was no significant correlation between ERP concentration and RDDP activity. PgR data were available on 36 of the patients; 17 (47%) were positive. No correlation between RDDP and PgR was apparent. Similarly, there was no correlation between RDDP and clinical stage of the disease.
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PMID:RNA-directed DNA polymerase activity in human breast cancer biopsy specimens. Relation to estrogen receptor protein. 620 38

A number of group II introns from eukaryotic organelles and prokaryotes contain open reading frames for polypeptides with homology to retroviral reverse transcriptases (RTs). We have used the yeast transposon (Ty) system to express ORFs for RTs from eukaryotic organelles. This includes the mitochondrial coxI intron i1 from the fungus Podospora anserina, the plastid petD intron from the alga Scenedesmus obliquus and the mitochondrial RTL gene from the alga Chlamydomonas reinhardtii. The ORFs were fused with the TYA ORF from the yeast retrotransposon Ty to produce virus-like particles in the recipient strains with detectable amounts of the RT-like polypeptides. Analysis of the heterologous gene products revealed biochemical evidence that the P. anserina intron encodes an RNA-directed DNA polymerase with properties typically found for RTs of viral or retrotransposable origin. In vitro assays showed that the intron encoded RT is sensitive to RT inhibitors such as N-ethylmaleimide and dideoxythymidine triphosphate but is insensitive against the DNA polymerase inhibitor aphidicolin. The direct biochemical evidence provided here supports the idea that intron encoded RTs are involved in intron transposition events.
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PMID:Reverse transcriptase activity of an intron encoded polypeptide. 751 30

The multifunctional HIV-1 RT (human immunodeficiency virus type 1-reverse transcriptase) enzyme possesses three main functions including the RNA- and DNA-directed DNA polymerases and the RNase H. The bisheteroarylpiperazine U-87201E inhibits the two polymerase functions but not the RNase H. Enzymatic kinetic studies of the HIV-1 RT-catalyzed RNA- and DNA-directed DNA polymerase activities were carried out in order to determine if the inhibitor interferes with either the template:primer or the deoxyribonucleotide triphosphate (dNTP)-binding sites of the enzyme. The data were analyzed using steady-state kinetics, considering that the polymerase reaction is ordered in that the template:primer is added first, followed by the dNTP and that the enzyme functions processively. The data were consistent with the model. The steady-state rate constants for the forward and backward reactions were of similar magnitude for both the RNA- and DNA-catalyzed DNA polymerases and suggest that both functions share the same substrate-binding sites. The dissociation constants for the enzyme-inhibitor and enzyme-substrate-inhibitor complexes were somewhat higher for the DNA-directed DNA polymerase function as compared to the RNA directed one. This indicates that U-87201E is a more potent inhibitor for the RNA-directed DNA polymerase than the DNA-directed DNA polymerase. The pattern of inhibition exerted by U-87201E was noncompetitive with respect to both the nucleic acid and nucleotide-binding sites of the RT enzyme for both the RNA- and DNA-directed DNA polymerases. Hence, U-87201E inhibits these functions by interacting with a site distinct from the template:primer and dNTP-binding sites. HIV-2 RT was insensitive to U-87201E, demonstrating the unique sensitivity of HIV-1 RT to this inhibitor.
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PMID:Steady-state kinetic studies with the non-nucleoside HIV-1 reverse transcriptase inhibitor U-87201E. 768 Oct 60

The quinoline U-78036 represents a new class of non-nucleoside human immunodeficiency virus (HIV)-1 reverse transcriptase inhibitors. The agent possesses excellent antiviral activity at nontoxic doses in HIV-1-infected lymphocytes grown in tissue culture. Enzymatic kinetic studies of the HIV-1 reverse transcriptase (RT)-catalyzed RNA-directed DNA polymerase function were carried out in order to determine whether the inhibitor interacts with the template-primer or deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. The data were analyzed using steady-state or Briggs-Haldane kinetics assuming that the template-primer binds to the enzyme first followed by the dNTP and that the polymerase functions processively. The calculated rate constants are in agreement with this model. The results show that the inhibitor acts as a mixed to noncompetitive inhibitor with respect to both the template-primer and the dNTP binding sites of the enzyme. Hence, U-78036 inhibits the RNA-directed DNA polymerase activity of RT by interacting with a site distinct from the template-primer and dNTP binding sites. Moreover, the potency of U-78036 is dependent on the base composition of the template-primer. The equilibrium constants for various enzyme-substrate-inhibitor complexes were at least seven times lower for the poly(rC).(dG)10-catalyzed system than the one catalyzed by poly(rA).(dT)10. In addition, the inhibitor does not impair the DNA-dependent DNA polymerase activity and the RNase H function of HIV-1 RT nor does it inhibit the RNA-directed DNA polymerase activity of the HIV-2, avian myoblastoma virus, and murine leukemia virus RT enzymes.
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PMID:The quinoline U-78036 is a potent inhibitor of HIV-1 reverse transcriptase. 768 7

U-31,355, or 4-amino-2-(benzylthio)-6-chloropyrimidine is an inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and possesses anti-HIV activity in HIV-1-infected lymphocytes grown in tissue culture. The compound acts as a specific inhibitor of the RNA-directed DNA polymerase function of HIV-1RT and does not impair the functions of the DNA-catalyzed DNA polymerase or the Rnase H of the enzyme. Kinetic studies were carried out to elucidate the mechanism of RT inhibition by U-31,355. The data were analyzed using Briggs-Haldane kinetics, assuming that the reaction is ordered in that the template:primer binds to the enzyme first, followed by the addition of dNTP, and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived that allows the calculation of all the essential forward and backward rate constants for the reactions occurring between the enzyme, its substrates, and the inhibitor. The results obtained indicate that U-31,355 acts as a mixed inhibitor with respect to the template:primer and dNTP binding sites associated with the RNA-directed DNA polymerase domain of the enzyme. The inhibitor possessed a significantly higher binding affinity for the enzyme-substrate complexes, than for the free enzyme and consequently did not directly affect the functions of the substrate binding sites. Therefore, U-31,355 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either inhibition of the phosphoester bond formation or translocation of the enzyme relative to its template:primer following the formation of the ester bond. Moreover, the potency of U-31,355 depends on the base composition of the template:primer in that the inhibitor showed a much higher binding affinity for the enzyme-poly (rC):(dG)10 complexes than for the poly (rA):(dT)10 complexes.
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PMID:The benzylthio-pyrimidine U-31,355, a potent inhibitor of HIV-1 reverse transcriptase. 860 69

We have previously reported that particles resembling retroviral particles and possessing an RNA-directed DNA polymerase activity can be prepared from platelets. Furthermore, we and others have shown that these particles are present at higher levels in patients with essential thrombocythemia and polycythemia vera. We show here that these particles package RNA molecules that encode HERV-K-related pol genes. A subset of the RNA molecules that are packaged are likely to encode the RNA directed DNA polymerase activity and, because these RNAs possess long/full-length open reading frames for the reverse transcriptase and RNaseH (also for part of the integrase domains in genomic clones) of HERV-K, we propose that these transcripts are indeed strong candidates for encoding the enzyme activity found in these particles. Moreover, by using a modification of the polymerase chain reaction-based reverse transcriptase assay in which activated DNA is added during cDNA synthesis to suppress DNA polymerase-mediated RNA-directed DNA synthesis, we have found that the particle-associated enzyme behaves like a retroviral reverse transcriptase, further supporting the conclusion that retrovirus-like, perhaps HERV-K sequences, encode this enzyme activity.
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PMID:Evidence for copurification of HERV-K-related transcripts and a reverse transcriptase activity in human platelets from patients with essential thrombocythemia. 935 71


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