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)

Muscle biopsy specimens were obtained from 48 human immunodeficiency virus-infected patients suffering from various neuromuscular symptoms. Microscopic examination by conventional and electron microscopy revealed a characteristic structural myopathy associated with mitochondrial changes in 13 patients, all of whom had received long-term zidovudine therapy. The mean cumulative dose they had received (498 +/- 145 gm) was significantly higher than that of the other 14 zidovudine recipients of the study. They suffered from a progressive, usually painful, proximal myopathy with pronounced wasting, normal-to-moderately elevated creatine kinase levels, and a myopathic electromyographic pattern. The condition usually improved after withdrawal of the drug. Assay of mitochondrial enzymes, including succinate-cytochrome c reductase, cytochrome c oxidase, and citrate synthase, showed a decline in respiratory chain capacity. Southern blot analysis of mitochondrial DNA showed no abnormality. It is likely that mitochondrial dysfunction, probably resulting from drug-induced inhibition of the mitochondrial DNA polymerase, is implicated in the pathogenesis of this complication of zidovudine therapy.
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PMID:Zidovudine myopathy: a distinctive disorder associated with mitochondrial dysfunction. 189 64

2',3'-dideoxyguanosine 5'-triphosphate (ddGTP) was found to be an efficient substrate for DNA polymerase beta when activated DNA was used as the template.primer. Under the optimized reaction conditions with activated DNA, the rate of the incorporation of ddGTP into DNA was almost equal to that of the corresponding normal substrate dGTP. The Km value for ddGTP (1.8 microM) was smaller than that for dGTP (7.8 microM). In contrast, ddGTP was not utilized as a substrate for DNA polymerase gamma with any of the activated DNA and (dC)n.(dG)12-18 as the template primer. Other DNA polymerases such as DNA polymerase alpha, E coli DNA polymerase I and retroviral reverse transcriptase could poorly utilize ddGTP as a substrate. Some of the kinetic properties of DNA polymerase beta revealed toward ddGTP are also described. Since DNA polymerase beta plays a role in DNA repair, the present results predict possible appearance of cytotoxicity or clinical side effect(s) of 2',3'-dideoxyguanosine (ddG), known as a potent inhibitor of human immunodeficiency virus, when ddG is administered to the patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex.
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PMID:Differential utilization of 2',3'-dideoxyguanosine 5'-triphosphate as a substrate for various DNA polymerases. 193 1

Several dideoxynucleosides, including 3'-azido-2',3'-dideoxythymidine (zidovudine, azidothymidine, AZT), 2',3'-dideoxycytidine (ddC), and 2',3'-dideoxyinosine (ddI), have been shown to be potent inhibitors of human immunodeficiency virus (HIV) replication in human T cells and macrophages. These compounds undergo anabolic phosphorylation within target cells to a 3'-triphosphate moiety; as triphosphates, they act at the level of HIV DNA polymerase (reverse transcriptase). AZT has been shown to reduce the morbidity and mortality of patients with severe HIV infection and to at least temporarily ameliorate certain cases of HIV-induced dementia. In phase 1 studies, ddC and ddI have been shown to induce immunologic and virologic improvements in patients with AIDS or related disorders; phase 2 studies of ddC and ddI are underway. The use of these drugs can be associated with toxicity. AZT can cause bone marrow toxicity or myositis with prolonged use, ddC can cause peripheral neuropathy at high doses, and ddI can cause sporadic pancreatitis and peripheral neuropathy at high doses. For each compound, however, a therapeutic window exists in which an anti-HIV effect can be attained without short-term toxicity in most patients. Dose-intensity appears to be an important determinant of the toxicity of dideoxynucleosides. Studies are underway to explore how the therapeutic profiles of these compounds may be enhanced by attention to scheduling or through the use of combination therapy.
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PMID:Initial clinical experience with dideoxynucleosides as single agents and in combination therapy. 207 27

In a previous paper, we determined that treatment of lymphocytes with nonviable preparations of human immunodeficiency virus type 1 (HIV-1) results in an impairment of the phosphatidylinositol/protein kinase C pathway, most likely due to an inhibition of the cleavage of phosphatidylinositol bisphosphate into inositol trisphosphate and diacylglycerol, mediated by phospholipase C. Here we show that one consequence of these changes is a reduced phosphorylation of nuclear matrix-associated DNA topoisomerase II, resulting in an inhibition of the activity of this enzyme. Antibodies to the viral proteins suppressed the inhibitory effects caused by the HIV-1 preparation. Furthermore, the phytohemagglutinin A-caused augmentation of nuclear matrix-associated DNA polymerase alpha and beta activities was found to be abolished by coincubation with the HIV preparation or with the HIV-1 gp120. The phytohemagglutinin A-enhanced matrix association and processivity of DNA polymerase alpha was determined to be reduced if the lymphocytes were in contact with HIV-1 preparation. These results suggest that the reduced proliferative response of lymphocytes to phytohemagglutinin A in the presence of disrupted HIV-1 preparation is due to inhibition of at least two, perhaps separate, pathways, one involving protein kinase C resulting in a reduced phosphorylation of DNA topoisomerase II and the other changing the state of matrix association of DNA polymerase alpha and beta.
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PMID:Effect of nonviable preparations from human immunodeficiency virus type 1 on nuclear matrix-associated DNA polymerase alpha and DNA topoisomerase II activities. 215 2

Because the risk factors for human immunodeficiency virus (HIV) infection and hepatitis B (HBV) are similar and therefore coinfection is not uncommon, a detailed histological and immunohistochemical study of chronic hepatitis B infection in a group of 20 HIV positive Caucasian males (who did not have AIDS) and 30 HIV negative controls were undertaken. Using both the conventional histological classification and the Knodell histological activity index it was shown that HIV negative patients were more likely to have active disease and also more scarring than HIV positive patients. Hepatitis B surface antigen (HBsAg) expression was not significantly different between the two groups but expression of hepatitis Be antigen (HBeAg) and HBV-DNA polymerase was greater in those who were HIV positive. HIV positive patients are therefore more likely to have immunohistochemical markers of active viral replication, although histologically, liver disease is less severe. These findings have important implications for assessing the biopsy specimens in this group of patients and for treatment strategies aimed at improving their immune function.
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PMID:Histological and immunohistochemical study of hepatitis B virus in human immunodeficiency virus infection. 233 17

To further clarify whether hepatitis B virus is cytopathic, the degree of hepatic histological activity was assessed and compared with levels of replicating virus in serum and liver of 74 untreated patients with chronic hepatitis B. Male homosexuals (n = 35) had significantly greater levels of DNA polymerase (P less than 0.05) and a trend toward higher hepatitis B virus DNA levels than heterosexuals (n = 39). Significantly greater DNA polymerase and hepatitis B virus DNA levels were observed in homosexuals infected with human immunodeficiency virus than in heterosexuals (P less than 0.005 and P less than 0.01, respectively) or human immunodeficiency virus-negative homosexuals (P less than 0.03 for both). Moreover, a trend was observed for higher grades of hepatitis B core antigen staining in the human immunodeficiency virus-infected population than in the human immunodeficiency virus-negative cohort. Hepatitis B virus DNA and DNA polymerase levels in the 74 patients were inversely related to total histological scores, and the degree of portal infiltrate and periportal necrosis bore an inverse relationship to peripheral markers of viral replication in human immunodeficiency virus-negative homosexuals and heterosexuals. Taken together, the data support the view that hepatitis B virus is not cytopathic because the amount of replicating virus does not directly correlate with the degree of histological activity.
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PMID:Relationship between histology, aminotransferase levels, and viral replication in chronic hepatitis B. 236 97

The thymidine analog 3'-azido-3'-deoxythymidine (BW A509U, azidothymidine) can inhibit human immunodeficiency virus (HIV) replication effectively in the 50-500 nM range [Mitsuya, H., Weinhold, K. J., Furman, P. A., St. Clair, M. H., Nusinoff-Lehrman, S., Gallo, R. C., Bolognesi, D., Barry, D. W. & Broder, S. (1985) Proc. Natl. Acad. Sci. USA 82, 7096-7100]. In contrast, inhibition of the growth of uninfected human fibroblasts and lymphocytes has been observed only at concentrations above 1 mM. The nature of this selectivity was investigated. Azidothymidine anabolism to the 5'-mono-, di-, and -triphosphate derivatives was similar in uninfected and HIV-infected cells. The level of azidothymidine monophosphate was high, whereas the levels of the di- and triphosphate were low (less than or equal to 5 microM and less than or equal to 2 microM, respectively). Cytosolic thymidine kinase (EC 2.7.1.21) was responsible for phosphorylation of azidothymidine to its monophosphate. Purified thymidine kinase catalyzed the phosphorylations of thymidine and azidothymidine with apparent Km values of 2.9 microM and 3.0 microM. The maximal rate of phosphorylation with azidothymidine was equal to 60% of the rate with thymidine. Phosphorylation of azidothymidine monophosphate to the diphosphate also appeared to be catalyzed by a host-cell enzyme, thymidylate kinase (EC 2.7.4.9). The apparent Km value for azidothymidine monophosphate was 2-fold greater than the value for dTMP (8.6 microM vs. 4.1 microM), but the maximal phosphorylation rate was only 0.3% of the dTMP rate. These kinetic constants were consistent with the anabolism results and indicated that azidothymidine monophosphate is an alternative-substrate inhibitor of thymidylate kinase. This conclusion was reflected in the observation that cells incubated with azidothymidine had reduced intracellular levels of dTTP. IC50 (concentration of inhibitor that inhibits enzyme activity 50%) values were determined for azidothymidine triphosphate with HIV reverse transcriptase and with immortalized human lymphocyte (H9 cell) DNA polymerase alpha. Azidothymidine triphosphate competed about 100-fold better for the HIV reverse transcriptase than for the cellular DNA polymerase alpha. The results reported here suggest that azidothymidine is nonselectively phosphorylated but that the triphosphate derivative efficiently and selectively binds to the HIV reverse transcriptase. Incorporation of azidothymidylate into a growing DNA strand should terminate DNA elongation and thus inhibit DNA synthesis.
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PMID:Phosphorylation of 3'-azido-3'-deoxythymidine and selective interaction of the 5'-triphosphate with human immunodeficiency virus reverse transcriptase. 243 Feb 86

The RNA-dependent DNA polymerase (RDDP) of human immunodeficiency virus (HIV) was purified from sucrose density gradient-banded virus by four successive procedures: anion exchange chromatography, cation exchange chromatography, affinity chromatography on oligo(dT)-cellulose and adsorption chromatography on hydroxyapatite. The enzyme preparation was free of cellular DNA-dependent DNA polymerase activity. The properties of HIV RDDP were determined with a variety of template-primers. Generally, the enzyme used Mg2+ for optimal activity except with (Cm)n X (dG)12-18 as template-primer. Kinetic data (Michaelis constant, Hill coefficient) were calculated for several substrates.
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PMID:Functional purification and enzymic characterization of the RNA-dependent DNA polymerase of human immunodeficiency virus. 243 65

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies.
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PMID:Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants. 244 94

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66


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