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)

During the fractionation of various enzymes concerned with DNA synthesis from the postmicrosomal supernatant fraction of various tissues, DNA polymerace [EC 2.7.7.7], thymidine kinase [EC 2.7.1.75], dTMP kinase [EC 2.7.4.9], deoxycytidine kinase [EC 2.7.1.74], and deoxycytidine monophosphokinase (dCMP kinase) [EC 2.7.4.14] were found in the pellet fraction of postmicrosomal supernatant. Further, the uridine kinase [EC 2.7.1.48] and aspartate transcarbamylase [EC 2.1.3.2] activities of postmicrosomal supernatant from various tissues were also present in this pellet fraction. The activities of DNA polymerase, thymidine kinase, uridine kinase, and aspartate transcarbamylase from normal and regenerating rat liver, and Yoshida sarcoma were higher in the pellet fraction than in the supernatant. On the other hand, the activities of dTMP kinase, dCMP kinase, and orotidine-5'-phosphate decarboxylase [EC 4.1.1.23] were lower in the pellet fraction than in the supernatant. The pellet fractions of regenerating rat liver and Yoshida sarcoma showed a remarkable incorporation of various precursors (thymidine, dTMP, deoxycytidine, and dCMP) into DNA in the presence of a suitable DNA template, ATP and all four deoxynucleoside 5'-triphosphates for DNA synthesis. Normal adult rat liver catalyzed a much smaller incorporation of all these precursors, except for dCMP.
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PMID:Intracellular distribution of various enzymes concerned with DNA synthesis from normal and regenerating rat liver, and Yoshida sarcoma. 113 86

2',3'-Dideoxy-3'-thiacytidine (cis-(+/-)-SddC) was found to have potent activity against hepatitis B virus and human immunodeficiency viruses in culture. Recent studies by us identified (-)-SddC as the stereoisomer responsible for the antiviral effect and showed that the cytotoxicity was mainly caused by (+)-SddC. Metabolism studies showed that these drugs were converted to their monophosphates, diphosphates, and triphosphates. The enzyme responsible for the formation of monophosphates was identified to be cytoplasmic deoxycytidine kinase in CEM cells. Uptake studies showed that the intracellular concentration of (-)-SddC and its metabolites was approximately 5-fold higher than that of (+)-SddC metabolites. (-)-SddCTP was more potent than (+)-SddCTP in inhibiting hepatitis B virus replication; (+)- and (-)-SddCTP exhibited minimal inhibition on polymerases alpha and delta, more inhibition on beta, and strong inhibition on gamma. In all cases, (+)-SddCTP was found to be more inhibitory than (-)-SddCTP to all four polymerases. (+)-SddCMP competed with dCTP for incorporation into DNA by DNA polymerase gamma and beta and served as a chain terminator; however, similar incorporation was not detected using other polymerases. The selective inhibition of DNA synthesis in isolated mitochondria by (+)- and (-)-SddCTP suggests a stereospecificity on the mitochondrial uptake of deoxynucleoside triphosphates.
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PMID:Biochemical pharmacology of (+)- and (-)-2',3'-dideoxy-3'-thiacytidine as anti-hepatitis B virus agents. 133 Oct 54

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate. 170 52

The main biochemical determinants involved in cytosine arabinoside (Ara-C) metabolism were studied in one lymphoblastic (Reh) and two myeloid (HL60 and K562) human leukemic cell lines exhibiting various sensitivities to Ara-C, Reh being the most and HL60 the least sensitive. The level of intracellular Ara-C accumulation and Ara-CTP formation was far more important in Reh cells than in myeloid cell lines but was not closely related to deoxycytidine kinase activity or to deoxycytidine triphosphate pool size. The level of Ara-C incorporated into DNA was similar in the three cell lines. Ara-CTP formation correlated better with the cytotoxicity to clonogenic cells than did Ara-C incorporation into DNA. DNA polymerase alpha was moderately inhibited to various degrees, depending on the cell line; this moderate inhibition does not seem sufficient to explain the inhibition of DNA synthesis. The activity of DNA ligase, the enzyme joining the Okazaki fragments, which was not detected in Reh cells, was strongly inhibited by Ara-C in HL60 and to a lesser degree, in K562 cells. The inhibition of DNA ligase probably also contributes to the inhibition of DNA synthesis and, thus, to the cytotoxic effect of Ara-C and may explain the smaller size of DNA fragments observed following Ara-C treatment. The variations in each critical determinant observed in these three cell lines increase the complexity and plurality of the mechanisms of Ara-C action.
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PMID:A study of the mechanisms of cytotoxicity of Ara-C on three human leukemic cell lines. 275 6

The acyclovir resistant mutant of varicella-zoster virus ACV-R (A 8) induced the same level of thymidine kinase activity in infected cells as the parent Kawaguchi strain. However, it induced less deoxycytidine kinase activity and did not induce phosphorylating activity for the nucleotide analogue, 9-(2 hydroxy-ethoxymethyl)-guanine-(acyclovir). Another acyclovir resistant mutant, ACV-R (A 4), which is cross-resistant to phosphonoacetate and is thought to be a viral DNA polymerase mutant, induced the same level of phosphorylating activities for thymidine, deoxycytidine and acyclovir as the parent strain. The altered substrate specificity of thymidine kinase induced by ACV-R (A 8) is concluded to confer resistance to acyclovir on ACV-R (A 8).
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PMID:Thymidine kinase with altered substrate specificity of acyclovir resistant varicella-zoster virus. 302 11

Peanut agglutinin (PNA), a D-galactose specific lectin, agglutinated 10-18% of lymphocytes isolated from tonsils of 3- to 6-yr-old children. PNA+ cells were found to be mainly B lymphocytes showing a 9.6 times higher specific activity of DNA polymerase alpha compared to the PNA- cells. The specific activity of deoxycytidine kinase as well as the incorporation of [5-3H]deoxycytidine were also much higher in PNA+ cell fraction than in PNA- fraction (7.7-fold and 6-fold, respectively). On the other hand, thymidine kinase activity and [5-3H]deoxythymidine incorporation were only 3.6 and 3.9 times higher, respectively. The data presented here show a high degree of DNA synthesis and preferential utilisation of [5-3H]deoxycytidine for DNA synthesis in this undifferentiated B lymphocyte fraction.
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PMID:Preferential utilisation of deoxycytidine by undifferentiated (peanut positive) tonsillar lymphocytes. 349 70

The total uptake, phosphorylation and incorporation of thymidine (dThd) and deoxycytidine (dCyd) were compared in intact and reversibly permeabilized human tonsillar lymphocytes. The total uptake of [3H]dThd was lower than that of [5-3H]dCyd, but almost all of [3H]dThd was incorporated into DNA. However, the main part of [5-3H]dCyd taken up by the lymphocytes was found in the pool as phosphorylated nucleoside (55%), and only a smaller part (13%) was incorporated into DNA. Phosphorylated nucleosides were determined by DEAE-cellulose sheets in the ethanol-soluble fraction of the cells. The reversible permeabilization of lymphocytes by Dextran T-150 destroys totally the [3H]dThd incorporation, while [5-3H]dCyd incorporation decreased only to 60% of intact cells. During permeabilization the phosphorylation of both nucleosides increased severalfold. After permeabilization all [3H]dThd was in dTMP form, while [5-3H]dCyd was also found in dCDP (3%) and dCTP (38%) form. In the meanwhile, 22% of thymidine kinase, 63% of deoxycytidine kinase and 98% of DNA polymerase activity were measured in permeabilized cells as compared to intact cells. The results suggest different relationships between the lymphocyte plasma membrane and the salvage pathways of the two pyrimidine nucleosides.
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PMID:Reversible permeabilization of lymphocytes destroys the incorporation of deoxythymidine but not of deoxycytidine. 394 81

The activities of the key enzymes of pyrimidine nucleotide and DNA syntheses in 43 human tumors and 28 normal human tissues were investigated. The activities of cytidine triphosphate synthetase, deoxycytidine monophosphate deaminase, uridine kinase, thymidine kinase, thymidine monophosphate kinase and DNA polymerase were markedly increased in tumor tissues, compared with those in the corresponding normal tissues, while the activities of deoxycytidine kinase, cytidine deaminase and deoxycytidine deaminase were only slightly increased. The use of thymidine and deoxyuridine as substrates of human pyrimidine nucleoside phosphorylase gave 1 to 2 orders of magnitude higher activity than that of uridine.
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PMID:Activities of various enzymes of pyrimidine nucleotide and DNA syntheses in normal and neoplastic human tissues. 628 2

The current study was undertaken to determine the relevance of leukemic blast cell proliferative activity, cellular parameters of Ara-C metabolism and the in vitro sensitivity to GM-CSF in association with the clinical response to TAD-9 induction therapy in 66 patients with de novo acute myeloid leukemia (AML). Proliferative activity was assessed by 3H-thymidine (3H-TdR) incorporation and thymidine kinase (TK) activity, parameters of Ara-C metabolism comprised the activities of deoxycytidine kinase (DCK) and DNA polymerase alpha (poly alpha) as well as Ara-CTP concentrations and 3H-Ara-C uptake into DNA. GM-CSF sensitivity was determined by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4 h concurrent exposure to GM-CSF and 3H-TdR (0.5 microCi/ml). The following results were obtained as expressed by median values and ranges: 3H-TdR incorporation: 1.07 pmol/10(5) cells (0.0-10.1), TK: 7.3 pmol/min/mg protein (1.3-56.0), DCK: 9.3 pmol/min/mg protein (0.77-47.1), poly alpha: 1.7 pmol/min/mg protein (0.00-28.9), Ara-CTP: 53.3 ng/10(7) cells (13.3-211.0), 3H-Ara-C uptake: 0.06 pmol/10(5) cells (0.0-0.57). 3H-Ara-C uptake was correlated with 3H-TdR incorporation (r = 0.74) and with the (S-phase dependent) activities of TK (r = 0.73) and poly alpha (r = 0.71, but not with DCK activity or intracellular Ara-CTP content. Blast cells of 37 from 55 analyzed patients were found to be sensitive to GM-CSF stimulation as defined by an increase in 3H-TdR incorporation > or = 1.5-fold over control values after the 48 h GM-CSF exposure. In vitro data were related with clinical response to TAD-9 induction therapy in 43 patients with newly diagnosed AML, taking the blast cell reduction at day 10 or 16 to < 5% or > or = 5% residual blasts as early parameter for adequate or inadequate response, respectively. While neither 3H-Ara-C uptake, nor intracellular Ara-CTP concentration, TK nor DCK activity were predictive for response, a high 3H-TdR incorporation and a high poly alpha activity were associated with adequate blast cell reduction. Median values of 3H-TdR incorporation were 2.26 pmol/10(5) cells for patients with adequate blast cell clearance and 0.80 pmol/10(5) cells for patients with inadequate blast cell clearance (P = 0.11), the respective values for poly alpha were 3.22 pmol/min/mg protein for responders and 1.1 pmol/min/mg protein for non-responders (P = 0.0085).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Blast cell proliferative activity and sensitivity to GM-CSF in vitro are associated with early response to TAD-9 induction therapy in acute myeloid leukemia. 747 75

Earlier studies have shown guanine arabinoside (ara-G) is an effective agent against growth of T-cell lines and freshly isolated human T-leukemic cells. However, poor water solubility of ara-G limits clinical use. 2-Amino-6-methoxypurine arabinoside (506U) is a water-soluble prodrug converted to ara-G by adenosine deaminase. 506U is not a substrate for deoxycytidine kinase, adenosine kinase, or purine nucleoside phosphorylase and is phosphorylated by mitochondrial deoxyguanosine kinase at a rate 4% that of ara-G phosphorylation. Mitochondrial DNA polymerase was the least sensitive to ara-GTP inhibition of the five human DNA polymerases tested. [3H]506U was anabolized to ara-G 5'-phosphates in CEM cells but not to phosphorylated metabolites of 506U. 506U was selective for transformed T over B cells and also inhibited growth in two of three monocytic lines tested. 506U given i.v. to cynomolgus monkeys was rapidly converted to ara-G; the ara-G had a half-life of approximately 2 h. 506U had in vivo dose-dependent efficacy against human T-cell tumors in immunodeficient mice. A Phase 1 trial of 506U against refractory hematological malignancies is now in progress at two study sites.
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PMID:2-Amino-6-methoxypurine arabinoside: an agent for T-cell malignancies. 761 70


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