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)

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 cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and alpha-polymerase. 310 55

1-beta-D-Arabinofuranosylcytosine (ara-C) is an effective antileukemic agent which acts as an inhibitor of DNA synthesis. The precise mechanism responsible for this inhibitory effect, however, remains unclear. The present work has examined the effects of the triphosphate derivative, ara-CTP, on purified DNA polymerase beta. These studies were performed on M13 phage DNA templates of defined sequence. The results demonstrate that ara-C is incorporated into DNA by DNA polymerase beta. The results also demonstrate that the incorporated ara-C residue acts as a relative chain terminator. Moreover, the relative chain terminating effects of ara-C are sequence specific. In this regard, DNA strand elongation was progressively slowed at sequences of two, three, and four contiguous sites for cytosine incorporation. We also demonstrate that the inhibitory effects of ara-C are reversed by competition with deoxycytidine-triphosphate for incorporation into the DNA strand. Taken together, these findings are consistent with structural differences of the incorporated arabinosyl moiety which alter reactivity of the 3'-terminus and thereby inhibit chain elongation. These findings also provide new insights regarding the inhibitory effects of ara-C on elongation of specific DNA sequences.
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PMID:Effects of 1-beta-D-arabinofuranosylcytosine incorporation on elongation of specific DNA sequences by DNA polymerase beta. 334 22

DNA primase (EC 2.7.7.6) produces an RNA oligomer of approximately 10 bases, which is required by DNA polymerase alpha (EC 2.7.7.7) for the initiation of DNA synthesis. We partially purified DNA primase from acute lymphocytic leukemia cells from patients using several chromatography columns. Poly(dT) and poly(dC), but not poly(dA) or poly(dG), were good templates for ribonucleoside triphosphate (rNTP)-dependent DNA synthesis (i.e., DNA primase activity), and they were used in the study of the effect of natural and arabinofuranosyl nucleoside triphosphates on DNA primase activity. The Km for GTP in the poly(dC) primase assay was approximately 175 microM. All noncomplementary natural rNTPs and deoxyribonucleoside triphosphates (dNTPs) inhibited poly(dC) primase activity to a similar extent (Ki values of ATP and CTP were 610 and 517 microM, respectively). 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP) and 9-beta-D-arabinofuranosyladenine 5'-triphosphate (araATP) were more potent inhibitors of poly(dC) primase activity than were CTP and ATP (Ki values were approximately 125 microM). araCTP, araATP, CTP, and ATP inhibited DNA primase activity in a manner competitive with GTP. The concentration required to inhibit poly(dC) DNA primase activity by 50% was determined for a number of arabinofuranosyl nucleoside triphosphate analogs, and the relative potency of inhibition of DNA primase activity was as follows: rNTP = dNTP = 5-aza-dCTP less than ara-5-azaCTP = araTTP = araATP = araCTP less than 2-fluoro-araATP = 2'-azido-2'-deoxy araCTP less than 2'-fluoro-araTTP = 2'-fluoro-5-iodo-araCTP = 2'-fluoro-5-methyl-araCTP. In the poly(dT) primase assay ATP did not follow classic Michaelis-Menten kinetics (ATP exhibited positive cooperativity with a Hill coefficient of 2.0). However, this assay was very sensitive to araCTP (apparent Ki of 25 microM). In summary, these experiments suggested that DNA primase is controlled by the levels of ribonucleoside triphosphates, and that the perturbation of these pools by any agent could lead to the inhibition of DNA primase and thereby inhibit DNA synthesis. Furthermore, aranucleoside triphosphate analogs directly inhibited DNA primase, and it is possible that this effect may contribute to the cytotoxicity of these compounds.
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PMID:Inhibition of DNA primase by nucleoside triphosphates and their arabinofuranosyl analogs. 380 92

Somatomotor cortex of mice with microcephaly induced by DNA polymerase inhibitor cytosine arabinoside (Ara-C), has been studied with a modified Golgi-Cox staining and a HRP retrograde tracing method. Microcephalic mice were prepared by prenatal injections of cytosine arabinoside on days 13.5 and 14.5 of pregnancy. Cytoarchitectonically, the cerebral cortices of adult microcephalic mice are characterized by atypical pyramidal cells with abnormal dendrites and irregular patterns of cellular lamination. Semiquantitative analyses of the abnormality of dendrites in Golgi-Cox preparation indicate that both the degree and direction of ramification are severely affected in Ara-C treated mice. In adult control cerebrum, original neurons of corticospinal tract labeled after HRP injection into the lumbar cord were situated in layer V. In the microcephalic brains, however, HRP labeled neurons, some of which had abnormal polarity, were scattered throughout all layers. This HRP study for corticospinal tract neurons also confirms the irregular pattern of the cortex in which only three layers are recognized.
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PMID:Neuroanatomical study of somatomotor cortex in microcephalic mice induced by cytosine arabinoside. 608 50

9-beta-D-Arabinofuranosyladenine (ara-A), 1-beta-D-arabinofuranosylcytosine (ara-C), and their 5'-triphosphates (ara-ATP and ara-CTP) were tested for ability to inhibit the hepatitis B virus (HBV)-associated deoxyribonucleic acid (DNA) polymerase. Ara-C did not inhibit the HBV DNA polymerase at the concentrations tested, ara-A did so by 50% at a concentration of 30 mM, with the inhibition noncompetitive with respect to deoxyadenosine 5-triphosphate (dATP). Ara-ATP and ara-CTP inhibited the DNA polymerase test competitively with respect to dATP and dCTP, respectively. Both compounds were also active after initiation of the DNA polymerase reaction. The inhibition caused by ara-ATP and ara-CTP was shown to be reversible, with no evidence that ara-ATP or ara-CTP was incorporated into the HBV DNA.
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PMID:Inhibition of hepatitis B virus deoxyribonucleic acid polymerase by the 5'-triphosphates of 9-beta-D-arabinofuranosyladenine and 1-beta-D-arabinofuranosylcytosine. 616 46

Ara-C should be converted to ara-CTP to inhibit DNA polymerase in the malignant cells but is rapidly inactivated to uracil arabinoside (ara-U) by cytidine deaminase in human tissue. Therefore, production as well as maintenance of ara-CTP in the cells is a function of both phosphorylation and deamination of ara-C, but is more dependent on the latter, because the deamination is several times superior to the former in terms of enzymatic activities. In chemotherapy with ara-C, the rate of the inactivation should be estimated for evaluating antitumor effect of the agent. Determination of serum or plasma deaminase activity can be a useful parameter of the inactivation. Attempts have been made to enhance the antitumor activity of ara-C by preventing deamination and a number of ara-C derivatives resistant to the deamination such as cyclocytidine, ara-C-5'-ester and acyl ara-C have been introduced. Cyclo-C gradually receives non-enzymatic hydrolysis to produce ara-C in neutral medium, which is useful for maintaining plasma ara-C level. Acyl ara-C such as behenoyl-ara-C (BHAC) is well incorporated into the cells and is highly distributed to lipophilic components such as membrane, microsome and mitochondria in the cells. The extremely gradual conversion of BHAC to ara-C in the cells is considered to be useful for maintaining effective intracellular concentration. A part of BHAC could be phosphorylated before deacylation. After intravenous administration of BHAC, the plasma drug concentrations are maintained significantly longer than those after the administration of the equivalent dose of ara-C. Therefore, BHAC is more resistant to the deamination than cyclo-C and the antitumor effect of the former is suspected to be milder but prolonged than that of ara-C or cyclo-C.
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PMID:[Chemotherapy of the malignancies from the viewpoint of pharmacology and biochemistry of cytosine arabinoside (ara-C) and its derivatives]. 619 11

Arabinocytidine and aphidicolin are inhibitors of alpha-DNA polymerase that have been shown to affect both normal DNA replication and repair synthesis in mammalian cells. In contradiction to the prevalent hypothesis that these inhibitors merely slow the polymerization rate at incision sites near lesions, our results suggest that the repair synthesis resistant to inhibitors is mediated by a separate pathway. Repair synthesis in contact-inhibited human cells following UV irradiation was inhibited 75-80% by arabinocytidine or aphidicolin, and most of the repair patches were not ligated into parental DNA, as judged by an enzymatic assay. However, the patches were not demonstrably shorter than those in untreated cells. Even following low-UV doses at which no inhibition of repair synthesis by the inhibitors was observed, a majority of the patches were not ligated. DNA polymerase beta is implicated in this alternate pathway, both by the known specificity of the inhibitors and by evidence from their sensitivity to S1 nuclease that the patches arise from displacement synthesis. The unligated patches are not degraded in vivo and eventually become ligated into parental DNA, very slowly in the presence of inhibitors but much more rapidly following their removal. Thus, under conditions of alpha-polymerase inhibition, a limited number of normal length repair patches are made, apparently by displacement synthesis, leaving displaced strands that remain substantially undegraded.
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PMID:Nature of DNA repair synthesis resistant to inhibitors of polymerase alpha in human cells. 642 5

1-beta-D-Arabinofuranosylcytosine (ara-C) incorporates into DNA, and the extent of this incorporation correlates significantly with inhibition of DNA synthesis. The incorporated ara-C residue provides a poor primer terminus for further chain elongation. There is a highly significant relationship between formation of (ara-C) DNA and loss of clonogenic survival. The present studies confirm that incorporation of ara-C into DNA, and not the competitive inhibition of DNA polymerase, is responsible for inducing lethal cellular events. The results also demonstrate that the incorporated ara-C residue is not excised from the DNA strand. Furthermore, the presistence of ara-C residues in DNA inhibits recovery of DNA synthesis following exposure to drug. The relative DNA chain-terminating effect of ara-C provides several mechanisms of action that explain internucleotide and chain terminus positioning of ara-C residues, reinitiation of previously replicated DNA segments, and DNA strand or chromosomal breaks. The precise mechanism of action is dependent upon dose scheduling of this drug.
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PMID:Effects of 1-beta-D-arabinofuranosylcytosine incorporation on eukaryotic DNA template function. 643 Dec 61

We studied the ability of 2'-deoxyguanosine (dGuo) to influence 1-beta-D-arabinofuranosylcytosine (ara-C) inhibition of soft agar cloning of the cultured human leukemia cell line K562. Ara-C alone inhibited cloning in concentrations of greater than 10 nM, with a steep drop in colony formation observed between 10 and 100 nM. dGuo and ara-C synergistically inhibited cloning; the combination of ineffective concentrations of dGuo (10-50 microM) and ara-C (less than or equal to nM) inhibited cloning by 40-70%. In K562 cells, dGuo is metabolized by both nucleoside kinase and purine nucleoside phosphorylase (PNP), resulting in augmentation of both the GTP pool (to more than 200% of control after a 3 hr incubation with 500 microM dGuo) and the dGTP pool (to more than 2700% of control after 3 hr with 500 microM dGuo). dGuo (50-500 microM) caused a decrease in the dCTP and dTTP pools and an increase in the dATP pool. Synergistic concentrations of dGuo plus 10 nM ara-C augmented the ara-CTP pool up to 800% of control after 3 hr to levels equivalent to those observed after incubation with 500 nM ara-C alone. Incorporation of 10 nM ara-CTP into DNA also increased in the presence of dGuo (up to a maximum of 300% of control), but only to a level that approximated the value observed with nM ara-C alone. The disparity between enlargement of the ara-CTP pool and augmentation of ara-C incorporation into DNA is consistent with the observation of Steinberg et al. [Cancer Res. 39, 4330 (1979)] that high concentrations of dGTP may inhibit DNA polymerase activity. Thus, synergy between dGuo and ara-C is multifactorial, possibly involving inhibition of DNA polymerase by elevated dGTP and ara-CTP pools and augmented incorporation of ara-C into DNA.
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PMID:Synergistic inhibition of human leukemia cell growth by deoxyguanosine and 1-beta-D-arabinofuranosylcytosine. 671 15


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