Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the association between incorporation of 1-beta-D-arabinofuranosylcytosine (ara-C) into herpes simplex virus type 1 (HSV-1) DNA and mutagenesis at selected genetic loci, we grew virus in the presence or absence of ara-C and compared the mutation frequencies. Both the forward mutation frequency of HSV-1 (KOS) to the tk- phenotype and the reversion frequency of a temperature-sensitive mutant to the non-temperature-sensitive (ts+) phenotype were significantly increased following growth in ara-C. When the same viruses were grown in an inhibitor of the HSV-1 DNA polymerase that is not incorporated into DNA, no significant increase in the frequency of tk- or ts+ particles was observed. Furthermore, HSV-1 araAr, a mutant resistant to ara-C on the basis of reduced incorporation of the analog into viral DNA, did not demonstrate an increase in the number of tk- particles following growth in ara-C. These results suggest that mutagenesis of HSV-1 following growth in ara-C correlates with incorporation of the nucleoside analog into viral DNA.
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PMID:Incorporation of 1-beta-D-arabinofuranosylcytosine into DNA and mutagenesis of herpes simplex virus type 1. 301 Aug 54

The DNA polymerase activity, and susceptibilities to 9-beta-D-arabinofuranosyladenine(ara-A) and 1-beta-arabinofuranosylcytosine(ara-C) of a phosphonoacetic acid resistant mutant (PAA-R) of varicella-zoster virus (VZV) selected in the presence of PAA were examined. The DNA polymerase activity of PAA-R was inhibited less than that of the parent strain by PAA in vitro. PAA-R was resistant to acyclovir and also to both ara-A and ara-C. The susceptibilities to ara-A and ara-C of four acyclovir resistant mutants selected in the presence of acyclovir, and also resistant to PAA, were examined. Two variants were resistant, one was slightly resistant, and one was sensitive to both drugs. These cross-resistances and susceptibilities of VZV variants to PAA, ACV, ara-A and ara-C should be considered in chemotherapy of VZV infections.
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PMID:Susceptibilities of phosphonoacetic acid and acyclovir resistant varicella-zoster virus mutants to 9-beta-arabinofuranosyladenine and 1-beta-arabinofuranosylcytosine. 302 9

The biological activities of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil (2'F-ara-FU), 1-(3'-deoxy-3'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil (3'F-ara-FU) and 1-beta-D-arabinofuranosylthymine (ara-T) were compared in human cytomegalovirus (HCMV)-infected and noninfected human fibroblasts. 2'F-ara-FU inhibited HCMV plaque formation (ED50, 16 microM for AD 169 strain) at lower concentrations than 3'F-ara-FU (ED50, 100 microM for AD 169). These nucleoside analogues are expected to be phosphorylated to their 5'-phosphate forms by cellular thymidine kinase in HCMV-infected cells. The thymidine kinase activities in the virus-infected and noninfected cells were compared. Cellular thymidine kinase was increased in the virus-infected cells and showed better phosphorylation of 2'F-ara-FU than did 3'F-ara-FU. HCMV DNA polymerase was purified using affinity column chromatography, and the inhibitory effect of the 5'-triphosphate derivatives of 2'F-ara-FU (2'F-ara-FUTP) and 3'F-ara-FU (3'F-ara-FUTP) against viral and host DNA polymerase alpha was examined. No significant difference in the effectiveness of inhibition was observed between viral DNA polymerase and host polymerase alpha. However, viral polymerase incorporated 2'F-ara-FUTP into newly synthesized DNA, whereas polymerase alpha did not utilize 2'F-ara-FUTP as a substrate. Thus, viral polymerase differs from host polymerase alpha in its recognition and utilization of 2'F-ara-FUTP. This difference may be important to the design of selective antiviral agents for HCMV.
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PMID:A proposed mechanism for the selective inhibition of human cytomegalovirus replication by 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-fluorouracil. 303 45

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

Prior reports demonstrated more than additive cytotoxic effects of cis-diamminedichloroplatinum(II) (CDDP) and 1-beta-D-arabinofuranosylcytosine (ara-C) in LoVo colon carcinoma cells. We have extended these findings by analyzing mechanisms that may underlie the effect of ara-C on CDDP-induced cytotoxicity. In contrast to a previous study, ara-C neither enhances DNA interstrand cross-link formation by CDDP nor affects the excision of platinum from DNA. Features peculiar to ara-C, such as its misincorporation into DNA, probably contribute since more than additive cytotoxic effects do not occur by combinations of CDDP with inhibitors of DNA synthesis that are not incorporated into DNA. Also, while ara-C does not significantly enhance the degree of inhibition of DNA synthesis caused by CDDP, the recovery of DNA synthesis after drug removal is significantly slowed when cells are exposed to both drugs. These findings contrast with those obtained with CDDP and aphidicolin (the latter agent resembles ara-C in competing with dCTP for binding to DNA polymerase alpha but, unlike ara-C, is not incorporated into DNA). Lastly, ara-C is incorporated into LoVo cell DNA undergoing replicative synthesis as well as into DNA undergoing repair synthesis after CDDP-induced induced DNA damage.
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PMID:Interactions of cis-diamminedichloroplatinum(II) with 1-beta-D-arabinofuranosylcytosine in LoVo colon carcinoma cells. 310 9

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

Cytotoxicity of arabinofuranosylcytosine (ara-C) has been related in vitro to the inhibition of the DNA polymerase activities by arabinosylcytosine triphosphate (ara-CTP) and the incorporation of ara-C into the DNA where, acting as a chain terminator, it slows the chain elongation. Induced in vitro cellular resistance to ara-C was shown to be secondary to altered deoxycytidine (dCyd) kinase activity, dCyd deaminase activity, or deoxynucleotides triphosphates (dNTP) pools. Recent studies reported no differences of ara-C metabolism in cells obtained from leukemic patients at diagnosis and at relapse after ara-C therapy, suggesting that unknown cellular biochemical determinants may be involved in acquisition of ara-C resistance. Using dialysed crude extracts of leukemic cells obtained from patients at diagnosis, we observed variable inhibition of their DNA polymerase activities by arabinosylcytosine monophosphate (ara-CMP) at 2 mmol/L (0% to 50% inhibition). In similar conditions, ara-CMP reduced the polymerase activities of human thymus extract by 35% and 55% in extract of HL-60 cells (cultured human promyelocytic cells). The ara-CMP factor responsible for inhibition of DNA polymerase activity was nondialysable, heat labile, proteinase K sensitive, and has an estimated molecular mass of 30 kilodalton by gel filtration. After partial purification, this protein had no DNA polymerase RNA polymerase activities. In presence of the regulator and ara-CMP at 2 mmol/L, we observed no inhibition of the HL-60 3'----5' and 5'----3' exonucleases activities, suggesting the regulator interaction being mainly with the DNA polymerases in presence of ara-CMP. The relevance of the presence or absence of this protein regarding the cell sensitivity to ara-C is under investigation.
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PMID:Inhibition of DNA polymerase-alpha by ara-CMP in the presence of a regulatory protein extracted from human promyelocytic leukemic cells (HL-60). 347 78

The dose-response relationship between extracellular concentration of cytosine arabinoside (ara-C) and intracellular formation of the putative active metabolites of ara-C [ara-C incorporation into DNA and intracellular pools of ara-C in triphosphate form (ara-CTP)] was investigated in blast cells obtained from patients with acute nonlymphocytic leukemia (ANLL) by exposing these cells in vitro to 10, 100, or 1,000 nmol/L of ara-C. We studied 23 untreated patients who subsequently achieved complete remission (CR) with a regimen using daunorubicin and conventional doses of ara-C (ara-C-sensitive group), and 30 patients judged to be ara-C-resistant either by failing initial induction therapy (16 patients) or by having relapsed on an ara-C-containing maintenance regimen (14 patients). In both patient groups, ara-C incorporation into DNA and intracellular ara-CTP both displayed statistically significant increases in response to increasing extracellular concentrations of ara-C (P = .0001 in both cases), with the rate of increase of ara-CTP greater than that of ara-C incorporation. Moreover, blast cells from all patients, even those who were most clinically resistant to ara-C, were able to form ara-CTP and to incorporate ara-C into DNA. Each tenfold increment in extracellular ara-C concentration caused an 8.5-fold increase in ara-CTP, but only a 3.6-fold increase in ara-C incorporation into DNA. Thus, the efficiency of incorporation of ara-C into DNA (defined as the ratio of ara-C incorporation to ara-CTP pools) decreased by 58% with each tenfold increment in the extracellular concentration of ara-C (P less than .0001), presumably as a result of the inhibitory effect of ara-CTP on DNA polymerase. Using an analysis of covariance, modest differences were found in the levels of the ara-C metabolite variables in the ara-C-sensitive group as compared with the resistant group. However, because there was considerable overlap in ara-C metabolite formation among the patient groups, it was not possible to predict clinical outcome by these in vitro assessments of ara-C metabolism.
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PMID:Metabolism of ara-C by blast cells from patients with ANLL. 371 4

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

Recent studies in our laboratory and others have demonstrated that DNA polymerase inhibitors such as the ara nucleosides, aphicolin and dideoxythymidine are potent inhibitors of the DNA excision repair process in confluent human fibroblasts as evidenced by the agent-dependent accumulation of single-strand interruptions in the DNA of UV-irradiated, but not in unirradiated, cellular DNA. In rapidly cycling cells, on the other hand, these agents are weak inhibitors at best but when used in combination with the ribonucleotide reductase inhibitor, hydroxyurea, a significant enhancement of inhibitory capacity is seen. In an attempt to better understand the mechanism of repair inhibition by DNA polymerase inhibitors, and the nature of this hydroxyurea enhancement, experiments were initiated in which the effects of a series of ribonucleotide reductase inhibitors on dNTP pools and on the DNA repair process were determined in both quiescent cultures and log-phase cultures of human fibroblasts. It was determined that hydroxyurea, deoxyadenosine, pyridine-2-carboxaldehyde thiosemicarbazone (TSC), pyrozoloimidazole (IMPY), 3,5-diamino-1,2,4-triazole (guanazole), 3,4,5-trihydroxy benzohydroxamic acid (THBA) and 3,4-dihydroxy benzohydroxamic acid (DHBA) are all effective inhibitors of the DNA repair process in confluent cells but not in log-phase cells. Moreover, the effects of these inhibitors can be reversed by the addition of certain combinations of deoxynucleosides. These reversal studies and the direct analysis of dNTP pool modulation by these compounds in log phase and confluent cultures support the notion that specific pool depletions rather than general imbalance of pools gives rise to the inhibition of the DNA excision repair process.
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PMID:Effects of nucleotide pool imbalances on the excision repair of ultraviolet-induced damage in the DNA of human diploid fibroblasts. 388 72


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