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)

We have demonstrated previously that 1-beta-D-arabinofuranosylcytosine (ara-C) incorporates specifically in cellular DNA and that the formation of (ara-C)DNA correlates significantly with inhibition of DNA synthesis and loss of clonogenic survival. Similar results have been obtained with 9-beta-D-arabinofuranosyl-adenine (ara-A). These findings have been extended by studying the incorporation of ara-C in DNA of a wild-type herpes simplex virus (HSV) and a mutant virus resistant to ara-C and ara-A. The results demonstrate that HSV resistance to ara-A is associated with formation of less (ara-C)DNA and less inhibition of DNA synthesis when compared to wild-type virus. This effect on formation of (ara-C)DNA is reversed upon exposure to higher (greater than 10(-6) M) ara-C concentrations, and this pattern of resistance corresponds to drug effect on virus plaque formation. The results also demonstrate a highly significant relationship between incorporation of ara-C in HSV DNA and inhibition of DNA synthesis for both viruses. Further, higher concentrations of ara-C that result in greater inhibition of DNA synthesis are associated with an increasing number of ara-C residues at the 3'-terminus of the DNA strand, thus suggesting that ara-C functions as a poor primer terminus for viral chain elongation. These results also suggest that HSV cross-resistance to ara-A and ara-C may be related to an altered viral DNA polymerase and that incorporation of ara-C in HSV DNA is at least one mechanism responsible for slowing viral synthesis and inducing lethal events.
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PMID:Incorporation of 1-beta-D-arabinofuranosylcytosine into DNA from herpes simplex virus resistant to 9-beta-D-arabinofuranosyladenine. 631 73

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2 X 10(6) Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 microM. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 microM, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 microM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.
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PMID:DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs. 632 Aug 90

We investigated, in a cloned hamster tracheal epithelial cell line HTE-B, the effects of inhibitors of DNA topoisomerase, novobiocin and nalidixic acid; of DNA polymerase, 1-beta-arabinofuranosylcytosine (ara-C) and 2',3'-dideoxythymidine; of ribonucleotide reductase, hydroxyurea; and of poly(ADP-ribose)synthetase, 3-aminobenzamide, upon the removal of benzo[a]pyrene adducted to DNA [B[a]P--DNA]. A substantial reduction in the rate of removal of the polycyclic hydrocarbon-adducts occurred when nalidixic acid was added to the HTE-B cells that had been previously incubated with B[a]P for 8 h. Novobiocin produced a similar, but less marked, effect. The rate of disappearance of the individual B[a]P--DNA adducts was measured by analysis of the h.p.l.c. profiles. Of the 5 major adducts observed under the h.p.l.c. conditions, 4 were reduced in control cells to 30% of the original levels by 24 h after removal of the B[a]P from the medium; adduct 5 was almost completely removed. In the presence of nalidixic acid, during the 24 h repair period, only the removal of adduct 5 was unimpaired; the removal of the other 4 adducts was significantly retarded. On the other hand, 3-aminobenzamide addition did not affect the rate of removal of B[a]P--DNA adducts from the HTE-B cells. We employed the combinations of ara-C and dideoxythymidine or ara-C and hydroxyurea to allow the accumulation of single strand breaks after incubation of the HTE-B cells with B[a]P. These breaks were assayed by alkaline elution analysis. Inclusion of these inhibitors during the 2 h after removal of the B[a]P from the medium resulted in the accumulation of 4-5 single strand breaks/10(10) daltons of HTE-B DNA. This compares with a minimum estimate of the number of adducts removed during this period of 3 adducts/10(7) daltons. This discrepancy may indicate that the majority of lesions are not repaired by a pathway sensitive to polymerase inhibitors. In the presence of 3-aminobenzamide, we routinely observed a 10% increase in the alkaline elution of the DNA obtained from B[a]P-treated cells (1-2 breaks/10(10) daltons). Our results indicate that an excision repair process may be involved in the removal of at least some of the B[a]P-induced damage to DNA. However, the repair of the multiple adducts is complex and may involve pathways other than classical excision repair.
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PMID:The influence of inhibitors on the repair of benzo[a]pyrene-damaged DNA in hamster tracheal epithelial cells. 632 Oct 50

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

It is known that mammalian cells repair X-ray-induced double-strand breaks (DSB). The mechanism of this repair is, however, as yet unknown but it is thought that the repair may involve recombination between homologous DNA strands. We have investigated the presence and kinetics of occurrence of recombinational molecular intermediates or 'heavy-heavy' (HH) hybrids in DNA of X-irradiated mouse Ehrlich ascites tumour cells unifiliarly substituted with bromodeoxyuridine. Purified DNA from density-labelled cells was analysed using isopyknic CsCl density centrifugation. After rebanding of the high-density material, the relative amount of HH-hybrid material was determined. When the cells were incubated after X-ray exposure, hybrids accumulated with an apparently biphasic kinetic; first a rapid accumulation directly after irradiation followed by a second, more slowly appearing peak. When DSB repair was inhibited by the nucleoside analogue 9-beta-D-arabinofuranosyladenine (ara-A), a powerful DNA polymerase inhibitor, the kinetics of the HH-hybrid formation were similar to those during the incubation after X-irradiation without ara-A. We interpret this as indicating that the first step in repair of DSB i.e. hybrid formation, occurs in the absence of DNA synthesis as predicted by the Resnick model of DSB repair. In an experiment in which the ara-A was washed away from the irradiated cells after 8-h treatment with the drug and replaced by fresh growth medium, so allowing DSB to repair, a similar HH-hybrid kinetic response was found to that occurring directly after irradiation in the absence of ara-A. The reasons for this are not yet clear. In this case, however, the response of the ara-A-treated cells after X-irradiation was much stronger than that in the untreated cells where only approximately 20% of the DSB remained. These kinetic results which show a temporary appearance of HH hybrids, indicate that a net exchange of genetic material does not take place; they therefore lend support to the postulated recombinational model of Resnick in which a temporary exchange between homologous DNA strands takes place during DSB repair.
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PMID:Kinetics of recombinational hybrid formation in X-irradiated mammalian cells: a possible first step in the repair of DNA double-strand breaks. 649 59

The triphosphates of acyclovir (ACV), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) and E-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) have been examined for their inhibitory effects on the endogenous DNA polymerase reactions of human hepatitis B virus (HBV) and woodchuck hepatitis virus (WHV). All three triphosphates (ACVTP, FIACTP and BVdUTP) inhibited the HBV and WHV DNA polymerases by competing with the corresponding natural substrates. FIACTP was the most potent inhibitor of HBV and WHV DNA polymerase while ACVTP was the least effective inhibitor. The inhibitory properties of these compounds were compared with those of the 5'-triphosphates of 1-beta-arabinofuranosyl-cytosine (ara-CTP) and 1-beta-arabinofuranosylthymine (ara-TTP). The 50% inhibitory doses for HBV and WHV DNA polymerases were in the following order: FIACTP less than BVdUTP less than ara-TTP less than ACVTP less than ara-CTP. BVdUTP appeared to be an efficient alternate substrate to dTTP for HBV DNA polymerase while FIACTP was much less efficient when substituted for dCTP. ACVTP did not act as an alternate substrate to dGTP and appeared to prevent DNA chain elongation.
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PMID:Inhibition of human and woodchuck hepatitis virus DNA polymerase by the triphosphates of acyclovir, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine and E-5-(2-bromovinyl)-2'-deoxyuridine. 654 55

Effects of various nucleoside analogues on X-ray-induced-PLD recovery (PLDR) were examined in plateau phase Chinese hamster HA-1 cells. Among the chemicals tested, 3'-dA (3'-deoxyadenosine) and ara-A (9-beta-D-arabinofuranosyladenine) were most potent inhibitors of PLDR at their slightly toxic doses. N6-butyryl-3'-dA and 3'-dG (3'-deoxyguanosine) were the most effective in suppressing PLDR at non-toxic doses. A specific inhibitor of DNA polymerase beta, 2', 3'-ddT (dideoxythymidine) was intermediately effective. However, possibly due to the lower intracellular incorporation or phosphorylation, 3'-deoxy-pyrimidine analogues and formycin B were less or non-effective. The enhancement of antitumor effect of cyclophosphamide by ara-A and 3'-dG was observed in SCC VII tumors in vivo. The involvement of DSB (or chromosome aberration) and SSB as well as base damage or crosslinks in PLD is suggested, since recently they have been shown not to be rejoined when treated with various agents such as hyperthermia and ara-A.
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PMID:PLDR inhibitors: their biological and clinical implications. 660 38

Inhibitory effects of 1-beta-D-arabinofuranosyluracil 5'-triphosphate (ara-UTP) and its 5-alkylated derivatives [1-beta-D-arabinofuranosylthymine 5'-triphosphate (ara-TTP): 1-beta-D-arabinofuranosyl-5-ethyluracil 5'-triphosphate (ara-EtUTP); 1-beta-D-arabinofuranosyl-5'-propyluracil 5'-triphosphate (ara-PrUTP); 1-beta-D-arabinofuranosyl-5-butyluracil 5'-triphosphate (ara-BuUTP)] on the activity of terminal deoxynucleotidyl-transferase (TdT) from calf thymus were examined. All these compounds inhibited TdT activity by competition with the natural substrate dTTP for the same substrate-binding site of the enzymes. The extent of inhibition by the inhibitor, however, decreased by introducing an alkyl group on the 5-position of the uracil nucleus, indicating the importance of inductive and steric effects of the 5-substituents on TdT. Of the four 5-alkylated ara-UTP's, ara-EtUTP was less inhibitory to TdT than the other compounds, and the potency of inhibition was restored by replacing 5-ethyl group with longer propyl or butyl group, suggesting that the hydrophobic effects of these 5-alkyl side chains are involved in the inhibitory action of the compounds. The results are compared and discussed with those of our previous report on the inhibition of DNA polymerase alpha and beta by these 5-alkylated ara-UTP's (Ono et al., 1981).
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PMID:Inhibition of terminal deoxynucleotidyltransferase by various 5-alkylated derivatives of 1-beta-arabinofuranosyluracil 5'-triphosphate: substituent effects on inhibitory action. 667 Nov 33

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

The HL-60 human leukemic promyelocyte can be induced to mature into terminally differentiated cells using certain nucleosides and chemotherapeutic agents. The mechanisms responsible for this induction of differentiation, however, remain unclear. We have monitored the effects of two specific inhibitors of DNA synthesis to determine whether slowing of DNA polymerization can induce HL-60 differentiation. The results demonstrate that cytosine arabinoside (ara-C) induces nonspecific esterase activity in HL-60 cells and increases surface expression of the monocyte antigen MY-4. The results also demonstrate that aphidicolin, an inhibitor of DNA polymerase which is not incorporated in DNA, induces similar phenotypic changes. The induction of differentiation by both agents was accompanied by loss of clonogenic potential as monitored by colony formation in methylcellulose. These observations suggest that terminal differentiation of HL-60 cells can be induced by drugs known to inhibit DNA synthesis.
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PMID:Induction of differentiation of human myeloid leukemia cells by inhibitors of DNA synthesis. 681 25


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