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:6.5.1.2 (DNA ligase)
2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light-induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Initial yield of thymine dimers and rate of removal of these lesions from cellular DNA appeared normal in polyamine-depleted cells. However, depleted cells exhibited retarded sealing of DNA strand breaks resulting from cellular repair processes, reduced repair synthesis and an increased sensitivity to UV killing. Incision at damaged sites was not affected since ara-C repair-dependent breaks accumulated in a normal fashion. Molecular analysis of inhibited repair sites by exonuclease III and T4 DNA ligase probes suggest that the strand interruptions consist of gaps rather than ligatable nicks, consistent with an interpretation of the repair defect being at the gap-filling stage rather than the ligation step. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggests that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair.
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PMID:Effect of polyamine depletion on DNA damage and repair following UV irradiation of HeLa cells. 228 46

The effects of the DNA synthesis inhibitor 9-beta-D-arabinofuranosyladenine (beta-ara A), a nucleoside analogue of desoxyadenine, on repair of DNA single and double strand breaks (ssb and dsb) were investigated in X-irradiated Ehrlich ascites tumour cells. Repair of ssb was followed using the unwinding method, and repair of dsb was measured with both the unwinding and the neutral sucrose centrifugation methods. Repair of ssb was inhibited in the presence of beta-ara A; however, even at high concentrations some repair took place. It is suggested that this proportion of the breaks (about 30 per cent) are joined by polynucleotide ligase, and do not require insertion of nucleotides. Dsb repair was strongly inhibited by beta-ara A, the inhibition being complete at high concentrations. It seems likely therefore that dsb repair has an absolute requirement for DNA polymerization. When cells were treated with beta-ara A (200 mumols/1, 2 hours) after irradiation dsb repair was inhibited; however, when the drug was washed away, repair of dsb returned. At 6 hours more breaks were found to have persisted in beta-ara A treated cells than in the untreated controls. Cells treated after X-irradiation with beta-ara A for 7 hours at 120 mumols/1 in conditioned medium and afterwards in fresh medium free of beta-ara A for 24 hours showed a higher number of residual dsb than control cells. It is suggested that these residual dsb may be relevant to the increased killing effect caused by adding beta-ara A to X-irradiated cultures.
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PMID:The effects of 9-beta-D-arabinofuranosyladenine on the repair of DNA strand breaks in X-irradiated Ehrlich ascites tumour cells. 698 7

Our working hypothesis states that DNA damage is a critical step in toxic cell death. The DNA hypothesis was tested in cultured mouse hepatocytes by examining whether inhibitors of DNA repair would increase dimethylnitrosamine toxicity and DNA damage in parallel. Inhibitors were chosen for selectivity toward DNA polymerase alpha (aphidicolin, myricetin), DNA ligase (ethidium bromide), or multiple repair enzymes (ara-C, doxorubicin). Dimethylnitrosamine caused concentration-dependent DNA damage at 6 hr and cell death at 24 hr (35% ALT release vs. 8.8% in control cultured hepatocytes). Each repair inhibitor increased dimethylnitrosamine-induced DNA damage and toxic cell death in parallel. Doxorubicin maximally elevated DNA fragmentation and toxicity (57% ALT release). Repair inhibitors alone failed to damage DNA or cause cell death in this model system. These data support the hypothesis that DNA damage is an early causal event in toxic cell death caused by alkylating hepatotoxicants.
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PMID:DNA as a critical target in toxic cell death: enhancement of dimethylnitrosamine cytotoxicity by DNA repair inhibitors. 799 86

Heavy metals, including zinc (Zn) and cadmium (Cd), are potentially important genotoxic agents in our environment. Here we report that human DNA ligase I, the major form of the enzyme in replicative cells, is a target for Zn and Cd ions. ZnCl2 at 0.8 mM caused complete inhibition of DNA ligase I activity, whereas only 0.04 mM CdCl2 was required to achieve a similar effect. Both metals affected all three steps of the reaction, namely, the formation of ligase-AMP intermediate, the transfer of the AMP to DNA and the ligation reaction that succeeds the formation of the AMP-DNA complex. Unlike F-ara-ATP and the natural protein inhibitor of DNA ligase-I, these metals may affect different domains of the enzyme. Moreover, these metal ions did not increase the rate of misligation of F-ara-A-modified DNA or mismatched DNA substrates, but considerable misligation was observed for the T:C mispairing. These data support the notion of high fidelity of the human DNA ligases and that the major action of these metal ions on the enzyme is their inhibitory function.
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PMID:Inhibition of human DNA ligase I activity by zinc and cadmium and the fidelity of ligation. 869 42

In the past decade, fludarabine has had a major impact in increasing the effectiveness of treatment of patients with indolent B-cell malignancies. This has come about in a variety of clinical circumstances, including use of fludarabine alone as well as in combinations with DNA-damaging agents or membrane-targeted antibodies. Other strategies have used fludarabine to reduce immunological function, thus facilitating non-myeloablative stem cell transplants. Fludarabine is a prodrug that is converted to the free nucleoside 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) which enters cells and accumulates mainly as the 5'-triphosphate, F-ara-ATP. The rate-limiting step in the formation of triphosphate is conversion of F-ara-A to its monophosphate, which is catalyzed by deoxycytidine kinase. Although F-ara-A is not a good substrate for this enzyme, the high specific activity of this protein results in efficient phosphorylation of F-ara-A in certain tissues. F-ara-ATP has multiple mechanisms of action, which are mostly directed toward DNA. These include inhibition of ribonucleotide reductase, incorporation into DNA resulting in repression of further DNA polymerisation, and inhibition of DNA ligase and DNA primase. Collectively these actions affect DNA synthesis, which is the major mechanism of F-ara-A-induced cytotoxicity. Secondarily, incorporation into RNA and inhibition of transcription has been shown in cell lines. With the standard dose of fludarabine (25 to 30 mg/m(2)/day given over 30 minutes for 5 days), plasma concentrations of about 3 micromol/L F-ara-A are achieved at the end of each infusion. Serial sampling of leukaemia cells from patients receiving these standard doses of fludarabine has demonstrated that the peak concentrations of F-ara-ATP are achieved 4 hours after start of fludarabine infusion. Although there is heterogeneity among individuals with respect to rate of F-ara-ATP accumulation, the peak concentrations are generally proportional to the dose of the drug. Knowledge of the plasma pharmacokinetics of its principal nucleoside metabolite F-ara-A, and the cellular pharmacology of the proximal active metabolite, F-ara-ATP, has provided some understanding of the activity of fludarabine when used as a single agent. Preclinical studies directed toward learning the mechanisms of action of this agent have formed the basis for several mechanism-based strategies for its combination and scheduling with other agents. As a single agent fludarabine has been effective for the indolent leukaemias. Biochemical modulation strategies resulted in enhanced accumulation of cytarabine triphosphate and led to the use of fludarabine for the treatment of acute leukaemias. Combination of fludarabine with DNA damaging agents to inhibit DNA repair processes has been highly effective for indolent leukaemias and lymphomas. The current review brings together knowledge of the mechanisms of fludarabine, the state of understanding of the plasma pharmacokinetics, and cellular pharmacodynamics of fludarabine nucleotides. This may be useful in the design of future therapeutic approaches.
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PMID:Cellular and clinical pharmacology of fludarabine. 1188 30

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.
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PMID:Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells. 1237 42

We examined the hypothesis that processes related to DNA recombination and repair are involved in learning and memory. Rats received intracerebroventricular (i.c.v.) infusions of the antimetabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) or its precursor cytosine arabinoside (ara-C) 30 min prior to conditioned taste aversion (CTA) training. Both ara-CTP and ara-C caused significant impairments in long-term memory (LTM) of CTA. Control experiments indicate that the effect of ara-CTP on CTA memory is related to interference with learning. Furthermore, as it was previously demonstrated for the protein synthesis inhibitor anisomycin, ara-CTP had no effect on CTA memory when it was injected 1 h after training. Importantly, although both ara-CTP and anisomycin significantly blocked LTM in the task, short-term memory (STM) measured 1 h after training was not affected by either of the drugs. Finally, ara-CTP had no effect on in vitro transcription, but it did effectively block nonhomologous DNA end joining (NHEJ) activity of brain protein extracts. We suggest that DNA ligase-mediated DNA recombination and repair processes are necessary for the expression of LTM in the brain.
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PMID:The antimetabolite ara-CTP blocks long-term memory of conditioned taste aversion. 1465 61

Genomic recombination requires cutting, processing, and rejoining of DNA by endonucleases, polymerases, and ligases, among other factors. We have proposed that DNA recombination mechanisms may contribute to long-term memory (LTM) formation in the brain. Our previous studies with the nucleoside analog 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP), a known inhibitor of DNA ligases and polymerases, showed that this agent blocked consolidation of conditioned taste aversion without interfering with short-term memory (STM). However, because polymerases and ligases are also essential for DNA replication, it remained unclear whether the effects of this drug on consolidation were attributable to interference with DNA recombination or neurogenesis. Here we show, using C57BL/6 mice, that ara-CTP specifically blocks consolidation but not STM of context fear conditioning, a task previously shown not to require neurogenesis. The effects of a single systemic dose of cytosine arabinoside (ara-C) on LTM were evident as early as 6 h after training. In addition, although ara-C impaired LTM, it did not impair general locomotor activity nor induce brain neurotoxicity. Importantly, hippocampal, but not insular cortex, infusions of ara-C also blocked consolidation of context fear conditioning. Separate studies revealed that context fear conditioning training significantly induced nonhomologous DNA end joining activity indicative of DNA ligase-dependent recombination in hippocampal, but not cortex, protein extracts. Finally, unlike inhibition of protein synthesis, systemic ara-C did not block reconsolidation of context fear conditioning. Our results support the idea that DNA recombination is a process specific to consolidation that is not involved in the postreactivation editing of memories.
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PMID:An inhibitor of DNA recombination blocks memory consolidation, but not reconsolidation, in context fear conditioning. 1670 4

The selection of nucleoside triphosphates by a polymerase is controlled by several energetic and structural features, including base pairing geometry as well as sugar structure and conformation. Whereas base pairing has been considered exhaustively, substantially less is known about the role of sugar modifications for both nucleotide incorporation and primer extension. In this study, we synthesized oligonucleotides containing 2'-fluoro-modified nucleosides with constrained sugar pucker in an internucleotide position and, for the first time, at a primer 3'-end. The thermodynamic stability of these duplexes was examined. The nucleoside 2'-deoxy-2'-fluoroarabinofuranosyluracil [U(2'F(ara))] favors the 2'-endo conformation (DNA-like), while 2'-deoxy-2'-fluororibofuranosyluracil [U(2'F(ribo))] favors the 3'-endo conformation (RNA-like). Oligonucleotides containing U(2'F(ara)) have slightly higher melting temperatures (T(m)) than those containing U(2'F(ribo)) when located in internucleotide positions or at the 3'-end and when correctly paired with adenine or mispaired with guanine. However, both modifications decrease the magnitude of DeltaH degrees and DeltaS degrees for duplex formation in all sequence contexts. In examining the thermodynamic properties for this set of oligonucleotides, we find entropy-enthalpy compensation is apparent. Our thermodynamic findings led to a series of experiments with DNA ligase that reveal, contrary to expectation based upon observed T(m) values, that the duplex containing the U(2'F(ribo)) analogue is more easily ligated. The 2'-fluoro-2'-deoxynucleosides examined here are valuable probes of the impact of sugar constraint and are also members of an important class of antitumor and antiviral agents. The data reported here may facilitate an understanding of the biological properties of these agents, as well as the contribution of sugar conformation to replication fidelity.
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PMID:Impact of sugar pucker on base pair and mispair stability. 1989 14

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