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 cytotoxicity of ara-C is believed to result from incorporation of ara-CTP into DNA and inhibition of DNA synthesis. Since complete inhibition of DNA synthesis would prevent further incorporation of ara-CTP, ara-C may have a self-limiting effect on its own cytotoxicity, particularly at the high concentrations typical of high-dose ara-C clinical protocols. In this study, the incorporation of [3H]-dThd and [3H]-ara-C into DNA were compared. Within 1 h of exposure of L5178Y cells to ara-C, the rate of [3H]-dThd incorporation into the acid-insoluble fraction was reduced by 98%. Despite this nearly complete block in [3H]-dThd incorporation, DNA synthesis was not completely inhibited since [3H]-ara-C continued to be incorporated for up to 6 h, although a plateau in ara-CDNA synthesis was observed between 2 and 3 h exposure when ara-CTP levels were maximal. The effect of ara-C on [3H]-dThd incorporation into DNA was due in part to an indirect effect of ara-C on the metabolism of intracellular [3H]-dThd to [3H]-dTTP. Within 30 min exposure to 10 microM ara-C, the rate of cellular [3H]-dTTP synthesis was slowed to only 15% of the control rate. This was not due to inhibition of [3H]-dThd transport, since the intracellular and extracellular concentrations of the nucleoside were equal. The effect of ara-C on [3H]-dTTP synthesis resulted from significant changes in deoxynucleoside 5'-triphosphate (dNTP) pools. dTTP, dATP, and dGTP levels were increased, whereas the dCTP concentration was decreased. When dThd kinase from L5178Y cells was assayed with increased dTTP levels induced by ara-C vs the dTTP level in control cells, its activity was reduced by 72%. Thus, the [3H]-dThd incorporation experiment overestimated the extent of inhibition of DNA synthesis by ara-C due to increased feedback inhibition of dThd kinase and increased competition for DNA polymerase between the elevated unlabeled dTTP pool and the decreased levels of [3H]-dTTP. In vitro assay of DNA polymerase in the presence of the ara-CTP concentration achieved after 0.5 or 3 h exposure to 10 microM ara-C (60 microM and 200 microM, respectively), plus the mixture of dNTPs found intracellularly at these times, resulted in 57% and 80% inhibition of the polymerase, respectively. This inhibition may account for the plateau in the accumulation of ara-CDNA that was observed at 3 h and suggests that ara-C incorporation may be self-limiting at high cellular concentrations of ara-CTP.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The effect of ara-C-induced inhibition of DNA synthesis on its cellular pharmacology. 231 Nov 69

Covalently closed-circular, superhelical SV4O DNA was used in all experiments. EcoRI endonuclease- and HpaII endonuclease-generated unit-length linear duplex DNAs were digested with S1 endonuclease under the conditions where single-stranded CNA was completely converted into the acid-soluble form. These were subjected to an end-to-end joining test with T4 DNA ligase. The ligation efficiency was significantly lower than that of the flush-ended linear duplex DNAs which were generated by both HpaI endonuclease digestion and the matching up of EcoRI-generated sticky end with Escherichia coli DNA polymerase I (Klenow fraction). However, the ligation efficiency of the S1-treated DNAs increased up to same level as the flush-ended DNA upon treatment with E. coli DNA polymerase I . Similar results were obtained in the case of S1 -generated unit-length linear duplex DNA. S1 does cleave both strands of superhelical DNA at unbasepaired sites.
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PMID:Efficiency of T4 DNA ligase-catalyzed end joining after S1 endonuclease treatment on duplex DNA containing single-stranded portions. 617 3