Abstract
The uncoupling of the calf thymus Topoisomerase I-mediated forward DNA cleavage reaction from the religation event by a rapid shift of cleavage temperature either from 37 °C to 0 °C or from 37 °C to 56 °C has been studied and utilized to elucidate the molecular mechanism by which camptothecin, a clinically relevant antineoplastic agent, influences the half reactions of the enzyme. Results of heating and cooling religation-inducing treatments have been compared: both temperature extremes reduce the amount of protein-linked DNA breaks to background levels, thereby affecting cleavage reversal. Camptothecin is found to stabilize the enzyme-DNA intermediate, by inhibition of the Topoisomerase I-mediated rejoining of cleaved DNA, even when the drug is added after formation of the complex. We conclude that:
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1.
Heating and cooling treatments show a pronounced effect on the DNA cleavage-religation equilibrium. The efficacy of cold is more pronounced than that of heat.
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2.
Reversal of the enzyme-DNA intermediate favors the DNA resealing versus the closed relaxed form.
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3.
Camptothecin affects the heat or cold induced religation: in fact in both cases the drug delays the religation step.
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Abbreviations
- CPT:
-
camptothecin
- DTT:
-
dithiothreitol
- PMSF:
-
phenylmethane sulphonyl fluoride
- SDS:
-
sodium dodecyl sulfate
- Topo I:
-
Topoisomerase I
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Carboni, M.C., Coderoni, S. Effect of CPT on the DNA cleavage/religation reaction mediated by calf thymus Topoisomerase I: evidence of an inhibition of DNA religation. Mol Biol Rep 20, 129–133 (1994). https://doi.org/10.1007/BF00990544
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DOI: https://doi.org/10.1007/BF00990544