On the mechanism of topoisomerase I inhibition by camptothecin

Evidence for binding to an enzyme-DNA complex

Robert P. Hertzberg, Mary Jo Caranfa, Sidney Hecht

Research output: Contribution to journalArticle

484 Citations (Scopus)

Abstract

Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H] camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.

Original languageEnglish (US)
Pages (from-to)4629-4638
Number of pages10
JournalBiochemistry
Volume28
Issue number11
StatePublished - 1989
Externally publishedYes

Fingerprint

Camptothecin
Type I DNA Topoisomerase
DNA
Enzymes
Superhelical DNA
DNA Cleavage
Catalysis
Alkaloids
DNA Breaks
Ultraviolet Rays
Dialysis
Ultraviolet radiation
Dilution
Plasmids
Assays
Stabilization
Derivatives

ASJC Scopus subject areas

  • Biochemistry

Cite this

On the mechanism of topoisomerase I inhibition by camptothecin : Evidence for binding to an enzyme-DNA complex. / Hertzberg, Robert P.; Caranfa, Mary Jo; Hecht, Sidney.

In: Biochemistry, Vol. 28, No. 11, 1989, p. 4629-4638.

Research output: Contribution to journalArticle

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