Published online 19 October 2004
Nucleic Acids Research, Vol. 32 No. 18 © Oxford University Press 2004; all rights reserved
Concerted bis-alkylating reactivity of clerocidin towards unpaired cytosine residues in DNA
Department of Pharmaceutical Sciences and 1 Department of Chemical Sciences, University of Padova, 35131 Padova, Italy and 2 Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, Baltimore, MD 21250, USA
* To whom correspondence should be addressed. Tel: +39049 827 5711/7; Fax: +39049 827 5366; Email: manlio.palumbo{at}unipd.it
Received July 8, 2004; Revised and Accepted September 30, 2004
Clerocidin (CL) is a topoisomerase II poison, which cleaves DNA irreversibly at guanines (G) and reversibly at cytosines (C). Furthermore, the drug can induce enzyme-independent strand breaks at the G and C level. It has been previously shown that G-damage is induced by alkylation of the guanine N7, followed by spontaneous depurination and nucleic acid cleavage, whereas scission at C is obtained only after treatment with hot alkali, and no information is available to explain the nature of this damage. We present here a systematic study on the reactivity of CL towards C both in the DNA environment and in solution. Selected synthetic derivatives were employed to evaluate the role of each chemical group of the drug. The structure of CL–dC adduct was then characterized by tandem mass spectrometry and NMR: the adduct is a stable condensed ring system resulting from a concerted electrophilic attack of the adjacent carbonyl and epoxide groups of CL towards the exposed NH2 and N3, respectively. This reaction mechanism, shown here for the first time, is characterized by faster kinetic rates than alkylation at G, due to the fact that the rate-determining step, alkylation at the epoxide, is an intramolecular process, provided a Schiff base linking CL and C can rapidly form, whereas the corresponding reaction of G N7 is intermolecular. These results provide helpful hints to explain the reversible/irreversible nature of topoisomerase II mediated DNA damage produced by CL at C/G steps.
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