Sequence-specific double-strand breakage of DNA by neocarzinostatin involves different chemical mechanisms within a staggered cleavage site

Abstract

Direct double-strand breaks in DNA have been implicated in cellular lethality of the antitumor antibiotic neocarzinostatin, but the mechanism of their formation has not been elucidated. Evidence is presented that neocarzinostatin causes sequence-specific direct double-strand breaks whose formation is strongly influenced by the activating thiol. Seven-fold more double-strand breaks result when glutathione rather than 2-mercaptoethanol is used to activate the drug to its putative diradical form, while the sequence specificity of cleavage remains the same. These data explain earlier inconsistencies in the ratios of double-strand to single-strand breaks obtained from in vitro and in vivo studies. Double-strand cleavage sites, occurring predominantly at GT steps, especially AGT·ACT, consist of trinucleotide sequences with a two-nucleotide 3′-stagger of the cleaved residues. The chemical structures of the cleavage sites suggest a model in which a neocarzinostatin-induced double-strand break results from abstraction of a C5′ hydrogen atom from the T of ACT and the C4′ hydrogen atom of the T of AGT by a single molecule of the diradical form of the drug. Single-strand breaks at these sites occur as separate events with attack at the C5' hydrogens. These findings permit the generalization that single-strand breaks produced by neocarzinostatin show a base preference but no clear sequence specificity, while bistranded lesions are sequence-specific in nature.