Solution structure of Co(III)-bleomycin-OOH bound to a phosphoglycolate lesion containing oligonucleotide: Implications for bleomycin-induced double-strand DNA cleavage

Bleomycin (BLM) is an antitumor antibiotic that is used clinically. Its maj or cause of cytotoxicity is thought to be related to BLM's ability to cause double-strand (ds) DNA cleavage. A single molecule of BLM appears to cleav e both strands of DNA in the presence of its required cofactors Fe2+ and ox ygen without dissociating from the helix. A mechanism for this process has been proposed based on a model structure of the hydroperoxide of Co(III)-BL M (CoBLM) bound sequence-specifically to an intact duplex containing a GTAC site, a hot spot for ds cleavage [Vanderwall, D. E., Lui, S. M., Wu, 'W., Turner, C. J., Kozarich, J. W., and Stubbe, J. (1997) Chem. Biol. 4, 373-38 7]. In this paper, we present a structural model for the second cleavage ev ent. Two-dimensional NMR spectroscopy and molecular modeling were carried o ut to study CoBLM bound to (CCAAAGXACTGGG).d(CCCAGTACTTTGG), where X repres ents a 3'-phosphoglycolate lesion next to a 5'-phosphate. Assignments of 72 9 NOEs, including 51 between the drug and the DNA and 126 within the BLM mo lecule, have been made. These NOEs in addition to 96 dihedral angle constra ints have been used to obtain a well-defined structural model for this comp lex. The model reveals that the bithiazole tail is partially intercalated b etween the T19 and the A20 of the duplex and that the metal binding domain is poised for abstraction of the T19 H4' in the minor groove. The modeling further reveals that the predominant conformation of the bithiazole protons is trans. Two cis conformations of these protons are also observed, and RO ESY experiments provide evidence for interconversion of all of these forms. The relationship of these observations to the model for ds cleavage is pre sented.