Chemical and structural characterization of the interaction of bleomycin A(2) with d(CGCGAATTCGCG)(2). Efficient, double-strand DNA cleavage accessible without structural reorganization

A detailed description of the interaction between Fe(I). bleomycin A(2) and the Dickerson-Drew dodecamer d(CGCGAATTCGCG)(2) is presented. The reaction between bleomycin and this substrate leads to DNA cleavage at two major si tes, adenosine(5) and cytidine(11), and two minor sites, cytidine(3) and th ymidine(8). The pattern and relative intensities of cleavage at these sites was not entirely consistent with what would be predicted based on the pref erence of the drug for cleavage at the pyrimidines of 5 ' -GC-3 ' and 5 ' - GT-3 ' sites. Insight into the origins of the apparent alteration of select ivity was provided by examination of the structure of the duplex which had been determined by X-ray crystallography. This indicated that the C4 ' hydr ogens of the two nucleotides located at the strongest cleavage sites, C-11 on one strand and A(5) on the other, were oriented toward each other in the minor groove. Two-dimensional NMR measurements and molecular dynamics mode ling indicated that a metalloBLM could bind to the duplex in an orientation that positioned the metal center roughly equally close to each of these hy drogen atoms. On the basis of this observation, it was proposed that these two residues represented a double-stranded BLM cleavage site. This hypothes is was tested through the study of the BLM-mediated cleavage of the related decamer duplex, d(CGCGAATTCG)(.)d(CGAATTCGCG), as well as the hairpin sequ ence d(CGCGAATTCGIIIITTTTCCCCCGAATTCGCG). By the use of the hairpin oligonu cleotide P-32-labeled alternately at the 5 ' and '3-ends, unequivocal evide nce was obtained for BLM-mediated double-strand cleavage. Quantitative anal ysis of the proportion of damage involving double-strand cleavage was effec ted by the use of the hairpin substrate: for damage initiated at the predom inant cleavage site (cytidin(31), analogous to cytidine(11) in the dodecanu cleotide), it is estimated that 43% of all damage leads to double-stranded lesions. The exceptional efficiency of double-strand cleavage observed in t his system must reflect the spatial proximity and orientation of the two su gar H's whose abstraction is required to produce double-stranded lesions.