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Chemical and Structural Characterization of the Interaction of Bleomycin A2 with d(CGCGAATTCGCG)2. Efficient, Double-Strand DNA Cleavage Accessible without Structural Reorganization
journal contribution
posted on 2001-08-18, 00:00 authored by Michael V. Keck, Richard A. Manderville, Sidney M. HechtA detailed description of the interaction between Fe(II)·bleomycin A2 and the Dickerson-Drew
dodecamer d(CGCGAATTCGCG)2 is presented. The reaction between bleomycin and this substrate leads to
DNA cleavage at two major sites, adenosine5 and cytidine11, and two minor sites, cytidine3 and thymidine8.
The pattern and relative intensities of cleavage at these sites was not entirely consistent with what would be
predicted based on the preference 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 selectivity was provided by examination of the structure
of the duplex which had been determined by X-ray crystallography. This indicated that the C4‘ hydrogens of
the two nucleotides located at the strongest cleavage sites, C11 on one strand and A5 on the other, were oriented
toward each other in the minor groove. Two-dimensional NMR measurements and molecular dynamics modeling
indicated that a metalloBLM could bind to the duplex in an orientation that positioned the metal center roughly
equally close to each of these hydrogen atoms. On the basis of this observation, it was proposed that these two
residues represented a double-stranded BLM cleavage site. This hypothesis was tested through the study of
the BLM-mediated cleavage of the related decamer duplex, d(CGCGAATTCG)·d(CGAATTCGCG), as well
as the hairpin sequence d(CGCGAATTCGIIIITTTTCCCCCGAATTCGCG). By the use of the hairpin
oligonucleotide 32P-labeled alternately at the 5‘ and 3‘-ends, unequivocal evidence was obtained for BLM-mediated double-strand cleavage. Quantitative analysis of the proportion of damage involving double-strand
cleavage was effected by the use of the hairpin substrate; for damage initiated at the predominant cleavage
site (cytidine31, analogous to cytidine11 in the dodecanucleotide), it is estimated that 43% of all damage leads
to double-stranded lesions. The exceptional efficiency of double-strand cleavage observed in this system must
reflect the spatial proximity and orientation of the two sugar H's whose abstraction is required to produce
double-stranded lesions.