Mapping of the cleavage-associated bleomycin binding site on DNA with a new method based on site-specific blockage of the minor groove with N2-isobutyrylguanine.

Abstract

Although the binding of various forms of bleomycin to DNA has been studied extensively, the transient nature of the activated bleomycin species which ultimately attacks DNA has largely precluded direct examination of its physical interactions with DNA. In an attempt to map the minimum binding site required for this species to effect DNA cleavage, several oligonucleotide duplexes were synthesized, each of which contained a single N2-isobutyrylguanine moiety at a specific position in the sequence. These duplexes were end-labeled, and sequence-specific bleomycin-induced cleavage was assessed in each strand of each duplex. Isobutyrylguanine substitution immediately 5' to a primary bleomycin target site suppressed bleomycin-induced cleavage by more than 10-fold. Substitution two bases 5' to a target site suppressed cleavage by about 4-fold, and substitution directly opposite the target site suppressed cleavage by 7-fold. Substitution immediately 3' to the target site, or at other more distant positions 3' or 5', had little or no effect. In cases where cleavage at a primary site was strongly suppressed, cleavage at the corresponding secondary site (the putative site of the second break in a bleomycin-induced double-strand break) was also inhibited, even when the secondary site was several bases away from the isobutyrylguanine substitution. The results suggest that the binding site required for bleomycin-induced DNA cleavage spans a region of approximately 2 or 3 bp in the minor groove, including the base associated with the sugar attacked and one or two bases to its 5' side. Computer-based molecular modeling indicated that these results are consistent with the predictions of recently proposed models in which the bithiazole is intercalated immediately 3' to the cleavage site, and the iron coordination site binds in the minor groove immediately 5' to the cleavage site. Both the empirical data and the modeling studies suggest that N2-isobutyrylguanine substitution effectively blocks the minor groove, but without significantly disturbing DNA secondary structure. Thus, it is proposed that site-specific incorporation of N2-isobutyrylguanine may provide a general method for mapping binding sites of minor groove-binding ligands on DNA.