NOVEL DNA PHOTOCLEAVING AGENTS WITH HIGH DNA-SEQUENCE SPECIFICITY RELATED TO THE ANTIBIOTIC BLEOMYCIN A(2)

We have designed and synthesized a series of novel DNA photocleaving a gents which break DNA with high sequence specificity. These compounds contain the non-diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analo gues of the DNA binding portion of the antibiotic bleomycin A(2). By u sing a variety of 5' or 3' P-32-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazol e compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce D NA cleavage 3' of the site. Analysis of cleavage sites on the compleme ntary DNA strand and inhibition of DNA breakage by distamycin A indica tes these bithiazole derivatives bind and attack the minor groove of D NA. A photoactive unlthlazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequen ce recognition of these derivatives appears to be determined by the th iazole moiety rather than the p-nitrobenzoyl group: use of a tetrameth ylene group in place of a dimethylene spacer shifted the position of D NA breakage by one base pair. Moreover, much less specific DNA photocl eavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spac er. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives c ontrasts with that of bleomycin A(2) which cleaves DNA most frequently at 5'-GPy-3' sequences. These results suggest that the cleavage speci ficity exhibited by bleomycin is not simply determined by its bithiazo le/sulphonium terminus, and the contributions from other features, e.g . its metal-chelating domain, must be considered. The novel thiazole-b ased DNA cleavage agents described here should prove useful as reagent s for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands.