Identification and cleavage site analysis of DNA sequences bound strongly by bleomycin.

A hairpin DNA library containing an 8-base pair sequence-randomized region was employed in a SELEX-type procedure to identify DNAs that bound strongly to bleomycin A5, the latter of which was immobilized on a solid support. Ten hairpin DNAs that bound BLM A5 strongly were identified and sequenced, and used to characterize DNA binding by the antitumor antibiotic. While all 10 selected hairpin DNAs bound to BLM strongly, they did exhibit a range of binding specificities. Further, while the binding specificity was generally the greatest for hairpin DNAs that contained at least one of the sequences (5′-GC-3′ and 5′-GT-3′) cleaved most frequently by Fe(II)·bleomycin, the hairpin DNA exhibiting the poorest binding specificity also contained a 5′-GT-3′ site. Four of the hairpin DNA substrates were 5′-32P end-labeled and used to assess the preferred sites of cleavage by Fe(II)·BLM. The substrate DNAs included two lacking any 5′-GT-3′ or 5′-GC-3′ site; these were cleaved at 5′-AA-3′ and (more strongly) at 5′-AT-3′...