Characterization of bleomycin-mediated cleavage of a hairpin DNA library.

A study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal-free BLM. The ability of Fe(II)·BLM to affect cleavage on both the 3' and 5' arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe·BLM A5-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5'-AT-3' dinucleotide sequence. This dinucleotide sequence and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates were apparent when examining the library of 10 hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe·BLM A5. The existence of multiple sites of cleavage on both the 3' and 5' arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method and gave results consistent with earlier reports of double-strand DNA cleavage but with a sequence selectivity that was different from those reported previously.