Characterization of bleomycin cleavage sites in strongly bound hairpin DNAs

The first complete, systematic study of DNA degradation by bleomycin under conditions analogous to those likely in a therapeutic setting has been carried out. Hairpin DNAs selected for their ability to bind strongly to BLM A ₅ were used to determine the relationship between high-affinity DNA binding sites and the cleavage efficiency and selectivity of BLM A₅ and deglycoBLM A₅ on these DNAs. Of the 10 hairpin DNAs examined, 8 contained at least one 5′-GC-3′ or 5′-GT-3′ cleavage site, which have traditionally been associated with strong cleavage by Fe•BLM. In the hairpin DNAs, these included the strongest cleavage sites for BLM A₅ and were generally among those for deglycoBLM A ₅. However, numerous additional cleavages were noted, many at sequences not usually associated with (deglyco)BLM-mediated cleavage. The remaining DNAs lacked the preferred (5′-GC-3′ or 5′-GT-3′) BLM cleavage sequences; however, strong cleavage was nonetheless observed at a number of unusual cleavage sites. The most prominent cleavage sequences were 5′-AT-3′, 5′-AA-3′, 5′-GA-3′, and 5′-TT-3′ treatment with Fe(II)•BLM A₅ or Fe(II)•deglycoBLM A₅ resulted in strong cleavage at these sequences. Additionally, in contrast with BLM A₅, which produced cleavage within the randomized and flanking invariant regions, deglycoBLM A₅ showed a preference for cleavage in the randomized region of the DNAs. Previous reports have established that deglycoBLM exhibits decreased DNA cleavage efficiency relative to BLM. This was also generally observed when comparing cleavage efficiencies for the strongly bound hairpin DNAs. However, some cleavage bands produced by Fe•deglycoBLM A₅ were stronger in intensity than those produced by BLM A₅ at concentrations optimal for both compounds. To investigate the chemistry of DNA degradation, selected hairpin DNAs were treated with n-butylamine following cleavage with Fe(II)•BLM A₅ or Fe(II)•deglycoBLM A ₅ to explore the alkali labile pathway of DNA degradation by BLM. While all 10 DNAs showed evidence of alkali labile products, five DNA hairpins afforded some products formed solely via the alkali labile pathway.