A Short DNA Sequence Confers Strong Bleomycin Binding to Hairpin DNAs

Abstract

Bleomycins A5 and B2 were used to study the structural features in hairpin DNAs conducive to strong BLM-DNA interaction. Two members of a 10-hairpin DNA library previously found to bind most tightly to these BLMs were subsequently noted to share the sequence 5'-ACGC (complementary strand sequence 5'-GCGT). Each underwent double-strand cleavage at five sites within, or near, an eight base pair region of the DNA duplex which had been randomized to create the original library. A new hairpin DNA library was selected based on affinity for immobilized Fe(III)·BLM A5. Two of the 30 newly identified DNAs also contained the sequence 5'-ACGC/5'-GCGT. These DNAs bound to the Fe(II)·BLMs more tightly than any DNA characterized previously. Surface plasmon resonance confirmed tight Fe(III)·BLM B2 binding and gave an excellent fit for a 1:1 binding model, implying the absence of significant secondary binding sites. Fe(II)·BLM A5 was used to assess sites of double-strand DNA cleavage. Both hairpin DNAs underwent double-strand cleavage at five sites within or near the original randomized eight base region. For DNA 12, four of the five double-strand cleavages involved independent single-strand cleavage reactions; DNA 13 underwent double-strand DNA cleavage by independent single-strand cleavages at all five sites. DNA 14, which bound Fe·BLM poorly, was converted to a strong binder (DNA 15) by insertion of the sequence 5'-ACGC/5'-GCGT. These findings reinforce the idea that tighter DNA binding by Fe·BLM leads to increased double-strand cleavage by a novel mechanism and identify a specific DNA motif conducive to strong BLM binding and cleavage.