DNA Methylation Reduces Binding and Cleavage by Bleomycin

In a recent study, we described the enhanced double-strand cleavage of hairpin DNAs by Fe·bleomycin (Fe·BLM) that accompanies increasingly strong binding of this antitumor agent and suggested that this effect may be relevant to the mechanism by which BLM mediates its antitumor effects. Because the DNA in tumor cells is known to be hypomethylated on cytidine relative to that in normal cells, it seemed of interest to study the possible effects of methylation status on BLM-induced double-strand DNA cleavage. Three hairpin DNAs found to bind strongly to bleomycin, and their methylated counterparts, were used to study the effect of methylation on bleomycin-induced DNA degradation. Under conditions of limited DNA cleavage, there was a significant overall decrease in the cleavage of methylated hairpin DNAs. Cytidine methylation was found to result in decreased BLM-induced cleavage at the site of methylation and to result in enhanced cleavage at adjacent nonmethylated sites. For two of the three hairpin DNAs studied, methylation was accompanied by a dramatic decrease in the binding affinity for Fe·BLM, suggesting the likelihood of diminished double-strand cleavage. The source of the persistent binding of BLM by the third hairpin DNA was identified. Also identified was the probable molecular mechanism for diminished binding and cleavage of the methylated DNAs by BLM. The possible implications of these findings for the antitumor selectivity of bleomycin are discussed

A Short DNA Sequence Confers Strong Bleomycin Binding to Hairpin DNAs

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 <b>12</b>, four of the five double-strand cleavages involved independent single-strand cleavage reactions; DNA <b>13</b> underwent double-strand DNA cleavage by independent single-strand cleavages at all five sites. DNA <b>14</b>, which bound Fe·BLM poorly, was converted to a strong binder (DNA <b>15</b>) 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