Metallobleomycin-mediated cleavage of DNA not involving a threading-intercalation mechanism

The DNA cleavage properties of metallobleomycins conjugated to three solid supports were investigated using plasmid DNA, relaxed covalently closed circular DNA, and linear duplex DNA as substrates. Cleavage of pBR322 and pSP64 plasmid DNAs by Fe(II)·BLM A₅-CPG-C₂ was observed with efficiencies not dissimilar to that obtained using free Fe(II)·BLM A₅. Similar results were observed following Fe(II)·BLM A₅-CPG-C₂-mediated cleavage of a relaxed plasmid, a substrate that lacks ends or negative supercoiling capable of facilitating strand separation. BLMs covalently tethered to solid supports, including Fe(II)·BLM A₅-Sepharose 4B, Fe(II)·BLM A₅-CPG-C₆, and Fe(II)·BLM A₅-CPG-C₂, cleaved a 5′-³²P end labeled linear DNA duplex with a sequence selectivity identical to that of free Fe(II)·BLM A₅; cleavage predominated at 5′-G₈₂T₈₃-3′ and 5′-G₈₄T₈₅-3′. To verify that these results could also be obtained using other metallobleomycins, supercoiled plasmid DNA and a linear DNA duplex were employed as substrates for Co(III)·BLM A₅-CPG-C₂. Free green Co(III)·BLM A₅ was only about 2-fold more efficient than green Co(III)·BLM A₅-CPG-C₂ in effecting DNA cleavage. A similar result was obtained using Cu(II)·BLM A₅-CPG-C₂ + dithiothreitol. In addition, the conjugated Co·BLM A₅ and Cu·BLM A₅ cleaved the linear duplex DNA with a sequence selectivity identical to that of the respective free metalloBLMs. Interestingly, when supercoiled plasmid DNA was used as a substrate, conjugated Fe·BLM A₅ and Co·BLM A₅ were both found to produce Form III DNA in addition to Form II DNA. The formation of Form III DNA by conjugated Fe·BLM A₅ was assessed quantitatively. When corrected for differences in the intrinsic efficiencies of DNA cleavage by conjugated vs free BLMs, conjugated Fe·BLM A₅ was found to produce Form III DNA to about the same extent as the respective free Fe·BLM A₅, arguing that this conjugated BLM can also effect double-strand cleavage of DNA. Although previous evidence supporting DNA intercalation by some metallobleomycins is convincing, the present evidence indicates that threading intercalation is not a requirement for DNA cleavage by Fe(II)·BLM A₅, Co(III)·BLM A₅, or Cu(I)·BLM A₅.