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.
ASJC Scopus subject areas
- Colloid and Surface Chemistry