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 A(5)-CPG-C-2 was observed with efficiencies not dissimilar to that obtained using free Fe(II). BLM As. Similar results were observed following Fe(II)BLM A(5)-CPG-C-6-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 A(5)-Sepharose 4B, Fe(II). BLM A(5)-CPG-C-6, and Fe(II). BLM A(5)-CPG-C-2, cleaved a 5 '-(32)p end labeled Linear DNA duplex with a sequence selectivity identical to that of free Fe(II). BLM As; cleavage predominated at 5 ' -G(82)T(83)-3 ' and 5 ' -G(84)T(85)-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 A(5)-CPG-C-2. Free green Co(m). BLM As 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 A(5)-CPG-C-2 + dithiothreitol. In addition, the conjugated Co . BLM As and Cu . BLM As 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 As and Co . BLM AS were both found to produce Form III DNA in addition to Form II DNA. The formation of Form m DNA by conjugated Fe . BLM As was assessed quantitatively. When corrected for differences in the intrinsic efficiencies of DNA cleavage by conjugated vs free BLMs, conjugated Fe . BLM As was found to produce Form m DNA to about the same extent as the respective free Fe . BLM As, 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 A(5), CO(III). BLM A(5), or Cu(I). BLM A(5).