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 cir cular DNA, and linear duplex DNA as substrates. Cleavage of pBR322 and pSP6 4 plasmid DNAs by Fe(II). BLM A(5)-CPG-C-2 was observed with efficiencies n ot dissimilar to that obtained using free Fe(II). BLM As. Similar results w ere observed following Fe(II)BLM A(5)-CPG-C-6-mediated cleavage of a relaxe d 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 wi th a sequence selectivity identical to that of free Fe(II). BLM As; cleavag e predominated at 5 ' -G(82)T(83)-3 ' and 5 ' -G(84)T(85)-3 '. To verify th at these results could also be obtained using other metallobleomycins, supe rcoiled 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 + dithiothrei tol. In addition, the conjugated Co . BLM As and Cu . BLM As cleaved the li near duplex DNA with a sequence selectivity identical to that of the respec tive 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 DN A 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 conj ugated BLM can also effect double-strand cleavage of DNA. Although previous evidence supporting DNA intercalation by some metallobleomycins is convinc ing, the present evidence indicates that threading intercalation is not a r equirement for DNA cleavage by Fe(II). BLM A(5), CO(III). BLM A(5), or Cu(I ). BLM A(5).