Cross-Linkage By ''Intact'' Bizelesin And Bisalkylation By The ''Separated Halves'' Of The Bizelesin Dimer: Contrasting Drug Manipulation Of Dna Conformation (5'-Taatta-3') Directs Alkylation Toward Different Adenine Targets


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Gel electrophoresis analysis of CPI-I bisalkylation of a 21-mer duplex containing 5'-TAA(2)TTA(1)-3' (the palindromic preferred cross-linking sequence of the (+)-CC-1065 analog Bizelesin) shows same-strand (strand one) alkylation of first A(1) and then A(2) instead of the anticipated symmetrical A(1) alkylation of strands one and two. Two-dimensional NMR analyses (NOESY and COSY) confirm the head-to-tail minor groove orientation of the same-strand-bound drugs, CPI-I contrasts sharply with Bizelesin (two CPI-I units linked tail-to-tail by a ureadiyl ''linker''), which symmetrically cross-links this sequence at A(1) (strands one and two), but only by first rearranging the duplex structure and consequently removing the duplex distortion stemming from monoadduct formation. CPI-I induces no such major DNA rearrangement prior to or during bisalkylation. Why does CPI-I react with the adenines of only a single strand? Two possible causes for the unexpected strand one A(2) alkylation are, first, retardation of strand two A(1) site's reactivity by focusing of monoadduct conformational distortion on this site and, second, elevation of A(2) reactivity above other competing adenine sites due to unusual monoadduct strand one A(2)T-step conformational properties. The relative importance of these two nonmutually exclusive factors was investigated using gel electrophoresis experiments: Time-course CPI-I bisalkylation studies were conducted on the AT-step sequence 5'-TAA(2)TTA(1)-3' and an A-tract sequence, 5'-TAA(2)AAA(1)-3', to see if the former sequence's AT-step flexibility, high base-pair opening rate, and unwinding capability (traits not shared by the latter sequence) controlled selection of the second target site. The observed parallel AT-step and A-tract sequence A(1) and A(2) bisalkylation patterns suggest that AT-step properties play at best a secondary role (compared to 5'-end TA-step behavior) in directing the second alkylation reaction to the AT-step site. rMD (solvated) simulations of the AT-step and A-tract monoadducts display distortion that is focused on this 5'-end TA-step site. While two-dimensional H-1 NMR spectra of the final bisadduct reveal no significant TA-step conformational distortion, they demonstrate that conformational adjustment at the A(2) ligand attachment site diminishes head-to-tail steric clash of the two drugs. These results suggest that the CPI-I monoadduct propagates bending distortion (to the 5'-side) through five base pairs toward the TA-step junction site. In the AT-step and A-tract sequences, neither adenine straddling this TA-step junction site is alkylated by CPI-I, suggesting that the base pairs forming the junction site are distorted away from a suitable orientation or are unable to assume a conformation suitable for alkylation. Consequently, the second alkylation occurs at a site (AT-step) that requires a modest displacement of the second ligand away from the already attached drug. The results and analysis of the data included in this paper provide important lessons for the design of inter- and intrastrand DNA-DNA cross-linkers.
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