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 Diffe

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• 作者：Frederick C. Seaman ; Jianxiong Chu ; and Laurence Hurley
• 刊名：Journal of the American Chemical Society
• 出版年：1996
• 出版时间：June 12, 1996
• 年：1996
• 卷：118
• 期：23
• 页码：5383 - 5395
• 全文大小：883K
• 年卷期：v.118,no.23(June 12, 1996)
• ISSN：1520-5126

文摘

Gel electrophoresis analysis of CPI-I bisalkylation of a 21-merduplex containing5'-TAA²TTA¹-3'(thepalindromic preferred cross-linking sequence of the (+)-CC-1065analog Bizelesin) shows same-strand (strand one)alkylation of first A¹ and then A² instead ofthe anticipated symmetrical A¹ alkylation of strands oneand two.Two-dimensional NMR analyses (NOESY and COSY) confirm thehead-to-tail minor groove orientation of thesame-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 atA¹ (strands one and two), but only by firstrearrangingthe duplex structure and consequently removing the duplex distortionstemming from monoadduct formation. CPI-Iinduces no such major DNA rearrangement prior to or duringbisalkylation. Why does CPI-I react with the adeninesof only a single strand? Two possible causes for the unexpectedstrand one A² alkylation are, first, retardationofstrand two A¹ site's reactivity by focusing of monoadductconformational distortion on this site and, second,elevationof A² reactivity above other competing adenine sites due tounusual monoadduct strand one A²T-stepconformationalproperties. The relative importance of these two nonmutuallyexclusive factors was investigated using gelelectrophoresis experiments: Time-course CPI-I bisalkylation studieswere conducted on the AT-step sequence 5'-TAA²TTA¹-3' and an A-tract sequence,5'-TAA²AAA¹-3', to see if the formersequence's AT-step flexibility, highbase-pair opening rate, and unwinding capability (traits not shared bythe latter sequence) controlled selection of thesecond target site. The observed parallel AT-step and A-tractsequence A¹ and A² bisalkylation patternssuggestthat AT-step properties play at best a secondary role (compared to5'-end TA-step behavior) in directing the secondalkylation reaction to the AT-step site. rMD (solvated)simulations of the AT-step and A-tract monoadducts displaydistortion that is focused on this 5'-end TA-step site. Whiletwo-dimensional ¹H NMR spectra of the finalbisadductreveal no significant TA-step conformational distortion, theydemonstrate that conformational adjustment at theA²ligand attachment site diminishes head-to-tail steric clash of the twodrugs. These results suggest that the CPI-Imonoadduct propagates bending distortion (to the 5'-side) through fivebase pairs toward the TA-step junction site.In the AT-step and A-tract sequences, neither adenine straddlingthis TA-step junction site is alkylated by CPI-I,suggesting that the base pairs forming the junction site are distortedaway from a suitable orientation or are unableto 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 thealready attached drug. The results andanalysis of the data included in this paper provide important lessonsfor the design of inter- and intrastrand DNA-DNA cross-linkers.