Arimondo Paola B, Angenault Stéphane, Halby Ludovic, Boutorine Alexandre, Schmidt Frédéric, Monneret Claude, Garestier Thérèse, Sun Jian-Sheng, Bailly Christian, Hélène Claude
Laboratoire de Biophysique, USM0503 Muséum National d'Histoire Naturelle, UMR8646 CNRS, UR565 INSERM, 43 Rue Cuvier, 75231 Paris Cedex 05, France.
Nucleic Acids Res. 2003 Jul 15;31(14):4031-40. doi: 10.1093/nar/gkg457.
Triple helix-forming oligonucleotides covalently linked to topoisomerase I inhibitors, in particular the antitumor agent camptothecin, trigger topoisomerase I-mediated DNA cleavage selectively in the proximity of the binding site of the oligonucleotide vector. In the present study, we have performed a systematic analysis of the DNA cleavage efficiency as a function of the positioning of the camptothecin derivative, either on the 3' or the 5' side of the triplex, and the location of the cleavage site. A previously identified cleavage site was inserted at different positions within two triplex site-containing 59 bp duplexes. Sequence-specific DNA cleavage by topoisomerase I occurs only with triplex conjugates bearing the inhibitor at the 3'-end of the oligonucleotide and on the oligopyrimidine strand of the duplex. The lack of targeted cleavage on the 5' side is attributed to the structural differences of the 3' and 5' duplex-triplex DNA junctions. The changes induced in the double helix by the triple-helical structure interfere with the action of the enzyme according to a preferred spatial organization. Camptothecin conjugates of oligonucleotides provide efficient tools to probe the organization of the topoisomerase I-DNA complex and will be useful to understand the functioning of topoisomerase I in living cells.
与拓扑异构酶I抑制剂，特别是抗肿瘤药物喜树碱共价连接的三链螺旋形成寡核苷酸，在寡核苷酸载体结合位点附近选择性地触发拓扑异构酶I介导的DNA切割。在本研究中，我们系统分析了喜树碱衍生物在三链体3'或5'侧的定位以及切割位点位置对DNA切割效率的影响。将一个先前确定的切割位点插入到两个含三链体位点的59 bp双链体中的不同位置。拓扑异构酶I的序列特异性DNA切割仅发生在寡核苷酸3'-末端且位于双链体寡嘧啶链上带有抑制剂的三链体缀合物中。5'侧缺乏靶向切割归因于3'和5'双链体-三链体DNA连接点的结构差异。三链螺旋结构在双螺旋中引起的变化根据优选的空间组织干扰酶的作用。寡核苷酸的喜树碱缀合物为探测拓扑异构酶I-DNA复合物的组织提供了有效的工具，并且将有助于理解拓扑异构酶I在活细胞中的功能。
