Pommier Y, Jenkins J, Kohlhagen G, Leteurtre F
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA.
Mutat Res. 1995 Sep;337(2):135-45. doi: 10.1016/0921-8777(95)00019-g.
DNA oligonucleotides containing a strong topoisomerase I cleavage site were used to study the DNA cleavage and strand transferase activities of calf thymus topoisomerase I (top1) in the absence and presence of camptothecin. A partially single-stranded oligonucleotide with only two nucleotides on the 3' side of the cleavage site (positions +1 and +2) was cleaved at the same position as the corresponding duplex oligonucleotide. However, cleavage in the absence of camptothecin was more pronounced than in the duplex oligonucleotide and was only partially reversible in the presence of 0.5 M NaCl, consistent with release of the dinucleotide 3' to the top1 break. Another reaction took place generating a larger DNA fragment which resulted from religation (strand transfer) of the 5'-hydroxyl terminus of the non-scissile DNA strand to the 3' end of the top1-linked oligonucleotide after loss of the +1 and +2 nucleotides. Top1 religation activity appeared efficient since only the last 5' base of the single-stranded DNA acceptor was complementary to the 3' tail of the donor DNA. Religation was not detectable with a double-stranded DNA acceptor, which is consistent with the persistence of top1-induced DNA double-strand breaks in camptothecin-treated cells. Camptothecin and other top1 inhibitors enhanced cleavage in both the partially single-stranded and the duplex oligonucleotides, indicating that they did not inhibit the induction of top1-mediated DNA cleavage but primarily blocked the religation step of the enzyme catalytic cycle. The top1 DNA strand transferase activity was reversibly inhibited by camptothecin and several derivatives, as well as saintopin. These results are discussed in terms of camptothecin-induced DNA recombinations.
含有强拓扑异构酶I切割位点的DNA寡核苷酸被用于研究在不存在和存在喜树碱的情况下小牛胸腺拓扑异构酶I（top1）的DNA切割和链转移酶活性。在切割位点（位置+1和+2）的3'侧只有两个核苷酸的部分单链寡核苷酸在与相应双链寡核苷酸相同的位置被切割。然而，在不存在喜树碱的情况下的切割比在双链寡核苷酸中更明显，并且在0.5M NaCl存在下仅部分可逆，这与二核苷酸3'释放到top1断裂处一致。发生了另一个反应，产生了一个更大的DNA片段，这是由于在失去+1和+2核苷酸后，非切割DNA链的5'-羟基末端与top1连接的寡核苷酸的3'末端重新连接（链转移）所致。top1重新连接活性似乎是有效的，因为只有单链DNA受体的最后一个5'碱基与供体DNA的3'尾巴互补。用双链DNA受体检测不到重新连接，这与喜树碱处理的细胞中top1诱导的DNA双链断裂的持续性一致。喜树碱和其他top1抑制剂增强了部分单链和双链寡核苷酸中的切割，表明它们没有抑制top1介导的DNA切割的诱导，而是主要阻断了酶催化循环的重新连接步骤。top1 DNA链转移酶活性被喜树碱及其几种衍生物以及圣托品可逆抑制。这些结果根据喜树碱诱导的DNA重组进行了讨论。
