Camptothecin (CPT) and its clinically important antitumor derivative topotecan (Tpt) were traditionally described as unique antitumor compounds exhibiting no affinity toward DNA alone or DNA topoisomerase I (top1) alone but interacting with both the enzyme and the DNA within the so-called ternary cleavable complexes. We present here the first experimental data on the molecular structure and geometry of Tpt-DNA complexes in solution. Tpt interacts with DNA within the DNA minor groove and demonstrates the preferential binding to GC-rich DNA. The flow linear dichroism (FLD) spectra show that the Tpt binds DNA only in lactone form and its chromophore forms the angle nearly 55°with the DNA long axis. Induced circular dichroism (CD) data independently confirm conclusions about Tpt preferable orientation drawn from the FLD experiments. The Raman spectroscopy data confirm the FLD and CD results and further demonstrate direct interactions of Tpt lactone ring with dG. The capability of Tpt to bind DNA in the minor groove of GC-rich DNA regions must be taken into account when considering molecular structure of ternary cleavable complexes of CPTs, DNA, and top1 in solution.
Topotecan (TPT), a water-soluble derivative of camptothecin, is a potent antitumor poison of human DNA topoisomerase I (top1) that stabilizes the cleavage complex between the enzyme and DNA. The role of the recently discovered TPT affinity to DNA remains to be defined. The aim of this work is to clarify the molecular mechanisms of the TPT-DNA interaction and to propose the models of TPT-DNA complexes in solution in the absence of top1. It is shown that TPT molecules form dimers with a dimerization constant of (4.0 +/- 0.7) x 10(3) M(-1) and the presence of DNA provokes more than a 400-fold increase of the effective dimerization constant. Flow linear dichroism spectroscopy accompanied by circular dichroism, fluorescence, and surface-enhanced Raman scattering experiments provide evidence that TPT dimers are able to bind DNA by bridging different DNA molecules or distant DNA structural domains. This effect may provoke modification of the intrinsic geometry of the cruciform DNA structures, leading to the appearance of new crossover points that serve as the sites of the top1 loading position. The data presume the hypothesis of TPT-mediated modulation of top1-DNA recognition before ternary complex formation.
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