弯曲DNA与人类I型拓扑异构酶相互作用机制及其作为抗肿瘤抑制剂的研究

Topoisomerase I (topo I) is an enzyme that releases the topological stress of DNA generated by replication and transcription and several other cellular processes and is critical for cell growth and proliferation. There are two subclasses of topo I, topo IA and topo IB, could been classified based on their different working mechanism. It has been demonstrated that Human topoisomerase I (htopo I), belonging to the topo IB subfamily, binds to DNA in a topology-dependent fashion with a strong preference for supercoiled DNA over relaxed DNA. Furthermore, it has been reported that topo IB acts preferentially on intrinsically curved DNA. Prior to thoroughly investigating the mode of action of htopo I, the specific structure of the substrate had to be identified. The endowed substrates of htopo I in organisms are negatively and positively supercoiled structures of DNA.In our preliminary study, a series of forcible curved DNA have been prepared and htopo I can distinguish different degree of DNA curvature as well as favor the highly curved DNA have been illustrated. Taking advantage of the outcomes of these previous studies, we propose in this research plan to systemic design and prepare a series of forcible curved DNA, intrinsically curved DNA and bulged DNA for investigation of the particular recognizable characteristic of htopo I... On the other hand, htopo I has been known as a potential target for cancer therapy. Several anticancer agents targeted to htopo I have been approved by FDA for clinical treatment. However, the therapeutic efficacy of these agents is limited by drug resistant and toxic side-effects. According to the results of htopo I recognize the forcible curved DNA and act preferentially on the intrinsically curved DNA, curved DNA can be used to design novel htopo I inhibitors with high therapeutic efficacy and low toxicity. We hope that implementation of the projects proposed in this research plan could provide beneficial information for design new DNA-based anticancer therapeutical agents that target htopo I.

人类I型拓扑异构酶（htopo I）是存在于人细胞核内的一种酶，参与DNA复制、转录和重组等重要的生命过程；并且是抗肿瘤药物研究的重要靶点。因此，深入了解该酶发挥作用的机制，具有深远的研究价值和科学意义。目前，htopo I识别底物的机制仍然没有得到阐明。有报道指出htopo I能与特定的DNA序列结合，而该特定的DNA序列正好是一段弯曲的DNA片段。在前期工作中，我们发现htopo I不但能识别强制性弯曲DNA，而且能区分强制性弯曲DNA的弯曲程度，与弯曲程度大的DNA有较强的相互作用。基于前人的研究和我们前期工作的基础上，本项目拟系统的设计和制备各种具有弯曲特征的DNA，全面了解各种弯曲DNA与htopo I的相互作用，探讨htopo I识别底物的机制。并研究以弯曲DNA为基础的新型高效、低毒的htopo I抑制剂，为抗肿瘤药物的研发提供理论依据和全新发展方向。

拓扑异构酶I是存在于细胞核内的一种酶，参与DNA复制、转录和重组等重要的生命过程。深入了解该酶发挥作用的机制，具有深远的研究价值。有研究指出人拓扑异构酶I (htopo I)能与特定的DNA序列结合，而该特定的序列正好是一段弯曲的DNA片段。我们的前期工作，也发现htopo I不但能识别强制性弯曲DNA，而且能区分强制性弯曲DNA的弯曲程度。因此，基于先前的研究结果，本项目设计和制备了mismatch和bulge两个系列具有弯曲特征的DNA，全面探讨它们与htopo I的相互作用，并发现它们能抑制细菌拓扑异构酶I的活性。首先，设计和制备了mismatch和bulge两个系列DNA，并对它们进行了结构验证。同时，由于bulge DNA结构比较独特，我们探讨了bulge系列DNA的电泳特性。研究发现bulge部分包含的碱基数目和碱基种类对bulge DNA的电泳迁移率都有一定的影响；而且，带正电荷的离子对bulge DNA的电泳特性也存在影响，随着离子浓度的增加，bulge弯曲程度减低，导致其迁移率逐渐增大。第二，探讨了mismatch DNA和bulge DNA与拓扑异构酶I的相互作用。研究发现，htopo I与mismatch和bulge系列DNA之间不产生发生相互作用，因为htopo I不能识别由mismatch和bulge引起的弯曲DNA结构。但是，意外的发现细菌拓扑异构酶I能较好的与mismatch DNA和bulge DNA发生相互作用。第三，研究了mismatch和bulge系列DNA对细菌拓扑异构酶I的抑制作用。研究发现本项目中设计的mismatch DNA和bulge DNA，对细菌拓扑异构酶I具有较好的抑制效果，并随着mismatch碱基的增加或bulge结构的增大，它们对该酶的抑制程度越强，其中抑制效果最强的IC50值为18.1nM。第四，初步探讨了mismatch和bulge系列DNA抑制细菌拓扑异构酶I的机理。通过设计延长反应时间的实验来探讨其抑制机理，在实验中发现，抑制的效果并没有随时间的延长而降低。因此，推测细菌拓扑异构酶I切断DNA突出单链后，并不能将其链接好而一直与该底物结合在一起，产生不可逆的损伤，导致抑制作用的发生。综上所述，本项目的研究结果为新型细菌拓扑异构酶I抑制剂的研发提供了理论依据和全新发展方向。

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