TOPOISOMERASES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。拓扑异构酶是一种重要的酶，它通过维持DNA超卷曲、解开DNA结和分解DNA链链来影响DNA代谢的几乎所有方面。这些酶在所有生物体中高度保守，是主要的药物靶点。我们目前的挑战集中在人类拓扑异构酶ii - α，一种2型拓扑异构酶，如何解开而不是缠结其DNA底物。尽管有广泛的生物化学数据可用于酶，没有任何完整的2型拓扑异构酶的原子结构信息。真核2型酶的单个结构域的原子结构是可用的，例如酵母和人类酶的atp酶结构域以及酵母酶的切割-宗教区域；然而，人类蛋白质中22%的c端没有可用的原子结构信息。电子冷冻显微镜和单粒子重建将用于确定在存在和不存在抗癌药物的情况下，单独或与其DNA底物配合的完整酶的三维结构。此外，分离反应中间体的重建将用于确定酶的构象变化。人类拓扑异构酶ii - α是一种340 kDa的同型二聚体，我们对该酶进行了初步的多次精细收敛重建，结果显示该颗粒的尺寸约为65′x 70′x 75′。通过酶本身的结构，与DNA的复合物，抗癌药物，以及每一个被捕获的动态中间体，我们将能够解释机器是如何通过其催化循环运动和进展的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Topoisomerases are essential enzymes that impact nearly every aspect of DNA metabolism through the maintenance of DNA supercoiling, untangling DNA knots, and resolving DNA catenanes. These enzymes are highly conserved in all organisms and are major pharmaceutical targets. Our current challenges focus on how human topoisomerase IIalpha, a type-2 topoisomerase, untangles rather than tangles its DNA substrates. Although there are extensive biochemical data available for the enzyme, there exists no atomic structure information on any complete type-2 topoisomerase. Atomic structures are available of individual domains of eukaryotic type-2 enzymes, such as the ATPase domain for both the yeast and human enzymes and the cleavage-religation region of the yeast enzyme; however, there is no atomic structure information available for 22% of the C-terminus of the human protein. Electron cryomicroscopy and single particle reconstruction will be used to determine the three-dimensional structure of the complete enzyme alone or complexed with its DNA substrate in the presence and absence of anticancer drugs. In addition, reconstructions of isolated reaction intermediates will be used to determine the conformational changes of the enzyme. Human topoisomerase IIalpha is a homodimer of 340 kDa and our initial multiple refinement convergence reconstructions of the enzyme alone show the dimensions of the particles to be roughly 65 ¿ x 70 ¿ x 75 ¿. With the structures of the enzyme alone, complexed with DNA, with anticancer drugs, and each trapped kinetic intermediate, we will be able to interpret how the machine moves and progresses through its catalytic cycle.