TOPOISOMERASES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 拓扑异构酶是一种重要的酶，通过维持DNA超螺旋、解开DNA结和分解DNA链烷来影响DNA代谢的几乎每个方面。 这些酶在所有生物体中高度保守，是主要的药物靶标。 我们目前的挑战集中在人类拓扑异构酶II α，2型拓扑异构酶，如何解开，而不是缠结其DNA底物。 虽然有广泛的生化数据可用于酶，有没有任何完整的2型拓扑异构酶的原子结构信息。 真核生物2型酶的单个结构域的原子结构是可用的，例如酵母和人酶的ATP酶结构域以及酵母酶的裂解-再连接区;然而，没有人蛋白质22%的C-末端的原子结构信息。 电子冷冻显微镜和单粒子重建将用于确定单独或与其DNA底物复合的完整酶在存在和不存在抗癌药物的情况下的三维结构。 此外，分离的反应中间体的重建将用于确定酶的构象变化。 人拓扑异构酶II α是340 kDa的同源二聚体，我们最初对该酶单独进行的多重精细收敛重建显示颗粒的尺寸约为65 A x 70 A x 75 A。 有了酶本身的结构、与DNA的复合物、抗癌药物以及每一个被捕获的动力学中间体，我们就能够解释机器是如何在催化循环中移动和前进的。

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 II alpha, 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 II alpha 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 A x 70 A x 75 A. 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.