TOPOISOMERASES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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链环，几乎影响DNA代谢的方方面面。这些酶在所有生物体中都高度保守，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IIα，一种2型拓扑异构酶，如何解缠而不是缠绕其DNA底物。尽管有广泛的生化数据可用来研究这种酶，但没有关于任何完整的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，例如酵母和人类酶的ATPase结构域以及酵母酶的裂解区域；然而，没有关于人类蛋白质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被用来确定在存在和不存在抗癌药物的情况下，完整的酶单独或与其DNA底物络合的三维结构。此外，分离的反应中间产物的重建将被用来确定酶的构象变化。人类拓扑异构酶IIα是一种340 kDa的同源二聚体，仅我们对该酶的初始多重精细化收敛重建就显示了颗粒的尺寸约为65A x 70A x 75A。根据酶的结构，与DNA、抗癌药物以及每个捕获的动力学中间体，我们将能够解释机器如何在其催化循环中移动和进展。

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.