STRUCTURAL STUDIES OF TYPE II TOPOISOMERASE FRAGMENTS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。我们计划收集两个拓扑异构酶II片段的本地数据集。II型拓扑异构酶调节DNA超螺旋并解决DNA代谢几乎每个阶段固有的拓扑危机。一般认为，DNA双链体是通过另一个DNA片段中的瞬时酶桥接双链断裂来实现各种拓扑变化的。因此，拓扑异构酶代表了真核和原核细胞的阿喀琉斯之踵。实际上，人拓扑异构酶II抑制剂用作广泛使用的化学疗法。小分子的这种抑制的机制基础在现象学上是很好理解的，但是这些药物中的任何一种与其真核拓扑异构酶靶点复合的结构都是缺乏的。为了更好地理解这些相互作用和正常的ATP水解耦合到DNA双链体运输的机械过程，我们已经结晶的ATP酶结构域的α亚型的人类拓扑异构酶II。家庭源衍射极限约为3 A。最近发表的酵母拓扑酶Ⅱ ATP酶结构应作为一个很好的搜索模型的分子置换。DNA促旋酶是唯一一种能将负超螺旋引入DNA的II型拓扑异构酶。这种细胞活性对原核生物的生存力至关重要，形成了有效和临床有效的抗生素促旋酶抑制剂的基础，例如，环丙沙星（Cipro）。 旋转酶所特有的负超螺旋感应偏置已经被追踪到旋转酶A的C-末端结构域。在文献中有诱人的暗示，这个结构域实际上以一种假定的结构将DNA包裹在它周围，让人想起核小体。 事实上，突变体的初始结构（我们去年在A-1收集的早期数据）以及随后的生物化学和生物物理测量证实了这一发现。我们希望在没有突变的情况下得到片段结构，并且在我们发表这些发现之前，现在已经有了与DNA结合的共晶体（40 bp双链体）。这些结构可以解释这类重要蛋白质的负超螺旋诱导偏好。

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. We plan to collect native data sets on two topoisomerase II fragments. Type II topoisomerases modulate DNA supercoiling and resolve topological crises intrinsic to virtually every phase of DNA metabolism. It is thought that a DNA duplex is passed through a transient enzyme-bridged double stranded break in another DNA segment in order achieve the divers topological changes. As such, topoisomerases represent an Achilles heel of eukaryotic and prokaryotic cells. Indeed, human topoisomerase II inhibitors serve as widely used chemotherapies. The mechanistic basis of this inhibition by small molecules is well understood phenomenologically, yet there is a paucity of structure of any of these drugs in complex with their eukarotic topoisomerase targets. Towards an improved understanding of these interactions and the normal ATP hydrolysis coupled to the mechanical process of DNA duplex transport, we have crystallized the ATPase domain of the alpha isoform of human topoisomerase II. Home source diffraction limits are around 3 A. The recently published yeast topo II ATPase structure should serve as an excellent search model for molecular replacement. DNA gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. This cellular activity is critical to prokaryotic viability, forming the basis of potent and clinically efficacious antibiotic gyrase inhibitors, e.g., ciprofloxacin (Cipro). The negative supercoil induction bias unique to gyrase has been traced to the C-terminal domain of gyrase A. There are tantalizing hints in the literature that this domain actually wraps DNA around it in a putative structure reminiscent of the nucleosome. Indeed the initial structure (our early data collected at A-1 last year) of a mutant and subsequent biochemistry and biophysical measurements have confirmed this finding. We would like to get the fragment structure in the absence of mutations and now have co-crystals with DNA bound (40 bp duplex) before we publish these findings. These structures can explain the negative supercoil induction preference for this important class of proteins.