STRUCTURAL STUDIES OF TYPE II TOPOISOMERASE FRAGMENTS

II型拓扑异构酶片段的结构研究

• 批准号: 7357715
• 负责人: GREGORY Lawrence VERDINE
• 金额: $ 1.31万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7357715
• 关键词: STRUCTURAL; STUDIES; TYPE; II; TOPOISOMERASE

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。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。据认为，DNA双链体通过另一个DNA片段中的瞬时酶桥双链断裂，以实现多种拓扑变化。因此，拓扑异构酶代表了真核和原核细胞的致命弱点。事实上，人类拓扑异构酶 II 抑制剂是广泛使用的化疗药物。小分子的这种抑制作用的机制基础在现象学上已被很好地理解，但这些药物中的任何一种与其真核拓扑异构酶靶点的复合结构都很少。为了更好地理解这些相互作用以及与 DNA 双链体运输的机械过程耦合的正常 ATP 水解，我们结晶了人拓扑异构酶 II α 亚型的 ATP 酶结构域。原源衍射极限约为 3 A。最近发表的酵母拓扑 II ATP 酶结构应作为分子替代的出色搜索模型。 DNA 旋转酶是唯一将负超螺旋引入 DNA 的 II 型拓扑异构酶。这种细胞活性对于原核生物的生存能力至关重要，形成了有效且临床有效的抗生素旋转酶抑制剂（例如环丙沙星（Cipro））的基础。 促旋酶特有的负超螺旋诱导偏向可追溯到促旋酶 A 的 C 末端结构域。文献中有诱人的暗示，该结构域实际上以一种让人想起核小体的推定结构将 DNA 包裹在其周围。 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已经证实了这一发现。在我们发表这些发现之前，我们希望获得没有突变的片段结构，并且现在已经有了与 DNA 结合的共晶（40 bp 双链体）。这些结构可以解释对这一类重要蛋白质的负超螺旋诱导偏好。