Control of DNA Topology

描述（由申请人提供）：该项目的长期目标是了解DNA拓扑异构酶的活性、调节和相互作用如何控制DNA拓扑结构并影响重要的细胞功能。通过捕获由IB型和IIA型拓扑异构酶形成的共价切割复合物来启动细胞杀伤的药物是有用的抗癌和抗菌药物。先前的结果表明IA型拓扑异构酶I切割复合物的积累可以触发细菌细胞死亡，但IA型细菌拓扑异构酶I的特异性抑制剂仍有待发现。下一个资助期的拟议研究活动与克服理解IA型拓扑异构酶机制的关键障碍和发现针对这类拓扑异构酶的新药有关。拓扑异构酶I通过切割双链体DNA中的单链并在切割的链重新连接之前使互补链通过断裂来催化DNA的松弛。在最近获得的共价切割复合物的晶体结构中，已经揭示了在相对于拓扑异构酶I切割的鉴定位点的-4位置处存在C核苷酸的分子基础。定点突变和生化分析将被用来测试这一假设，即与此C核苷酸的相互作用是重要的松弛超螺旋催化的大肠杆菌。coli DNA拓扑异构酶I。结果将揭示在进化过程中C-核苷酸识别的保守性是否与生理功能所需的DNA缠绕活性的效率有关。DNA链在链通过过程中如何被酶引导的分子机制仍然是理解整个酶催化机制的关键障碍。部分DNA双链体分子被E.在一条链上的单个位点处的大肠杆菌拓扑异构酶I将用作测定全长酶的晶体结构以及用于定位通过链上的位点特异性蛋白质-DNA相互作用的底物。这些结果将提供细菌拓扑异构酶I如何催化去除过量负超螺旋的重要功能的分子基础，通过使互补链通过单链初始切割后的断裂。遗传学研究将测试以下假设：在晶体结构中观察到的非共价蛋白质-DNA相互作用的某些扰动负责将切割的DNA底物的3 '-OH部分保持在用于DNA再连接的位置，这可能导致DNA切割中间体的积累，不仅导致拓扑异构酶I松弛活性的丧失，而且还可能触发细菌细胞死亡途径。重组E. coli或Y.鼠疫杆菌拓扑异构酶I，可以导致这种扰动的非共价相互作用与DNA将被确定的遗传选择的显性致死作用，在E。大肠杆菌，并进行了生化表征。这些实验的成功将为发现以拓扑异构酶I为靶点的新型抗菌药物提供非常有用的信息。

DESCRIPTION (provided by applicant): The long term goals of this project are to understand how the activity, regulation and interactions of DNA topoisomerases control DNA topology and affect vital cellular functions. Drugs that initiate cell killing by trapping the covalent cleavage complex formed by type IB and type IIA topoisomerases are useful anti-cancer and anti-bacterial drugs. Previous results have shown that accumulation of type IA topoisomerase I cleavage complex can trigger bacterial cell death but specific inhibitors of type IA bacterial topoisomerase I remain to be discovered. The proposed research activities for the next funding period are relevant for both overcoming the critical barrier in the understanding of the mechanism of type IA topoisomerases and discovery of novel drugs targeting this class of topoisomerases. Topoisomerase I catalyzes the relaxation of DNA by cleaving a single strand in duplex DNA and passing the complementary strand through the break before religation of the cleaved strand. The molecular basis for the presence of a C nucleotide at the -4 position relative to the identified sites of cleavage by topoisomerase I has been revealed in the recently obtained crystal structure of the covalent cleavage complex. Site-directed mutagenesis and biochemical analysis will be used to test the hypothesis that interactions with this C nucleotide are important for relaxation of supercoiling catalyzed by E. coli DNA topoisomerase I. The results will reveal if the conservation of the C-nucleotide recognition during evolution is linked to the efficiency of the DNA winding activity required for physiological function. The molecular mechanism of how the passing DNA strand is guided by the enzyme during strand passage remains the critical barrier for progress in the understanding the overall enzyme catalytic mechanism. Partial DNA duplex molecules that are cleaved by E. coli topoisomerase I at a single site on one strand will be used for as substrates for determination of crystal structure of the full length enzyme as well as for mapping of site-specific protein-DNA interactions on the passing strand. The results will provide the molecular basis of how bacterial topoisomerase I catalyzes the important function of removal of excess negative supercoils by passing the complementary strand through the break after initial cleavage of a single strand. Genetic studies will test the hypothesis that certain perturbations of the non-covalent protein-DNA interactions observed in the crystal structure to be responsible for holding the 3'-OH portion of the cleaved DNA substrate in position for DNA religation could result in accumulation of DNA cleavage intermediate, leading to not only loss of topoisomerase I relaxation activity, but also potentially triggering the bacterial cell death pathway. Mutations in recombinant E. coli or Y. pestis topoisomerase I that can result in such perturbations of the non-covalent interactions with DNA will be identified by genetic selection for dominant lethal effects in E. coli and characterized biochemically. Success in these experiments would provide very useful information for discovery of novel antibacterial drugs targeting topoisomerase I specifically.