DNA Topoisomerases and DNA Topology in E. coli

Zechiedrich拓扑异构酶是普遍存在的必需酶，其使DNA链彼此通过以影响核酸代谢的几乎每个方面。 拓扑异构酶IV已被P.I.以及其它一些在子染色体分离和DNA超螺旋调控中所必需的基因。杆菌 本项目的目标是继续使用P.I.开发的测定方法，DNA拓扑异构酶的作用和分子机制，并评估未解决的拓扑结构在E.杆菌使用位点特异性重组和拓扑异构酶抑制的生物化学和遗传方法与解结测定使我们能够测试拓扑异构酶IV解结DNA和细胞中的持久结是致命的假设。 具体而言，本项目的目标是：（1）确定哪种拓扑异构酶在体内解开DNA;（2）确定拓扑异构酶IV如何区分不同的拓扑形式;（3）评估未解决的DNA结的后果。 E.研究大肠杆菌是因为它在拓扑异构酶方面有很好的特征，并且已知基因组序列的生化和遗传分析是直接的。研究结果将揭示拓扑异构酶如何解开而不是缠结DNA，以及它们如何区分有问题的DNA结或链烷与有益的DNA超螺旋。

0090880ZechiedrichTopoisomerases are ubiquitous, essential enzymes that pass DNA strands through each other to affect nearly every aspect of nucleic acid metabolism. Topoisomerase IV has been shown by the P.I. and others to be required for segregation of daughter chromosomes and modulation of DNA supercoiling in E. coli. The objectives of this project are to continue determining, using assays developed by the P.I., the roles and molecular mechanisms of DNA topoisomerases and to assess the consequences of unresolved topological structures in E. coli. Biochemical and genetic approaches with unknotting assays using site-specific recombination and topoisomerase inhibition allow us to test the hypotheses that topoisomerase IV unknots DNA and that persistent knots in the cell are lethal. Specifically, the objectives of this project are: (1) To ascertain which topoisomerase unknots DNA in vivo; (2) To determine how topoisomerase IV distinguishes different topological forms; and (3) To assess the consequences of unresolved DNA knots. E. coli is studied because it is well characterized with regard to topoisomerases, and biochemical and genetic analyses are straightforward with the genome sequence known. Results from the research will reveal how topoisomerases untangle rather than tangle DNA and how they distinguish problematic DNA knots or catenanes from beneficial DNA supercoils.