DNA Topoisomerases and DNA Topology in E. coli

Zechiedrich拓扑异构酶是普遍存在的基本酶，它使DNA链相互传递，几乎影响核酸新陈代谢的方方面面。P.I.和其他研究人员已经证明，拓扑异构酶IV是分离子代染色体和调节大肠杆菌中DNA超级卷曲所必需的。这个项目的目标是继续利用P.I.开发的分析方法来确定DNA拓扑异构酶的作用和分子机制，并评估大肠杆菌中未分解的拓扑结构的后果。使用定点重组和拓扑异构酶抑制的解结分析的生化和遗传学方法使我们能够检验拓扑异构酶IV解结DNA和细胞中持续打结是致命的假设。具体地说，该项目的目标是：(1)确定体内哪些拓扑异构酶能解开DNA；(2)确定拓扑异构酶IV如何区分不同的拓扑形式；以及(3)评估未分解的DNA结的后果。研究大肠杆菌是因为它在拓扑异构酶方面有很好的特征，而且生物化学和遗传分析是直接的，基因组序列是已知的。这项研究的结果将揭示拓扑异构酶如何解开而不是缠绕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.