Single-Molecule DNA Topology

 描述（由申请人提供）：II型拓扑异构酶（II型拓扑异构酶）活性对于维持基因组的拓扑状态以及所有生命系统的生存至关重要。在所有已知的生物体中至少存在一种II型拓扑异构酶基因，这一事实就更加突出了。靶向II型拓扑活性是许多抗癌和抗微生物药物的基础，使该策略成为过去三十年中最成功的一般治疗方法之一。然而，II型拓扑异构酶机制的许多详细方面仍然未知或表征不佳，随着对现有拓扑异构酶抑制剂的耐药性不断增加，未来的药物开发需要更好地了解拓扑异构酶酶学方面。本项目的目标是开发一个全面的数学模型的拓扑状态在打结和/或超螺旋DNA，解释II型拓扑异构酶机制的重要细节。我们将使用最先进的单分子荧光技术来测试这些模型的预测。这些都是具有挑战性的，但可以实现的目标，其结果将提高我们对DNA酶学和抗拓扑异构酶药物活性的基本理解。这个建议建立在我们在理解DNA环力学和DNA拓扑结构方面取得的基本进展的基础上，以发展复杂核蛋白组装体结构的理论描述。我们的方法涉及融合的数学结理论与半解析和数值模型的DNA构象的统计热力学。我们建议研究的行动II型酶环状DNA的拓扑状态之间的转换定义的结类型和连接数在打结，超螺旋DNA。由此产生的拓扑状态的概率分布强烈依赖于II型酶诱导的转换率，从而提供了一个探针的II型拓扑异构酶的酶机制。

 DESCRIPTION (provided by applicant): Type-II topoisomerase (type-II topo) activity is essential for maintaining the topological state of genomes and hence the survival of all living systems. This fact is underscored by the existence of at least one type-II topoisomerase gene in all known organisms. Targeting of type-II topo activity is the basis for many anticancer and anti-microbial drugs, making this strategy one of the most successful general therapeutic approaches in the last thirty years. However, many detailed aspects of type-II topo mechanisms remain unknown or poorly characterized and better insight into aspects of topoisomerase enzymology will be needed for future drug development as increasing resistance to existing topoisomerase inhibitors evolves. The objective of the present project is to develop a comprehensive mathematical model of topological states in knotted and/or supercoiled DNA that explains important details of type-II-topoisomerase mechanisms. We will test predictions of these models by using state-of-the-art single-molecule fluorescence techniques. These are challenging, but achievable, goals, the outcome of which will improve our basic understanding of DNA enzymology and anti-topoisomerase drug activity. This proposal builds on fundamental progress that we have made in understanding DNA-loop mechanics and DNA topology to develop a theoretical description of the structure of complex nucleoprotein assemblies. Our approach involves a fusion of mathematical knot theory with semi-analytical and numerical models of the statistical thermodynamics of DNA conformations. We propose to study the action of type-II enzymes on circular DNA in terms of transitions between topological states defined by knot type and linking number in knotted, supercoiled DNA. The resulting probability distributions of topological states strongly depend on rates of type-II enzyme-induced transitions, and thus provide a probe of enzymatic mechanisms of type-II topoisomerases.