Dynamics of Protein-DNA Interactions in DNA Replication

描述(申请人提供)复制酶中的功能主题从原核生物到真核生物都是保守的。DNA聚合酶使用环形滑动夹子来实现复制基因组所需的加工能力。细菌、酵母和人类的滑动夹在结构上有显著的相似之处。这些钳子通过多亚单位钳位加载器的活动组装到DNA上，将ATP的水解与钳位加载的机械任务结合在一起。这一过程要求蛋白质-蛋白质和蛋白质-DNA相互作用在钳位加载反应过程中发生变化。ATP的结合和水解促进了钳制加载器的构象变化，从而调节了这些相互作用。我们建议定义动态的蛋白质-蛋白质和蛋白质-DNA相互作用，这是伽马复合体钳制加载器将大肠杆菌线圈DNA聚合酶IIIβ钳制到DNA上所需的。基于实时荧光的分析将被用来直接监测溶液中蛋白质-蛋白质和蛋白质-DNA的相互作用，并将这些与ATP结合和水解相关联。这种方法使我们的实验室有独特的能力来定义在钳制加载反应期间发生在毫秒时间尺度上的蛋白质-蛋白质和蛋白质-DNA相互作用的变化。我们建议使用这些实验来测试夹具加载的动力学模型，通过定义夹具加载器和夹具之间的相互作用，通过定义核苷酸依赖的步骤，并通过使用FRET来测量夹具的打开和关闭。关于这些复制体的大部分研究都来自于对大肠杆菌系统的研究，在人类系统中也看到了许多相似之处。E.COIL模型系统的优点是可以高产率地获得稳定的蛋白质用于体外分析。

DESCRIPTION (provided by applicant) Functional themes in replicases have been conserved from prokaryotes to eukaryotes. DNA polymerases use ring-shaped sliding clamps to achieve the processivity required to replicate a genome. There are remarkable structural similarities in sliding clamps from bacteria, yeast, and man. These clamps are assembled onto DNA by the activity of multisubunit clamp loaders that couple hydrolysis of ATP to the mechanical task of clamp loading. This process requires that protein-protein and protein-DNA interactions change during the clamp loading reaction. Binding and hydrolysis of ATP promote conformational changes in the clamp loader that modulate these interactions. We propose to define the dynamic protein-protein and protein-DNA interactions that are required for the gamma complex clamp loader to assemble the Escherichia coil DNA polymerase III beta clamp onto DNA. Real time fluorescence-based assays will be used to monitor protein-protein and protein-DNA interactions directly in solution and correlate these with ATP binding and hydrolysis. This approach gives our laboratory the unique ability to define changes in the protein-protein and protein-DNA interactions that occur on a millisecond timescale during the clamp loading reaction. We propose to use these experiments to test a kinetic model for clamp loading by defining interactions between the clamp loader and clamp, by defining nucleotide-dependent steps, and by using FRET to measure clamp opening and closing. Much of what has been learned about these replisomes has come from studies of the E. coli system and many analogies are seen in the human system. An E. coil model system offers the advantage that stable proteins can be obtained in high yields for in vitro analysis.