Dynamics of Protein-DNA Interactions in DNA Replication

复制酶的功能主题从原核生物到真核生物都是保守的。DNA聚合酶使用环状滑动夹来实现复制基因组所需的加工速度。细菌、酵母和人类的滑动夹在结构上有显著的相似之处。这些夹通过多亚基夹加载器的活性组装到DNA上，这些多亚基夹加载器将ATP的水解与夹加载的机械任务结合起来。这一过程要求蛋白质-蛋白质和蛋白质- dna相互作用在夹片加载反应中发生变化。ATP的结合和水解促进了调节这些相互作用的钳加载器的构象变化。我们建议定义动态蛋白质-蛋白质和蛋白质-DNA相互作用，这是伽马复合物钳加载器将埃希氏菌线圈DNA聚合酶III β钳组装到DNA上所需的。实时荧光检测将用于监测蛋白质-蛋白质和蛋白质- dna直接在溶液中的相互作用，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具有独特的能力来定义在夹片加载反应期间在毫秒时间尺度上发生的蛋白质-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钳加载的动力学模型，通过定义钳加载器和钳之间的相互作用，通过定义核苷酸依赖的步骤，并通过FRET来测量钳的打开和关闭。我们对这些复制体的了解大多来自对大肠杆菌系统的研究，在人类系统中也看到了许多类似的情况。E. coil模型系统的优点是可以在体外分析中获得高产量的稳定蛋白质。

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.