Mechanism and Evolutionary Design of DNA Polymerase Clamp Loaders.

项目摘要/摘要 我们提出的研究试图了解复合体的进化起源和适应能力 分子机器。很难理解蛋白质是如何发挥作用的，而蛋白质的功能依赖于微调 许多成分之间的协作性，随着有机体的进化而适应和改变。也就是说，如何 进化满足了高性能的需求，同时保持了多样化和 适应性专业化？为了深入了解这些过程，我们计划进行高通量突变 以及一组与DNA复制有关的蛋白质的功能研究。 DNA的高速复制依赖于被称为滑动夹的蛋白质，滑动夹是环绕DNA的蛋白质 并且可以沿着双螺旋快速扩散，而不会与之解离。因为滑动夹具形成 封闭的圈子，他们自己不容易与DNA联系在一起。滑动夹具打开并加载 通过被称为钳位加载器的ATP驱动的分子机器进行DNA复制的起始点。夹持式装载机 是一个进化上古老的依赖于ATP的分子机器家族的成员，称为AAA+ATPase， 它们是一组不同的蛋白质，将三磷酸腺苷的结合和水解转化为对 蛋白质。因为DNA聚合酶钳位加载器在它们的三个方面都很好地被理解- 空间结构，它们也是理解分子内力传递的极佳模型 更广泛地说，是复杂蛋白质机器的进化。这项提议的中心目标是发展 对这种复杂蛋白质机器的机制和进化分歧的理解，导致 对于我们预测和控制细胞系统在正常和疾病中行为的能力的进步 各州。 T4噬菌体(T4)是一种感染大肠杆菌的小病毒。T4基因组编码它自己的 DNA复制蛋白，包括滑动夹和夹加载器，与其密切相关的蛋白质 在包括人类细胞在内的真核细胞中的类似物。我们已经开发并验证了一种强大的高- T4噬菌体(T4)钳位加载系统的吞噬功能测定。这个平台开启了许多 在适当的生物学背景下研究机制和设计原则的途径。我们将使用高- 在钳位加载器中进行吞吐量突变以定位突变敏感性和变构偶联并检查 在一个非常不同的AAA+ATPase中这些性质的保守，一种控制转录的蛋白质 细菌。我们将使用对基因组序列进行训练的统计模型来推断和 在夹具加载器中的氨基酸之间，并在生物学背景下测试这些推论。这样做的目的是 项目代表了使用新的检测系统来了解钳夹加载器和AAA+的统一工作主体 机制，并测试新出现的基于序列的模型在理解和工程方面的潜力 复杂的大分子机器。

Project Summary/Abstract Our proposed research seeks to understand the evolutionary origin and capacity to adapt of complex molecular machines. It is challenging to comprehend how protein function, which depends on finely tuned cooperativity between many components, is adapted and altered as an organism evolves. That is, how does evolution satisfy the demands of high performance while maintaining the capacity for diversification and adaptive specialization? To gain insight into these processes we plan to carry out high-throughput mutagenesis and functional studies of a set of proteins involved in DNA replication. High-speed DNA replication relies on proteins known as sliding clamps, which are proteins that encircle DNA and can diffuse rapidly along the double-helix without dissociating from it. Because the sliding clamps form closed circles, they do not readily associate with DNA on their own. Sliding clamps are opened and loaded onto the start sites of DNA replication by ATP-driven molecular machines called clamp loaders. Clamp loaders are members of an evolutionarily ancient family of ATP-dependent molecular machines called AAA+ ATPases, which are a diverse set of proteins that transduce ATP binding and hydrolysis into mechanical action on proteins. Because the DNA polymerase clamp-loaders are very well understood in terms of their three- dimensional structures, they are excellent models for understanding intramolecular force transmission as well as, more generally, the evolution of complex protein machines. The central goal of this proposal is to develop an understanding of the mechanisms and evolutionary divergence of such complex protein machines, leading to advances in our ability to predict and control the behaviors of cellular systems in both normal and disease states. The T4 bacteriophage (T4) is a small virus that infects the E. coli bacterium. The T4 genome encodes its own DNA replication proteins, including a sliding clamp and clamp loader, proteins that are closely related to their counterparts in eukaryotic cells, including human cells. We have developed and validated a powerful high- throughput functional assay for the T4 bacteriophage (T4) clamp loader system. This platform opens up many avenues to investigate mechanism and design principles in a proper biological context. We will use high- throughput mutagenesis to map mutational sensitivity and allosteric coupling in the clamp loader and examine the conservation of these properties in a very divergent AAA+ ATPase, a protein that controls transcription in bacteria. We will use statistical models trained on genome sequences to infer the essential constraints on and between amino acids in clamp loaders and test these inferences in a biological context. The aims of this project represent a unified body of work to use new assay systems to understanding clamp loader and AAA+ mechanism, and to test the potential of emerging sequence-based models for understanding and engineering complex macromolecular machines.