Mechanism and Evolutionary Design of DNA Polymerase Clamp Loaders.

DNA 聚合酶夹钳装载机的机制和进化设计。

• 批准号: 10587243
• 负责人: JOHN KURIYAN
• 金额: $ 33.71万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587243
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Amino Acid Sequence; Amino Acids; Architecture; Bacteria; Bacteriophage T4; Bacteriophages; Base Sequence; Behavior Control; Binding; Biochemical; Biological; Biological Assay; Cells; Closure by clamp; Communication; Complex; Computer Models; Conserved Sequence; Coupling; DNA; DNA Binding; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Diffuse; Disease; Dissociation; Engineering; Escherichia coli; Eukaryotic Cell; Evolution; Family; Family member; Genetic Epistasis; Genome; Genomics; Goals; Homologous Gene; Human; Hydrogen Bonding; Hydrolysis; Libraries; Link; Machine Learning; Macromolecular Complexes; Maps; Mechanics; Modeling; Molecular Machines; Mutagenesis; Mutation; Nature; Organism; Orthologous Gene; Pattern; Performance; Process; Property; Protein Engineering; Proteins; Research; Set protein; Site; Slide; Speed; Statistical Models; Stimulus; System; Testing; Training; Transcription Initiation; Transcriptional Regulation; Variant; Virus; Work; cell behavior; design; enhancer binding protein; experimental study; fitness; high throughput screening; insight; macromolecular assembly; member; mutant; novel; preservation; protein complex; protein function; protein oligomer; response; three dimensional structure; transmission process

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.

项目概要/摘要 我们提出的研究旨在了解复杂的进化起源和适应能力 分子机器。理解蛋白质的功能是具有挑战性的，这取决于精细调节 许多组成部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 进化满足高性能的需求，同时保持多样化的能力和 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诱变 以及一组参与 DNA 复制的蛋白质的功能研究。 DNA 的高速复制依赖于称为滑动钳的蛋白质，这种蛋白质环绕 DNA 并且可以沿着双螺旋快速扩散而不与其分离。因为滑动夹具形成 封闭的圆圈，它们本身不容易与 DNA 结合。滑动夹具打开并加载 通过 ATP 驱动的分子机器（称为夹具装载机）到达 DNA 复制的起始位点。夹钳装载机 是进化上古老的 ATP 依赖性分子机器家族的成员，称为 AAA+ ATP 酶， 它们是一组不同的蛋白质，可将 ATP 结合和水解转化为机械作用 蛋白质。因为 DNA 聚合酶夹钳装载机在其三个方面已得到很好的理解： 维度结构，它们也是理解分子内力传递的优秀模型 更一般地说，是复杂蛋白质机器的进化。该提案的中心目标是发展 对这种复杂蛋白质机器的机制和进化分歧的理解，导致 提高我们预测和控制细胞系统在正常和疾病状态下的行为的能力 州。 T4 噬菌体 (T4) 是一种感染大肠杆菌的小病毒。 T4基因组编码自己的 DNA复制蛋白，包括滑动夹和夹加载器，这些蛋白与其密切相关 真核细胞（包括人类细胞）中的对应物。我们开发并验证了强大的高 T4 噬菌体 (T4) 夹具装载系统的通量功能测定。这个平台开放了很多 在适当的生物学背景下研究机制和设计原理的途径。我们将使用高 通量诱变以绘制夹具加载器中的突变敏感性和变构耦合并检查 这些特性在一种非常不同的 AAA+ ATP 酶中的保守性，AAA+ ATP 酶是一种控制转录的蛋白质 细菌。我们将使用在基因组序列上训练的统计模型来推断和 夹装载机中氨基酸之间的关系，并在生物学背景下测试这些推论。本次活动的目的 该项目代表了一个统一的工作主体，使用新的分析系统来理解夹具装载机和 AAA+ 机制，并测试新兴的基于序列的模型在理解和工程方面的潜力 复杂的高分子机器。