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.

项目总结/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，这取决于微调的 许多组成部分之间的协同性随着有机体的进化而适应和改变。那就是， 进化满足了高性能的需求，同时保持了多样化的能力， 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。 高速DNA复制依赖于被称为滑动夹的蛋白质，这是一种环绕DNA的蛋白质 并且可以沿着双螺旋快速扩散而不与其解离。 封闭的圆圈，它们本身不容易与DNA结合。滑动夹打开并加载 在DNA复制的起始位点上，由ATP驱动的分子机器，称为钳加载器。夹钳装载机 是一个进化上古老的ATP依赖性分子机器家族的成员，称为AAA+ ATP酶， 这是一组不同的蛋白质，它们使ATP结合并水解成机械作用， proteins.因为DNA聚合酶钳装载器在它们的三个方面都很好地被理解- 三维结构，它们也是理解分子内力传递的极好模型 更普遍地说，是复杂蛋白质机器的进化。该提案的核心目标是发展 了解这种复杂蛋白质机器的机制和进化分歧， 我们预测和控制正常和疾病中细胞系统行为的能力的进步 states. T4噬菌体（T4）是一种感染大肠杆菌的小病毒。大肠杆菌T4基因组编码自己的 DNA复制蛋白，包括滑动钳和钳装载器，与它们的DNA复制密切相关的蛋白质。 在真核细胞中的对应物，包括人类细胞。我们已经开发并验证了一个强大的高- T4噬菌体（T4）夹加载系统的通量功能测定。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- 通量诱变，以绘制夹加载器中的突变敏感性和变构偶联，并检查 这些性质在一个非常不同的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基因组序列的基本约束， 并在生物学背景下测试这些推论。其目的是 项目代表了一个统一的工作主体，使用新的分析系统来了解夹钳装载器和AAA+ 机制，并测试新兴的基于序列的模型的理解和工程的潜力 复杂的高分子机器