Structural basis for RPA and DNA primase functions

RPA 和 DNA 引物酶功能的结构基础

• 批准号: 8237799
• 负责人: WALTER J. CHAZIN
• 金额: $ 33.62万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8237799
• 关键词: Address; Affinity; Amino Acid Sequence; Architecture; Bacteria; Binding; Biochemical; Biological Assay; Biological Models; Cells; Collaborations; Complex; Computer Simulation; Crystallography; DNA; DNA Binding; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; Defect; Dependence; Disease; Enzymes; Fluorescence Polarization; Foundations; Genome; Genomic Instability; Goals; Hand; Human; Hybrids; Knowledge; Lead; Length; Link; Maintenance; Malignant Neoplasms; Maps; Modeling; Molecular; Mutation; NMR Spectroscopy; Neutrons; Nucleotides; Pathway interactions; Peptide Sequence Determination; Polymerase; Primer Extension; Process; Proteins; RNA; RNA chemical synthesis; RNA primers; Replication Initiation; Replication Origin; Research; Roentgen Rays; Role; SS DNA BP; Simian virus 40; Single-Stranded DNA; Site; Structure; System; Work; base; design; helicase; insight; mutant; programs; protein complex; protein structure; replication factor A

DESCRIPTION (provided by applicant): Replication of DNA is a complex multi-step process essential to cell propagation and survival, which proceeds via the action of multi-protein machines. Understanding the machinery at the replication fork has high impact because it is the most critical site for propagation and maintenance of the genome. While considerable progress has been made in elucidating the mechanisms of DNA replication from studies of bacteria and archae, information on replication in humans is lacking because the protein sequences and structures are not conserved. The long-term goal of our research is to understand the action of the DNA replication machinery in humans. Our research currently focuses on understanding the initiation of the step known as DNA priming. We have shown active loading of human replication protein A (RPA) onto single-stranded DNA (ssDNA) created by the SV40 helicase at the origin of replication and involvement of RPA in the transition to DNA priming. After the DNA is unwound, an initial primer is synthesized on the ssDNA template by primase. The studies proposed here are designed to generate insight into how RPA and primase function together to initiate synthesis of the primer strand. Aim 1 investigates the structure of RPA in different DNA-bound states using a combination of small angle X-ray and neutron scattering (SAXS, SANS) and NMR spectroscopy. Aim 2 addresses the role of interactions with RPA in promoting the loading of primase on the template using a combination of biochemical mapping and structural analyses by NMR, modeling, SAXS and SANS. Once primase is loaded on the DNA template, it synthesizes a ~10 nucleotide primer and then transfers the primed template to DNA polymerase a for primer extension. The means by which primase recognizes the template and counts the length of the primer remains a complete mystery. Aim 3 proposes to elucidate the structural basis for these processes by determining x-ray crystal structures of primase in different DNA bound states. Together, these results will inform the structural basis for the hand-off of ssDNA from RPA to DNA primase and counting of the RNA primer, which are critical steps in the replication of DNA. PUBLIC HEALTH RELEVANCE: Faithful replication and maintenance of our genomes requires the action of complex multi-protein machinery. Defects in components of this machinery lead to mutation and ultimately cancer and other diseases associated with genomic instabilities. Investigating the structure and coordinated action of the complex of proteins involved in initiating replication in humans will provide detailed mechanistic insight into the action of the multi-protein machinery at the replication fork, the most critical site for propagation and maintenance of the genome.

描述(由申请人提供)：DNA的复制是一个复杂的多步骤过程，对细胞的繁殖和生存至关重要，它通过多蛋白质机器的作用进行。了解复制分叉的机制具有很大的影响，因为它是基因组传播和维护的最关键位置。虽然从细菌和古生物的研究中已经在阐明DNA复制机制方面取得了相当大的进展，但由于蛋白质序列和结构不保守，因此关于人类复制的信息很少。我们研究的长期目标是了解DNA复制机制在人类中的作用。我们目前的研究重点是了解被称为DNA启动的步骤的启动。我们已经展示了在复制的起始处，人类复制蛋白A(RPA)主动负载到由SV40解旋酶产生的单链DNA(SsDNA)上，并参与了向DNA启动的转变。DNA解开后，用底物酶在单链DNA模板上合成一条初始引物。这里提出的研究旨在深入了解RPA和Primase如何共同作用于启动引物链的合成。目的1结合小角X射线和中子散射(SAXS，SANS)和核磁共振波谱研究RPA在不同DNA结合状态下的结构。目的2利用生化图谱和核磁共振、建模、SAXS和SANS的结构分析相结合的方法，研究与RPA的相互作用在促进Primase在模板上的负载方面的作用。一旦将底物酶装载到DNA模板上，它就会合成一个~10个核苷酸的引物，然后将该模板转移到DNA聚合酶a上进行引物延伸。Primase如何识别模板并计算引物的长度仍然是一个完全的谜。目的3建议通过测定不同DNA结合状态下Primase的X射线晶体结构来阐明这些过程的结构基础。综上所述，这些结果将为单链DNA从RPA传递到DNA引物和RNA引物的计数提供结构基础，这是DNA复制的关键步骤。 与公共健康相关：我们基因组的忠实复制和维护需要复杂的多蛋白质机制的行动。这种机制组件的缺陷会导致突变，最终导致癌症和其他与基因组不稳定性相关的疾病。研究参与启动人类复制的蛋白质复合体的结构和协调作用，将提供对复制叉处多蛋白质机制作用的详细机制洞察，复制叉处是基因组传播和维护的最关键位置。