AAV Rep-DNA Complexes Underlaying Site-specific Integration

AAV Rep-DNA 复合物是位点特异性整合的基础

• 批准号: 8143333
• 负责人: Carlos R Escalante
• 金额: $ 28.86万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8143333
• 关键词: Animal Viruses; Architecture; Binding; Binding Sites; Biochemical; Cell Cycle; Cells; Chromosomes; Chromosomes, Human, Pair 19; Complex; DNA; DNA biosynthesis; DNA-Binding Proteins; Dependovirus; Development; Elements; Event; Gene Transduction Agent; Genetic Recombination; Genome; Goals; Human Chromosomes; Hydrolysis; Kinetics; Knowledge; Lead; Life Cycle Stages; Mediating; Modeling; Nature; Nonstructural Protein; Pathogenicity; Process; Property; Proteins; Protocols documentation; Reaction; Recruitment Activity; Regulation; Replication Initiation; Research Proposals; Resolution; Roentgen Rays; Role; Site; Specificity; Structure; Traumeel S; Virus; Virus Replication; X-Ray Crystallography; base; design; gene therapy; helicase; insight; latent infection; melting; new technology; novel; public health relevance; research study; site-specific integration; stoichiometry; structural biology; tissue tropism; tool; viral DNA

DESCRIPTION (provided by applicant): Adeno-Associated Virus type 2 (AAV-2) is unique among animal viruses in its ability to establish a latent infection by integrating site-specifically into a locus of chromosome 19. Moreover, because of its apparent non- pathogenicity, broad cell and tissue tropism and ability to infect in a cell-cycle independent fashion, AAV has emerged as one of the most promising vectors for gene therapy. A number of studies indicate that AAV- mediated site-specific integration requires only three players: a) the AAV Rep78 or Rep68 non-structural proteins, b) an AAV DNA element containing a Rep Binding Site (RBS) and c) the cellular AAVS1 integration site. During a decisive event, these players come together to form a protein-DNA complex that serves as the starting point for an integration process that is considered to be parallel to the AAV replication initiation process. This pre-integration complex is dependent on the ability of Rep68/78 proteins to bind the integration site AAVS1, promote its melting and catalyze a strand-specific nicking reaction that leaves a Rep68/78 protein covalently bound to the integration site. Although a general view of this mechanism has been achieved through the study of AAV DNA replication, little is known regarding the architecture of the Rep-AAVS1 complex and the steps that lead to its formation. The long-term goal of this proposal is to establish a structural, biophysical and biochemical framework required to understand the mechanism of AAV-mediated site-specific integration. Our approach is to couple structural studies using X-ray crystallography, cryo-EM and small angle X-ray scattering (SAXS) with biophysical, kinetic and functional studies to get a complete understanding of the events that lead to site-specific integration. We expect that the results obtained in this research proposal will serve both as a starting point to generate accurate models to understand this unique mechanism and also will give us the knowledge and tools to potentially design Rep proteins with novel specificities that could be used to develop new technologies in the gene therapy field. PUBLIC HEALTH RELEVANCE: This study will contribute significantly toward understanding the mechanism of AAV mediated site-specific integration. In addition, it will contribute to our understanding of origin DNA replication initiation. Knowledge gained from this proposal will serve as the basis for designing novel gene therapy vectors that could be targeted to a specific region in the chromosome.

描述（由申请人提供）：腺相关病毒2型（AAV-2）在动物病毒中是独一无二的，它能够通过特异性地整合到19号染色体的位点来建立潜伏感染。此外，由于AAV具有明显的非致病性，广泛的细胞和组织亲和性以及以细胞周期独立的方式感染的能力，AAV已成为最有希望的基因治疗载体之一。许多研究表明，AAV介导的位点特异性整合只需要三个参与者：A) AAV Rep78或Rep68非结构蛋白，b)含有Rep Binding Site （RBS）的AAV DNA元件和c)细胞AAVS1整合位点。在一个决定性的事件中，这些参与者聚集在一起形成一个蛋白质- dna复合物，作为整合过程的起点，被认为与AAV复制起始过程平行。这种整合前复合物依赖于Rep68/78蛋白结合整合位点AAVS1、促进其融化和催化链特异性缺口反应的能力，从而使Rep68/78蛋白与整合位点共价结合。尽管通过对AAV DNA复制的研究已经对这一机制有了一个大致的认识，但关于Rep-AAVS1复合体的结构和导致其形成的步骤却知之甚少。本提案的长期目标是建立一个结构、生物物理和生化框架，以了解aav介导的位点特异性整合机制。我们的方法是将x射线晶体学、低温电镜和小角度x射线散射（SAXS）的结构研究与生物物理、动力学和功能研究相结合，以全面了解导致位点特异性整合的事件。我们期望在这项研究计划中获得的结果将作为一个起点，以产生准确的模型来理解这种独特的机制，也将为我们提供知识和工具，以潜在地设计具有新特异性的Rep蛋白，可用于开发基因治疗领域的新技术。