Structural and Mechanistic Insights into AAV Rep Mediated Site-Specific Integration and Packaging

对 AAV 代表介导的特定站点集成和打包的结构和机制见解

• 批准号: 10365359
• 负责人: Carlos R Escalante
• 金额: $ 34.39万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10365359
• 关键词: Binding; Binding Sites; Biochemical; Biological Assay; Biophysics; Capsid; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Binding; DNA Binding Domain; DNA biosynthesis; Data; Dependovirus; Event; Family; Funding; Gene Transduction Agent; Generations; Genetic Conjugation; Genome; Goals; Human Genome; Kinetics; Length; Life; Life Cycle Stages; Mediating; Methodology; Methods; Molecular; Nature; Organism; Phase; Play; Process; Property; Proteins; Reaction; Replication Origin; Resolution; Role; Single-Stranded DNA; Site; Specificity; Structure; System; Testing; Time; Transact; Transcriptional Regulation; Transgenes; Viral Packaging; Virus; Work; X-Ray Crystallography; endonuclease; experimental study; gene therapy; helicase; improved; in vivo; insight; integration site; melting; novel; particle; reconstruction; site-specific integration; stem; stoichiometry; structural biology; vector

PROJECT SUMMARY Adeno-Associated Virus (AAV) has evolved the property to integrate into its host genome exploiting a unique mechanism shared by organisms in all domains of life to process single stranded DNA during DNA replication, bacterial conjugation and special cases of transposition. The large AAV Rep proteins Rep68/Rep78 are the key players that carry out this process using two multifunctional domains that include an origin binding domain (OBD) that binds DNA specifically and belongs to the family of HUH endonucleases; and a SF3 helicase domain. Integration is contingent on the binding of Rep68/Rep78 to the integration site AAVS1, promote its melting and perform a strand-specific transesterification reaction in the single-stranded region that has been extruded during the melting step. Little is known about the molecular details of this process and our long-term goal is to determine the molecular mechanism of site-specific integration mediated by AAV Rep proteins. In this proposal we will focus on uncovering the mechanisms of Rep mediated DNA melting, nicking and packaging using a combination of structural biology methods that include X-ray crystallography, and single- particle reconstruction Cryo-EM coupled to biochemical, biophysical and in vivo assays.

项目总结 腺相关病毒(AAV)已进化出整合到其宿主基因组中的特性，利用独特的 在DNA复制过程中，生命所有领域的生物体都有处理单链DNA的机制， 细菌接合和特殊情况的转座。大的AAV Rep蛋白Rep68/Rep78是 使用包括起源绑定域的两个多功能域执行这一过程的关键角色 (OBD)，与DNA特异结合，属于hH内切酶家族；以及SF3解旋酶 域。整合取决于Rep68/Rep78与整合位点AAVS1的结合，促进其 熔化并在单链区域中执行链特异性的酯交换反应 在熔化步骤中挤出。关于这一过程的分子细节以及我们长期的 目的是确定AAV Rep蛋白介导的位点特异性整合的分子机制。在……里面 在这项提案中，我们将重点揭示Rep介导的DNA熔化、划痕和 使用结构生物学方法的组合进行包装，包括X射线结晶学和单一- 粒子重建低温电子显微镜结合生化、生物物理和活体分析。