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

项目摘要 腺相关病毒（AAV）已经进化出整合到其宿主基因组中的特性，其利用独特的免疫调节机制， 在DNA复制过程中，生物体在所有生命领域中共有的处理单链DNA的机制， 细菌接合和特殊的转座情况。大的AAV Rep蛋白Rep 68/Rep 78是重组的AAV Rep蛋白。 使用两个多功能结构域（包括一个原始结合结构域）执行这一过程的关键参与者 (OBD)特异性结合DNA并属于HUH内切核酸酶家族的蛋白质;和SF 3解旋酶 域整合取决于Rep 68/Rep 78与整合位点AAVS 1的结合，促进其整合。 熔融并在已被聚合的单链区域中进行链特异性酯交换反应。 在熔融步骤中挤出。我们对这一过程的分子细节和我们的长期研究知之甚少。 目的是确定由AAV Rep蛋白介导的位点特异性整合的分子机制。在 本建议我们将集中于揭示Rep介导的DNA解链，切口和 包装使用结构生物学方法的组合，包括X射线晶体学，和单- 颗粒重建Cryo-EM耦合到生物化学，生物物理和体内测定。

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.