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

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.