Adeno-Associated Virus (AAV) Rep proteins use a unique mechanism to integrate into its host genome using a strand-transfer reaction and also to process single stranded DNA during DNA replication. 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 DNA melting and nicking using a combination of structural biology methods that include X-ray crystallography, small-angle X-ray scattering (SAXS) and single-particle reconstruction electron microscopy coupled to biochemical, biophysical and in vivo assays.
This work will offer significant insights into the molecular mechanism by which AAV Rep proteins are able to assemble, melt and nick DNA as a preamble to the site-specific integration reaction. A through understanding of this process is crucial to improve the current AAV based gene therapy vectors and to generate vectors with novel specificities.
