DOI
https://doi.org/10.25772/ACW6-HR39
Defense Date
2014
Document Type
Thesis
Degree Name
Master of Science
Department
Physiology
First Advisor
Carlos Escalante
Abstract
Multiple DNA transactions are at the center of almost all processes regulating the AAV life cycle. A common feature shared by all transactions is the binding of the large AAV Rep proteins Rep78/Rep68 onto DNA sites harboring multiple GCTC repeats. AAV mediated site-specific integration is contingent upon the formation of a productive complex between Rep78/Rep68 and the AAVS1 site located at chromosome 19. In order to understand the mechanistic details of the initial assembly process we carried out equilibrium binding experiments of Rep68 and its individual domains with a 42-mer AAVS1 site. Results show that although Rep68 binds AAVS1 with high affinity (69 nM), both the OBD and helicase individual domains bind DNA weakly with affinities of >>60μM and 22μM respectively under our experimental conditions. Mutant Rep68 proteins that have a defective oligomerization interface bind DNA poorly suggesting that productive binding requires both the concerted interaction of the individual domains with DNA and oligomerization. Moreover, we show that a minimal number of two repeats is required to form a stable complex. In addition, initial studies were done to characterize the interaction between Rep68 and the viral ITR DNA sequences using AUC and electron microscopy.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-18-2014