Base Flipping in Lesion Search and Recognition by the Human Thymine DNA Glycosylase Revealed by Single Molecule Imaging

Abstract

To elucidate the poorly characterized process of damage search before base extrusion by glycosylases in base excision repair (BER), we studied damage detection by the human thymine DNA glycosylase (hTDG) by single molecule atomic force microscopy (AFM) imaging. Our results indicate that TDG exploits the intrinsic flexibility of its target sites as an initial recognition criterion. Enhanced flexibility likely also directly serves to selectively facilitate extrusion of potential candidate target sites from the DNA duplex for extrahelical base interrogation by TDG. Importantly, our data support an equilibrium between search (slightly bent DNA conformation) and interrogation (flipped base, strongly bent DNA) states during TDG's DNA lesion scanning process, indicating continuous base flipping in lesion search. Our AFM and fluorescence DNA base flipping data indicate stabilization of the extrahelical target base via an arginine finger in the protein, leading to prolonged residence time in the catalytic site and enhanced excision probability. Comparison of our results for hTDG with data on a second glycosylase, hOGG1, which recognizes structurally less flexible DNA lesions, support the generalizability of our proposed lesion search and recognition mechanism to a common strategy employed by BER glycosylases.