title: The origins of high-affinity enzyme binding to an extrahelical DNA base.
abstract: Base flipping is a highly conserved strategy used by enzymes to gain catalytic access to DNA bases that would otherwise be sequestered in the duplex structure. A classic example is the DNA repair enzyme uracil DNA glycosylase (UDG) which recognizes and excises unwanted uracil bases from DNA using a flipping mechanism. Previous work has suggested that enzymatic base flipping begins with dynamic breathing motions of the enzyme-bound DNA substrate, and then, only very late during the reaction trajectory do strong specific interactions with the extrahelical uracil occur. Here we report that UDG kinetically and thermodynamically prefers substrate sites where the uracil is paired with an unnatural adenine analogue that lacks any Watson-Crick hydrogen-bonding groups. The magnitude of the preference is a striking 43000-fold as compared to an adenine analogue that forms three H-bonds. Transient kinetic and fluorescence measurements suggest that preferential recognition of uracil in the context of a series of incrementally destabilized base pairs arises from two distinct effects: weak or absent hydrogen bonding, which thermodynamically assists extrusion, and, most importantly, increased flexibility of the site which facilitates DNA bending during base flipping. A coupled, stepwise reaction coordinate is implicated in which DNA bending precedes base pair rupture and flipping.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Krosky
authors.forename: Fenhong
authors.initials: F
authors.lastname: Song
authors.forename: James T
authors.initials: JT
authors.lastname: Stivers
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics; D051981:Uracil-DNA Glycosidase
chemical_list: D004247:DNA; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase