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.

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

“…That the protein significantly stabilizes a base separated state (compare with SI Fig. 8) is consistent with findings for other DNA-repair proteins (4,7,8,10,26). For the active-site entry step, it was necessary to substitute the distance between the O 6 atom of the flipping base and the C ␣ atom of Ala-154 (d 5 ) for d 3 because Gua lacks the methyl carbon.…”

Uracil DNA glycosylase uses DNA hopping and short-range sliding to trap extrahelical uracils

“…3A). Experimentally, the reactions are first quenched at various times with a potent and specific inhibitor of UNG (18,19), and the uracil excision products are then converted to unique dsDNA fragments AB, BC, A, and C by using abasic site endonuclease and restriction endonuclease nicking reactions (SI Methods). The central aspect of this method is that the single-excision intermediates (fragments AB and BC) are consumed when UNG transfers successfully to the second site and excises the uracil.…”

Extrahelical Damaged Base Recognition by DNA Glycosylase Enzymes

“…A systematic study of this question has been undertaken for UNG (Scheme 1, reaction 1). In these studies, DNA duplexes containing a single U/X or T/X base pair were used, where X was a adenine analogue capable of forming one, two or three hydrogen bonds with the opposing U or T (Figure 3a) [19][20][21]. [As further elaborated below, UNG can bind T or U in an extrahelical mode, but the shared binding site for T and U is not the active site pocket that only accomodates U.]…”

“…The reaction coordinate for uracil flipping by UNG. The microscopic rate constants have been calculated by combining NMR [19,34] and rapid kinetic measurements [24,25] [20,25], final flipped state (pdb 1EMH) [33]. Since the rates by neccesity were obtained using different substrates and by extrapolation of the base pair opening rates to 25 °C, the values should only be considered best approximations.…”