The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes hydrolytic cleavage of the N-glycosidic bond of premutagenic uracil residues in DNA by flipping the uracil base from the DNA helix. The mechanism of base flipping and the role this step plays in site-specific DNA binding and catalysis by enzymes are largely unknown. The thermodynamics and kinetics of DNA binding and uracil flipping by UDG have been studied in the absence of glycosidic bond cleavage using substrate analogues containing the 2'-α and 2'-β fluorine isomers of 2'-fluoro-2'-deoxyuridine (U(β), U(α)) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP). Activity measurements show that DNA containing a U(β) or U(α) nucleotide is a 107-fold slower substrate for UDG (t( 1/2 ) ≃ 20 h), which allows measurements of DNA binding and base flipping in the absence of glycosidic bond cleavage. When UDG binds these analogues, but not other DNA molecules, a 4-8-fold 2-AP fluorescence enhancement is observed, as expected for a decrease in 2-AP base stacking resulting from enzymatic flipping of the adjacent uracil. Thermodynamic measurements show that UDG forms weak nonspecific complexes with dsDNA (KD(ns) = 1.5 μM) and binds ~25-fold more tightly to U(β) containing dsDNA (K(D)(app) ≃ 50 nM). Thus, base flipping contributes less than ~2 kcal/mol to the free energy of binding and is not a major component of the > 106-fold catalytic specificity of UDG. Kinetic studies at 25 °C show that site-specific binding occurs by a two-step mechanism. The first step (E + S mutually implies ES) involves the diffusion- controlled binding of UDG to form a weak nonspecific complex with the DNA (K(D) ≃ 1.5-3 μM). The second step (ES mutually implies E'F) involves a rapid step leading to reversible uracil flipping (k(max) ≃ 1200 s-1). This step is followed closely by a conformational change in UDG that was monitored by the quenching of tryptophan fluorescence. The results provide evidence for an enzyme-assisted mechanism for uracil flipping and exclude a passive mechanism in which the enzyme traps a transient extrahelical base in the free substrate. The data suggest that the duplex structure of the DNA is locally destabilized before the base-flipping step, thereby facilitating extrusion of the uracil. Thus, base flipping contributes little to the free energy of DNA binding but contributes greatly to specificity through an induced-fit mechanism.
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