Jt. Stivers et al., Kinetic mechanism of damage site recognition and uracil flipping by Escherichia coli uracil DNA glycosylase, BIOCHEM, 38(3), 1999, pp. 952-963
The DNA repair enzyme uracil DNA glycosylase (UDC) catalyzes hydrolytic cle
avage of the N-glycosidic bond of premutagenic uracil residues in DNA by fl
ipping the uracil base from the DNA helix. The mechanism of base flipping a
nd the role this step plays in site-specific DNA binding and catalysis by e
nzymes are largely unknown. The thermodynamics and kinetics of DNA binding
and uracil flipping by UDG have been studied in the absence of glycosidic b
ond cleavage using substrate analogues containing the 2'-alpha and 2'-beta
fluorine isomers of 2'-fluoro-2'-deoxyuridine (U-beta, U-alpha) positioned
adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP). A
ctivity measurements show that DNA containing a U-beta or U-alpha nucleotid
e is a 10(7)-fold slower substrate for UDG (t(1/2) approximate to 20 h), wh
ich 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 expect
ed for a decrease in 2-AP base stacking resulting from enzymatic flipping o
f the adjacent uracil. Thermodynamic measurements show that UDG forms weak
nonspecific complexes with dsDNA (K-D(ns) = 1.5 mu M) and binds similar to
25-fold more tightly to U-beta containing dsDNA (K-D(app) approximate to 50
nM). Thus, base flipping contributes less than similar to 2 kcal/mol to th
e free energy of binding and is not a major component of the > 10(6)-fold c
atalytic specificity of UDG. Kinetic studies at 25 degrees C show that site
-specific binding occurs by a two-step mechanism. The first step (E + S <->
ES) involves the diffusion-controlled binding of UDG to form a weak nonspe
cific complex with the DNA (K-D approximate to 1.5-3 mu M). The second step
(ES <-> E'F) involves a rapid step leading to reversible uracil flipping (
k(max) approximate to 1200 s(-1)). This step is followed closely by a confo
rmational change in UDG that was monitored by the quenching of tryptophan f
luorescence. The results provide evidence for an enzyme-assisted mechanism
for uracil flipping and exclude a passive mechanism in which the enzyme tra
ps a transient extrahelical base in the free substrate. The data suggest th
at the duplex structure of the DNA is locally destabilized before the base-
flipping step, thereby facilitating extrusion of the uracil. Thus, base fli
pping contributes little to the free energy of DNA binding but contributes
greatly to specificity through an induced-fit mechanism.