Kinetic mechanism of damage site recognition and uracil flipping by Escherichia coli uracil DNA glycosylase.
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'-alpha and 2'-beta fluorine isomers of 2'-fluoro-2'-deoxyuridine (Ubeta, Ualpha) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP). Activity measurements show that DNA containing a Ubeta or Ualpha nucleotide is a 10(7)-fold slower substrate for UDG (t1/2 approximately 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 (KDns = 1.5 microM) and binds approximately 25-fold more tightly to Ubeta containing dsDNA (KDapp approximately 50 nM). Thus, base flipping contributes less than approximately 2 kcal/mol to the free energy of binding and is not a major component of the >10(6)-fold catalytic 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 left and right arrow ES) involves the diffusion-controlled binding of UDG to form a weak nonspecific complex with the DNA (KD approximately 1.5-3 microM). The second step (ES left and right arrow E'F) involves a rapid step leading to reversible uracil flipping (kmax approximately 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.
Stivers
J T
JT
Center for Advanced Research in Biotechnology, University of Maryland, National Institute for Standards and Technology, Rockville 20850, USA.
Pankiewicz
K W
KW
Watanabe
K A
KA
D001665:Binding Sites; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005467:Floxuridine / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D046911:Macromolecular Substances; D009699:N-Glycosyl Hydrolases / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013816:Thermodynamics; D014498:Uracil / Q000737:chemistry*; D051981:Uracil-DNA Glycosidase
D046911:Macromolecular Substances; D005467:Floxuridine; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase