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J. Biol. Chem., Vol. 277, Issue 22, 19424-19432, May 31, 2002
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From the Uracil-DNA glycosylase catalyzes the excision of
uracils from DNA via a mechanism where the uracil is extrahelically
flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has
not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of
excision at the active site during catalysis. Stopped-flow fluorometry
monitoring the 2-aminopurine fluorescence defined the kinetics of
uracil flipping. Parallel experiments detecting the protein
fluorescence showed a slower Leu191 insertion step
occurring after nucleotide flipping but before excision. The inserted
Leu191 acts as a doorstop to prevent the return of the
flipped-out uracil residue, thereby facilitating the capture of the
uracil in the active site and does not play a direct role in
"pushing" the uracil out of the DNA helix. The results define for
the first time the proper sequence of events during a catalytic cycle
and establish a "pull-push", as opposed to a "push-pull",
mechanism for nucleotide flipping.
Presteady-state Analysis of a Single Catalytic Turnover by
Escherichia coli Uracil-DNA Glycosylase Reveals a
"Pinch-Pull-Push" Mechanism*
§,,, and Department of Biochemistry and Biophysics,
Oregon State University and the ¶ Department of Environmental and
Molecular Toxicology and the Environmental Health Science Center,
Oregon State University, Corvallis, Oregon 97331
*
This work was supported by National Institutes of Health
Grants GM32823 and ES00210 (to D. W. M.) and GM58771 (to I. W.). This is Technical Report 11872 from the Oregon Agricultural
Experimental Station.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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