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J. Biol. Chem., Vol. 276, Issue 45, 42347-42354, November 9, 2001

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Turning On Uracil-DNA Glycosylase Using a Pyrene Nucleotide Switch^*

Yu Lin Jiang, Keehwan Kwon, and James T. Stivers

From the Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185

Base flipping is a highly conserved process by which enzymes swivel an entire nucleotide from the DNA base stack into their active site pockets. Uracil DNA glycosylase (UDG) is a paradigm enzyme that uses a base flipping mechanism to catalyze the hydrolysis of the N-glycosidic bond of 2'-deoxyuridine (2'-dUrd) in DNA as the first step in uracil base excision repair. Flipping of 2'-dUrd by UDG has been proposed to follow a "pushing" mechanism in which a completely conserved leucine side chain (Leu-191) is inserted into the DNA minor groove to expel the uracil. Here we report a novel implementation of the "chemical rescue" approach to show that the weak binding affinity and low catalytic activity of L191A or L191G can be completely or partially restored by substitution of a pyrene (Y) nucleotide wedge on the DNA strand opposite to the uracil base (U/A to U/Y). These results indicate that pyrene acts both as a wedge to push the uracil from the base stack in the free DNA and as a "plug" to hinder its reinsertion after base flipping. Pyrene rescue should serve as a useful and novel tool to diagnose the functional roles of other amino acid side chains involved in base flipping.

The atomic coordinates and the structure factors (code 1SSP) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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