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Originally published In Press as doi:10.1074/jbc.M005095200 on August 10, 2000

J. Biol. Chem., Vol. 275, Issue 43, 33449-33456, October 27, 2000
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Separating Substrate Recognition from Base Hydrolysis in Human Thymine DNA Glycosylase by Mutational Analysis*

Ulrike Hardeland, Marc Bentele, Josef Jiricny, and Primo SchärDagger

From the Institute of Medical Radiobiology, University of Zürich and the Paul Scherrer Institute, August Forel Strasse 7, Zürich 8008, Switzerland

Human thymine DNA glycosylase (TDG) was discovered as an enzyme that can initiate base excision repair at sites of 5-methylcytosine- or cytosine deamination in DNA by its ability to release thymine or uracil from G·T and G·U mismatches. Crystal structure analysis of an Escherichia coli homologue identified conserved amino acid residues that are critical for its substrate recognition/interaction and base hydrolysis functions. Guided by this revelation, we performed a mutational study of structure function relationships with the human TDG. Substitution of the postulated catalytic site asparagine with alanine (N140A) resulted in an enzyme that bound mismatched substrates but was unable to catalyze base removal. Mutation of Met-269 in a motif with a postulated role in protein-substrate interaction selectively inactivated stable binding of the enzyme to mismatched substrates but not so its glycosylase activity. These results establish that the structure function model postulated for the E. coli enzyme is largely applicable to the human TDG. We further provide evidence for G·U being the preferred substrate of TDG, not only at the mismatch recognition step of the reaction but also in base hydrolysis, and for the importance of stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.

* This study was supported by grants from the Schweizeriche Krebsliga (to U. H., J. J., P. S.) and the Swiss National Science Foundation (to M. B., P. S.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ277958 and AJ277789.

Dagger To whom correspondence should be addressed: Tel: 41-1-634-8926; Fax: 41-1-634-8904; E-mail: schaer@imr.unizh.ch.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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