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Papers In Press, published online ahead of print August 10, 2000
J. Biol. Chem, 10.1074/jbc.M005095200
Submitted on June 13, 2000
Revised on July 20, 2000
Accepted on August 10, 2000

Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis

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

Institute of Medical Radiobiology, University of Zurich, Zurich CH-8008

Corresponding Author: schaer{at}imr.unizh.ch

Human thymine DNA glycosylase (TDG) was identified 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 GoT and GoU 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 M269 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 GoU 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.


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