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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-34314

Hardeland, U; Bentele, M; Jiricny, J; Schär, P (2000). Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis. Journal of Biological Chemistry, 275(43):33449-33456.

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Abstract

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.

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
DDC:570 Life sciences; biology
Language:English
Date:2000
Deposited On:09 Jul 2010 08:27
Last Modified:17 Jul 2014 12:43
Publisher:American Society for Biochemistry and Molecular Biology
ISSN:0021-9258
Additional Information:This research was originally published in: Hardeland, U; Bentele, M; Jiricny, J; Schär, P (2000). Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis. Journal of Biological Chemistry, 275(43):33449-33456. © the American Society for Biochemistry and Molecular Biology.
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:10.1074/jbc.M005095200
PubMed ID:10938281
Citations:Web of Science®. Times Cited: 80
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Scopus®. Citation Count: 84

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