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Thermodynamic and kinetic characterization of binding stoichiometry and mismatch repair activity by human thymine DNA glycosylase
Author(s) -
Morgan Michael Thomas,
Drohat Alexander C.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.836.15
Subject(s) - dna glycosylase , dna , protein subunit , thymine , dna repair , biochemistry , biophysics , chemistry , ap site , base excision repair , binding site , substrate (aquarium) , stereochemistry , biology , microbiology and biotechnology , gene , ecology
Human thymine DNA glycosylase (TDG) is involved in efficiently repairing G:T and other mismatches through the base excision repair pathway. Our recent crystal structure revealed the catalytic core of TDG bound to abasic DNA in a 2:1 (protein:DNA) complex. The N‐terminal domain of TDG promotes the 2:1 complex and enhances the activity for G:T lesions. Here we investigate the relationship between 2:1 binding and T removal. Our work seeks to understand the mechanisms of this dimerization event, the substrate characteristics that favor 1:1 or 2:1 binding, the role of the N‐terminus in mediating such an event, and how the second subunit affects activity of the productively bound subunit. We find that, as compared with U, T removal is more sharply diminished when the substrate is only capable of binding a single copy of TDG. Furthermore, we find that both subunits are catalytically active, and the activity depends on the number of bases between two mispairs. ITC experiments show that TDG binding is significantly less favorable for DNA that forms a 1:1 versus a 2:1 complex , and this effect is greater for G:T versus G:U substrates. Supported by NIH grant to ACD.