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Steady‐state kinetics of human thymine DNA glycosylase (hTDG) using a coupled‐enzyme assay: Implications for the mechanism of hTDG stimulation by human AP endonuclease 1
Author(s) -
Fitzgerald Megan,
Drohat Alexander C.
Publication year - 2007
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.21.5.a290-d
Subject(s) - dna glycosylase , ap site , uracil dna glycosylase , dna , base excision repair , chemistry , enzyme kinetics , ap endonuclease , thymine , biochemistry , uracil , dna repair , enzyme , biophysics , microbiology and biotechnology , active site , biology
DNA glycosylases initiate base excision repair by removing damaged or mismatched bases, producing an abasic (AP) site in the DNA. Many glycosylases bind the AP DNA product tightly, impeding enzymatic turnover. Human thymine DNA glycosylase (hTDG), which recognizes G·T mispairs and other mutagenic lesions, exhibits severe product inhibition, precluding the use of steady‐state kinetics to study its catalytic mechanism. To overcome this problem, we developed a coupled enzyme assay where the second enzyme, human AP endonuclease (hAPE1), stimulates the turnover of hTDG. The coupled reaction is monitored continuously by fluorescence spectroscopy using DNA containing 2‐aminopurine adjacent to the target base. We determined the steady‐state kinetic parameters for hTDG, and its catalytic core (hTDG‐core, Phe 111 to Val 308 ) against a G·U substrate. We find that hAPE1 enhances the steady state turnover ( k cat ) of hTDG and hTDG‐core by about 15‐fold and 100‐fold, respectively, and that hAPE1 actively displaces AP DNA from hTDG and hTDG‐core. Thus, the 110 N‐terminal residues and 102 C‐terminal residues of hTDG are not required for its stimulation by hAPE1. Using the coupled assay, we show that hTDG‐core is not inhibited by the substrate bases uracil or thymine at up to 5 mM concentrations. In contrast, hTDG‐core is inhibited by duplex DNA containing a stable dU analog and by DNA containing a central CpG site.