Premium
Mutational analysis of a putative base flipping residue R275 in human thymine DNA glycosylase
Author(s) -
Maiti Atanu,
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.836.14
Subject(s) - dna glycosylase , ap site , chemistry , dna , biochemistry , base excision repair , thymine , ap endonuclease , nucleotide , dna repair , base pair , microbiology and biotechnology , biology , gene
Thymine DNA glycosylase (TDG) is a base excision repair enzyme that excises T from G·T mispairs and removes other lesions, exhibiting specificity for damage at CpG sites. DNA glycosylases use nucleotide flipping to find lesions and cleave the base‐sugar bond. Typically, a bulky side chain penetrates the DNA, filling the space vacated by the flipped nucleotide. Our crystal structure of human TDG catalytic domain bound to abasic DNA shows that an arginine (R275) plugs the helical gap and contacts two phosphates flanking the abasic sugar (Maiti A, et al ., Proc Natl Acad Sci 105 : 8890‐8895). Here, we examine the role of R275 in the catalytic mechanism of TDG. Isothermal titration calorimetry (ITC) experiments show that substrate (analog) binding affinity is significantly lower for R275A‐hTDG versus hTDG, and lower still for R275L‐hTDG. Single turnover kinetics experiments show that k max is significantly lower for R275A‐hTDG versus hTDG, and lower still for R275L‐hTDG. Our results are surprising, because two related enzymes, mismatch‐specific uracil glycosylase and uracil DNA glycosylase have leucine at the position corresponding to R275 of hTDG. We find that mutation of R275 has no effect on steady‐state turnover, k cat , suggesting that R275 cannot account for the exceedingly slow product release observed for hTDG. This work was supported by an NIH grant (to ACD).