Mutant of human thymine DNA glycosylase (TDG) residues that can selectively cleave 5-carboxycytosine in DNA for use in assays to identify drugs that modulate activity of the enzyme.
- Is very specific - shows no activity with uracil and only residual activity with other substrates.
- Has higher activity at a lower pH.
- Remains specific even in presence of excess genomic DNA.
Thymine DNA glycosylase (TDG) catalyzes the repair of mismatched DNA by recognizing and then excising the damaged base. Specifically, these enzymes favor the removal of thymine, uracil, or 5-hydroxymethyluracil from mismatched pairs resulting from spontaneous deamination of cytosine derivatives. In addition, TDG can remove 5-formylcytosine and 5-carboxylcytosine (5caC). If mismatched DNA is not repaired, DNA abnormalities and carcinogenesis can occur.
Emory researchers have generated a mutant human TDG that contains an asparagine-to-aspartate (N157D) mutation capable of selectively cleaving 5caC with a rate comparable to the wild-type glycosylase. Unlike the native protein, this mutant shows no activity towards uracil and only residual activity towards other substrates. In addition, it remains highly specific even in the presence of excess genomic DNA. The ability of the mutant strain to cleave 5caC selectively could allow for the genomic locations of 5caC to be identified at single-base resolution.
Publication: Hashimoto et al. (2013) Journal of Molecular Biology