Abstract
The recombinant thymine-DNA glycosylase (TDG) from Aeropyrum pernix ( A. pernix) was expressed in Escherichia coli. The enzymatic activity of recombinant A. pernix TDG (ApeTDG) was characterized using oligonucleotides containing a thymine/uracil base as substrate. ApeTDG had distinct glycosylase activity on T/G mismatch. The optimal temperature and pH for thymine removal were 65–70 °C and pH 7.0–8.5, respectively. High concentration of NaCl inhibited the thymine removal. Divalent ions had different influence on the thymine removal by ApeTDG. Ca 2+ and Mg 2+ had no inhibition on the enzymic activity, but Ni 2+, Co 2+, Cu 2+, Mn 2+, and Zn 2+ completely inhibited the excision reaction. As derived from a hyperthermophilic archaea, ApeTDG protein was heat-resistant at 75 °C. ApeTDG also had a relatively weak DNA glycosylase activity on uracil base, with the following order: U/C > U/G ≈ U/T > U/U ≈ U/I ≈ U/AP ≈ U/- > U/A. Additional mismatch located at 3′ of T/G had less inhibition on the thymine removal than that located at 5′ of T/G, and two additional mismatches located at each side of T/G completely inhibited the excision of thymine. Together, these data suggest that ApeTDG is a TDG protein with weak UDG activity.
Published Version
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