Expression and characterization of thymine-DNA glycosylase from Aeropyrum pernix.

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 degrees 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 degrees C. ApeTDG also had a relatively weak DNA glycosylase activity on uracil base, with the following order: U/C>U/G approximately U/T>U/U approximately U/I approximately U/AP approximately 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.

Evidence for three thymine DNA glycosylases in human cell extracts: substrate specificities of thymine DNA glycosylase activities.

BACKGROUND: Purified human thymine DNA glycosylase (TDG) recognizes a G: T mispair in a CpG sequence context more strongly than in any other, in addition to its inactivity toward 2-aminopurine: T or 2,6 diaminopurine: T pairs. We investigated the multiplicity of TDG to establish a better relationship between in vitro G: T mismatch incision and in vivo repair of a G: T to a G: C pair. MATERIAL/METHODS: Cell-free extract was prepared from A1235-MR4 human glioma cells grown in tissue culture. Fractions containing TDG activities were separated on a strong anion-exchange column. 45-bp DNA containing a single G: T or altered G: T mispair was prepared for measuring mismatch-specific strand-incision. RESULTS: The extract yielded three fractions containing TDG activities. Each was further purified on a sizing column to exclude a relationship between a small fragment and TDG activity. While the substrate activity range of fraction III, eluting at the highest salt concentration, was the same as the known TDG, fractions eluted at medium and low concentrations were distinct: fractions I and II reacted with substrates of known TDG and DNA containing or 2-aminopurine: T (2,6-diaminopurine: T) base pairs. Modified m4T mispaired with G in DNA was acted on by fraction I and not II or III, suggesting fraction I activity is distinct. Each fraction showed strong activity on DNA with G: U and G: T mispairs in the CpG sequence context. CONCLUSIONS: The unique range of each TDG activity corresponding to the three fractions indicates that human cells possibly express three distinct TDGs.