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.
Liu
Xi-Peng
XP
School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Li
Chun-Peng
CP
Hou
Jing-Li
JL
Liu
Yu-Fen
YF
Liang
Ru-Bing
RB
Liu
Jian-Hua
JH
D041522:Aeropyrum / Q000201:enzymology* / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013696:Temperature; D013941:Thymine / Q000737:chemistry / Q000378:metabolism; D044502:Thymine DNA Glycosylase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D019843:Archaeal Proteins; D044502:Thymine DNA Glycosylase; D013941:Thymine