Expression and characterization of thymine-DNA glycosylase from Aeropyrum pemix
Expression and characterization of thymine-DNA glycosylase from Aeropyrum pemix
复制标题
Aeropyrum pernix 胸腺嘧啶 DNA 糖基化酶的表达和表征。
DOI：
10.1016/j.pep.2009.10.006
复制
发表时间：
2010-03-01
影响因子：
1.6
通讯作者：
Liu, Jian-Hua
中科院分区：
文献类型：
--
作者：
Liu, Xi-Peng;Li, Chun-Peng;Liu, Jian-Hua
从Aeropyrum pernix（A. pernix）在大肠杆菌中表达。重组A. pernix TDG（ApeTDG）的特征在于使用含有胸腺嘧啶/尿嘧啶碱基的寡核苷酸作为底物。ApeTDG对T/G错配有明显的糖基化活性。胸腺嘧啶去除的最佳温度和pH分别为65-70 ℃和7.0-8.5。高浓度NaCl抑制胸腺嘧啶的去除。二价离子对ApeTDG去除胸腺嘧啶有不同程度的影响。Ca ~（2+）、Me ~（2+）对酶活性无抑制作用，Ni ~（2+）、Co ~（2+）、Cu ~（2+）、Mn ~（2+）、Zn ~（2+）对酶切反应有完全抑制作用。ApeTDG蛋白来源于极端嗜热古菌，在75 ℃下耐热。ApeTDG对尿嘧啶碱基也有较弱的DNA糖基化酶活性，其顺序为：U/C > U/G approximate U/T > U/U approximate U/I approximate U/AP approximate U/- > U/A。位于T/G 3'端的错配对胸腺嘧啶去除的抑制作用小于位于T/G 5'端的错配，位于T/G两侧的两个错配完全抑制胸腺嘧啶的去除。总之，这些数据表明ApeTDG是具有弱UDG活性的TDG蛋白。(C)2009 Elsevier Inc. All rights reserved.
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. Ca2+ and Me2+ had no inhibition on the enzymic activity, but Ni2+, Co2+, Cu2+ Mn2+, and Zn2+ 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 approximate to U/T > U/U approximate to U/I approximate to U/AP approximate to 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. (C) 2009 Elsevier Inc. All rights reserved.