Neddermann P, Jiricny J
Department of Biochemistry, Istituto di Richerche di Biologia Molecolare P. Angeletti, Rome, Italy.
J Biol Chem. 1993 Oct 5;268(28):21218-24.
G/T mispairs that arise in the DNA of higher eukaryotes as a result of spontaneous hydrolytic deamination of 5-methylcytosine to thymine must be corrected to G/C pairs. We describe here the purification to apparent homogeneity of the enzyme that initiates this repair process by excising the mispaired thymine from the hetero-duplex to generate an apyrimidinic site. The enzymatic activity could be attributed to a 55-kDa polypeptide, which was purified from extracts of HeLa cells by a combination of conventional and DNA-affinity chromatography. The enzyme is a mismatch-specific thymine-DNA N-glycosylase, capable of hydrolyzing the carbon-nitrogen bond between the sugar-phosphate backbone of the DNA and a mispaired thymine. In addition to the G/T, the enzyme can remove thymine also from C/T and T/T mispairs in the order G/T >> C/T > T/T. It has no detectable endonucleolytic activity on apyrimidinic sites and does not catalyze the removal of thymine from A/T pairs or from single-stranded DNA.
高等真核生物DNA中由于5-甲基胞嘧啶自发水解脱氨生成胸腺嘧啶而出现的G/T错配必须校正为G/C对。我们在此描述了一种酶的纯化过程，该酶通过从异源双链体中切除错配的胸腺嘧啶以产生无嘧啶位点来启动这一修复过程，并将其纯化至表观均一。该酶活性可归因于一种55 kDa的多肽，它是通过传统色谱法和DNA亲和色谱法相结合，从HeLa细胞提取物中纯化得到的。该酶是一种错配特异性胸腺嘧啶-DNA N-糖苷酶，能够水解DNA糖磷酸骨架与错配胸腺嘧啶之间的碳氮键。除了G/T错配，该酶还能按照G/T >> C/T > T/T的顺序从C/T和T/T错配中去除胸腺嘧啶。它对无嘧啶位点没有可检测到的核酸内切酶活性，也不催化从A/T对或单链DNA中去除胸腺嘧啶。
