Dai Qing, Lu Xingyu, Zhang Liang, He Chuan
Department of Chemistry and Institute for Biophysical Dynamics, the University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.
Tetrahedron. 2012 Jul 1;68(26):5145-5151. doi: 10.1016/j.tet.2012.04.031. Epub 2012 Apr 14.
As an important step of the active demethylation of 5-methylcytosine (5mC), human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC) from double-stranded DNA (dsDNA). Here, we present synthesis of DNA oligos containing a 2'-deoxy-2'-fluoro-D-arabinofuranosyl-5-carboxylcytidine (F-5caC) modification that act as hTDG inhibitors. The glycosylase activity assay showed that F-5caC oligos were resistant to excision by the hTDG catalytic domain (hTDG(cat), residues 111-308) and they could inhibit the excision of DNA oligos containing 5caC. The electrophoretic mobility shift assay confirmed that DNA oligos containing F-5caC could bind well with unmodified hTDG(cat) to form a stable complex, which makes it possible to obtain the crystal structure of the complex to reveal details on how hTDG(cat) recognizes the DNA substrate.
作为5-甲基胞嘧啶（5mC）主动去甲基化的重要步骤，人胸腺嘧啶DNA糖基化酶（hTDG）能有效地从双链DNA（dsDNA）中切除5-羧基胞嘧啶（5caC）。在此，我们展示了含有2'-脱氧-2'-氟-D-阿拉伯呋喃糖基-5-羧基胞苷（F-5caC）修饰的DNA寡核苷酸的合成，这些寡核苷酸可作为hTDG抑制剂。糖基化酶活性测定表明，F-5caC寡核苷酸对hTDG催化结构域（hTDG(cat)，第111-308位氨基酸残基）的切除具有抗性，并且它们能够抑制含有5caC的DNA寡核苷酸的切除。电泳迁移率变动分析证实，含有F-5caC的DNA寡核苷酸能与未修饰的hTDG(cat)良好结合，形成稳定的复合物，这使得获得该复合物的晶体结构以揭示hTDG(cat)识别DNA底物的细节成为可能。
