真菌DNA去甲基化途径关键双加氧酶CcTET的底物选择性识别和催化机制研究

DNA甲基化与去甲基化动态平衡在生命体生长发育中具有关键作用。其中DNA主动去甲基化主要通过双加氧酶TET对5-甲基胞嘧啶(5mC)逐级氧化来实现，其表达和功能异常与多种疾病的发生发展密切相关。然而由于缺乏化学生物学研究工具和检测技术，人类TET难以重组表达，多种核酸氧化产物无法精确区分和检测，其生物功能尚未阐明，探针发现工作进展缓慢。申请人以本课题组前期在真菌中发现的DNA去甲基化酶CcTET为研究对象，利用化学生物学和结构生物学交叉技术，解析了CcTET和CcTET-5mC-DNA复合物结构，初步阐明了CcTET的DNA底物识别机制。在此基础上，拟基于已阐明分子机制，进一步设计和筛选具有更高催化活性或能催化单一氧化产物的突变体，并阐明其核酸修饰产物选择性的分子机制。这将为深入研究人类TET酶的调控机制和DNA去甲基化的生物功能提供新的化学生物学研究工具，并奠定前期理论基础。

The dynamic balance of DNA methylation and demethylation plays a pivotal role in the growth and development of life. The DNA active demethylation is mainly mediated by the continuous oxidation of 5-methylcytosine by the ten-eleven translocation dioxygenases (TET). The abnormal expression or function of TET highly associates with numerous diseases. However, due to the lack of chemical biology research tools and technique, the human TET could not be recombinantly expressed and studied, and the various nucleic acid products by Tet oxidation could not be time-resolvedly monitored and detected, its biofunction has not been elucidated. The chemical probe discovery has also been delayed. In this study, the applicant focuses on a fugus-version TET enzyme (CcTET) that was previously discovered by our group from Coprinopsis cinerea. The applicant has solved the crystal structures of CcTET and CcTET-5mC-DNA complex, and elucidated the DNA recognition mechanism of CcTET. The applicant will further design and screen CcTET mutants that either can significantly enhance its oxidation activity or produce single oxidation product, and uncover the selective and catalytic mechanisms. This work will provide new chemical biology research tools for functional study against human TET and DNA demethylation, and lay the preliminary theoretical foundation.