5-Formylcytosine and 5-Carboxylcytosine Significantly Reduce the Catalytic Activity of Hhal DNA Methyltransferase

DNA methylation is an essential epigenetic modification, and found to be dynamically changed due to the ob- servation of active DNA demethylation. During active demethylation, 5-methylcytosine （5mC） was oxidized step- wise by ten-eleven translocation （TET） enzymes into 5-hydroxymethylcytosine （5hmc）, 5-formylcytosine （5fC）, and 5-carboxylcytosine （5caC）. Then, the subsequent excision of 5fC and 5caC combined with base excision repair further restored cytosine, which completes the demethylation process. Here, we report that 5-formylcytosine and 5-carboxylcytosine significantly reduce the activity of HhaI DNA methyltransferase to methylate target cytosines when present on the hemi-modified sequence of the complementary DNA. This finding demonstrates that 5fC and 5caC function as more than intermediates for active DNA demethylation.

ColEl-Hhal片断DNA编码产物的体外合成

在S-30无细胞转录翻译联对系统中,应用小量S-30抽提液,即在每毫升反应混合液内,S-30蛋白质含量从6.6毫克降低至1毫克,不仅对DNA模板具有较高合成能力,而且对降低内源蛋白质合成起了显著作用。以Col El—Hhal的六个片断为模板,在S-30转录翻译联对系统进行蛋白合成,各片段DNA都编码了其相应的蛋白质。体外合成的蛋白产物应用Colicin抗血清的沉淀试验得到验证。