Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India.
Biochem Biophys Res Commun. 2019 Oct 8;518(1):38-43. doi: 10.1016/j.bbrc.2019.08.005. Epub 2019 Aug 9.
Uracil is not always a mistakenly occurring base in DNA. Uracils in DNA genomes are known to be important in the life cycles of Bacillus subtilis phages (PBS1/2) and the malarial parasite, Plasmodium falciparum; and have been implicated in the development of fruit fly and antibody maturation in B-lymphocytes. Availability of a sensitive, specific and robust technique for the detection uracils in genes/genomes is essential to understand its varied biological roles. Mycobacterium smegmatis UdgX (MsmUdgX), identified and characterised in our laboratory, forms covalent complexes with the uracil sites in DNA in a specific manner. MsmUdgX cleaves the glycosidic bond between uracil and the deoxyribose sugar in DNA to produce uracilate and oxocarbenium ions. The oxocarbenium ion is then captured into a covalent complex by the nucleophilic attack of a histidine side chain of MsmUdgX. Here, we describe the use of a fusion protein, mCherry tagged MsmUdgX (mChUdgX), which combines the property of MsmUdgX to covalently and specifically bind the uracil sites in the genome, with the sensitivity of fluorescent detection of mCherry as a reporter. We show that both the purified mChUdgX and the Escherichia coli cell-extracts overexpressing mChUdgX provide high sensitivity and specificity of detecting uracils in DNA.
尿嘧啶并不总是 DNA 中错误出现的碱基。芽孢杆菌噬菌体（PBS1/2）和疟原虫等生物的 DNA 基因组中的尿嘧啶，在其生命周期中具有重要作用；而且，尿嘧啶在果蝇的发育和 B 淋巴细胞中抗体的成熟过程中也有涉及。因此，开发一种灵敏、特异且稳健的方法来检测基因/基因组中的尿嘧啶对于理解其多样化的生物学功能至关重要。我们实验室发现并鉴定了分枝杆菌尿嘧啶-DNA 糖基化酶 X（MsmUdgX），它以特定的方式与 DNA 中的尿嘧啶位点形成共价复合物。MsmUdgX 会切断 DNA 中尿嘧啶与脱氧核糖之间的糖苷键，生成尿嘧啶和氧碳正离子。然后，氧碳正离子通过 MsmUdgX 中组氨酸侧链的亲核攻击被捕获到共价复合物中。在这里，我们描述了一种融合蛋白 mCherry 标记的 MsmUdgX（mChUdgX）的应用，它结合了 MsmUdgX 共价且特异性地结合基因组中尿嘧啶位点的特性，以及 mCherry 作为报告分子进行荧光检测的敏感性。我们表明，纯化的 mChUdgX 和过表达 mChUdgX 的大肠杆菌细胞提取物都能提供检测 DNA 中尿嘧啶的高灵敏度和特异性。
