Development of mCherry tagged UdgX as a highly sensitive molecular probe for specific detection of uracils in DNA
Development of mCherry tagged UdgX as a highly sensitive molecular probe for specific detection of uracils in DNA
复制标题
DOI：
10.1016/j.bbrc.2019.08.005
复制
发表时间：
2019-10-08
影响因子：
3.1
通讯作者：
Varshney, Umesh
中科院分区：
文献类型：
--
作者：
Datta, Madhurima;Aroli, Shashanka;Varshney, Umesh
尿嘧啶并不总是DNA中错误出现的碱基。DNA基因组中的尿嘧啶在枯草杆菌噬菌体(PBS1/2)和疟疾原虫恶性疟原虫的生活史中起重要作用，并与果蝇的发育和B淋巴细胞中抗体的成熟有关。一种灵敏、特异和可靠的检测基因/基因组中尿嘧啶的技术对于了解尿嘧啶的各种生物学作用是至关重要的。污垢分枝杆菌UdgX(MsmUdgX)是我们实验室鉴定和鉴定的，它以特定的方式与DNA中的尿嘧啶形成共价复合体。MsmUdgX裂解尿嘧啶和DNA中的脱氧核糖之间的糖苷键，产生尿酸和氧卡宾离子。然后，氧卡宾离子被MsmUdgX的组氨酸侧链的亲核攻击捕获到共价络合物中。在这里，我们描述了一种融合蛋白mCherry标记的MsmUdgX(MChUdgX)的使用，它结合了MsmUdgX与基因组中尿嘧啶位点共价结合的特性，以及作为报告分子对mCherry的荧光检测的敏感性。我们发现，纯化的mChUdgX和高表达mChUdgX的大肠杆菌细胞提取物都提供了检测DNA中尿嘧啶类药物的高度敏感性和特异性。(C)2019 Elsevier Inc.保留所有权利。
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. (C) 2019 Elsevier Inc. All rights reserved.