Wilkinson Adam, Sayer Heather, Bullard Desmond, Smith Andrew, Day Jonathan, Kieser Tobias, Bowater Richard
Phico Therapeutics, Ltd., Babraham Hall, Babraham, Cambridge, United Kingdom.
Proteins. 2003 May 15;51(3):321-6. doi: 10.1002/prot.10361.
Sequencing of the genomes of Mycobacterium tuberculosis H37Rv and Streptomyces coelicolor A3(2) identified putative genes for an NAD(+)-dependent DNA ligase. We have cloned both open reading frames and overexpressed the protein products in Escherichia coli. In vitro biochemical assays confirm that each of these proteins encodes a functional DNA ligase that uses NAD(+) as its cofactor. Expression of either protein is able to complement E. coli GR501, which carries a temperature-sensitive mutation in ligA. Thus, in vitro and in vivo analyses confirm predictions that ligA genes from M. tuberculosis and S. coelicolor are NAD(+)-dependent DNA ligases.
结核分枝杆菌H37Rv和天蓝色链霉菌A3(2)基因组测序鉴定出了假定的NAD(+)依赖性DNA连接酶基因。我们克隆了这两个开放阅读框，并在大肠杆菌中过表达了蛋白质产物。体外生化分析证实，这些蛋白质中的每一个都编码一种以NAD(+)作为辅因子的功能性DNA连接酶。任一蛋白质的表达都能够互补携带ligA温度敏感突变的大肠杆菌GR501。因此，体外和体内分析证实了来自结核分枝杆菌和天蓝色链霉菌的ligA基因是NAD(+)依赖性DNA连接酶的预测。
