Wilkinson Adam, Smith Andrew, Bullard Desmond, Lavesa-Curto Manuel, Sayer Heather, Bonner Alexandra, Hemmings Andrew, Bowater Richard
Phico Therapeutics Ltd, Babraham Hall, Babraham, Cambridge, CB2 4AT, UK.
Biochim Biophys Acta. 2005 May 20;1749(1):113-22. doi: 10.1016/j.bbapap.2005.03.003.
NAD(+)-dependent DNA ligases are essential enzymes in bacteria, with the most widely studied of this class of enzymes being LigA from Escherichia coli. NAD(+)-dependent DNA ligases comprise several discrete structural domains, including a BRCT domain at the C-terminus that is highly-conserved in this group of proteins. The over-expression and purification of various fragments of E. coli LigA allowed the investigation of the different domains in DNA-binding and ligation by this enzyme. Compared to the full-length protein, the deletion of the BRCT domain from LigA reduced in vitro ligation activity by 3-fold and also reduced DNA binding. Using an E. coli strain harbouring a temperature-sensitive mutation of ligA, the over-expression of protein with its BRCT domain deleted enabled growth at the non-permissive temperature. In gel-mobility shift experiments, the isolated BRCT domain bound DNA in a stable manner and to a wider range of DNA molecules compared to full LigA. Thus, the BRCT domain of E. coli LigA can bind DNA, but it is not essential for DNA nick-joining activity in vitro or in vivo.
NAD(+) 依赖性 DNA 连接酶是细菌中的必需酶，这类酶中研究最广泛的是来自大肠杆菌的 LigA。NAD(+) 依赖性 DNA 连接酶包含几个离散的结构域，包括 C 端的 BRCT 结构域，该结构域在这组蛋白质中高度保守。大肠杆菌 LigA 各种片段的过表达和纯化使得能够研究该酶在 DNA 结合和连接中的不同结构域。与全长蛋白相比，从 LigA 中缺失 BRCT 结构域使体外连接活性降低了 3 倍，并且也降低了 DNA 结合能力。使用携带 ligA 温度敏感突变的大肠杆菌菌株，缺失其 BRCT 结构域的蛋白质的过表达使得在非允许温度下能够生长。在凝胶迁移率变动实验中，与完整的 LigA 相比，分离的 BRCT 结构域以稳定的方式结合 DNA，并且能结合更广泛的 DNA 分子。因此，大肠杆菌 LigA 的 BRCT 结构域可以结合 DNA，但对于体外或体内的 DNA 切口连接活性不是必需的。
