PMID:15848142
| Citation |
Wilkinson, A, Smith, A, Bullard, D, Lavesa-Curto, M, Sayer, H, Bonner, A, Hemmings, A and Bowater, R (2005) Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA). Biochim. Biophys. Acta 1749:113-22 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1016/j.bbapap.2005.03.003 |
| Keywords |
Base Sequence; Binding Sites; DNA/chemistry; DNA/metabolism; DNA Ligases/chemistry; DNA Ligases/genetics; DNA Ligases/metabolism; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Molecular Sequence Data; NAD/chemistry; NAD/metabolism; Protein Structure, Tertiary; Sequence Deletion; Substrate Specificity |
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