Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA)

✍ Scribed by Adam Wilkinson; Andrew Smith; Desmond Bullard; Manuel Lavesa-Curto; Heather Sayer; Alexandra Bonner; Andrew Hemmings; Richard Bowater

• Book ID: 104003415
• Publisher: Elsevier Science
• Year: 2005
• Tongue: English
• Weight: 598 KB
• Volume: 1749
• Category: Article
• ISSN: 1570-9639
• DOI: 10.1016/j.bbapap.2005.03.003

✦ Synopsis

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.