Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10537962 | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics | 2005 | 10 Pages |
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.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Adam Wilkinson, Andrew Smith, Desmond Bullard, Manuel Lavesa-Curto, Heather Sayer, Alexandra Bonner, Andrew Hemmings, Richard Bowater,