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

A Wilkinson, A Smith, D Bullard, M Lavesa-Curto, H Sayer, A Bonner, A Hemmings, R Bowater

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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.
Original languageEnglish
Pages (from-to)113-122
Number of pages10
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1749
Issue number1
DOIs
Publication statusPublished - 20 May 2005

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