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

Wilkinson, Adam, Smith, Andrew, Bullard, Desmond, Lavesa-Curto, Manuel, Sayer, Heather, Bonner, Alexandra, Hemmings, Andrew ORCID: and Bowater, Richard ORCID: (2005) Analysis of ligation and DNA binding by Escherichia coli DNA ligase (LigA). Biochimica et Biophysica Acta - Proteins and Proteomics, 1749 (1). pp. 113-122. ISSN 1570-9639

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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.

Item Type: Article
Faculty \ School: Faculty of Science > School of Biological Sciences
UEA Research Groups: Faculty of Science > Research Groups > Plant Sciences
Faculty of Science > Research Groups > Molecular Microbiology
Faculty of Science > Research Groups > Chemistry of Life Processes
Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry
Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017)
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Depositing User: EPrints Services
Date Deposited: 01 Oct 2010 13:37
Last Modified: 24 Oct 2022 02:11
DOI: 10.1016/j.bbapap.2005.03.003

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