Clone, purify and 1D nuclear magnetic resonance spectroscopy of the BRCT domain of E. coli DNA Ligase LigA

Abstract EN

DNA ligases are essential enzymes in all domains of life.

The role of these enzymes are to bind DNA ends, nicked DNA and join broken nucleic acid strands.

The tertiary structure of E.

coli DNA ligase LigA has four main domains: nucleotidyltransferase, oligomer-binding, Helix-hairpin-Helix and BRCT domain.

One of the main objectives of my previous study was to explore the potential DNA ligase LigA as possible antibiotic targets by using a molecular docking programme called Molecular Operating Environment (MOE) (in silico) just in the first three domains of the tertiary structure of NAD+-dependent DNA ligase of E.

coli LigA protein.

Fortunately, it was found that four compounds out of the eight (5-Azacytidine, Geneticin, Chlorhexidine and Imidazolidinyl Urea) did inhibit the activity of E.

coli LigA protein in silico, in vitro and then in vivo experiments after purify the native LigA protein.

Importantly, the tertiary structure of this small BRCT domain has not been solves before.

It does not appear in the crystal structure of 2OWO (PDB) that solved by Nandakumar et al., 2007.

In this paper, the project was carried out to clone, express and purify the 88 amino acid of the forth domain (BRCT) and doing initial NMR experiment (1D NMR spectrum) to check the folding of this domain.

It was found and determined that the BRCT domain of E.

coli DNA ligase LigA is folded accurately, which is increased and supported the possibility as antibiotic target in all domains of E.

coli LigA protein in the future.