AbstractDNA ligases are essential enzymes across all three domains of life. Their role is to bind nicked DNA or DNA ends and re-join broken nucleic acid strands. In bacteria, there are two key pathways that DNA ligases are involved in: DNA replication during cell division and DNA repair for breaks in DNA that occur as a result of damage. In E.coli, there are two genes for DNA ligases: LigA, whose structure has been published and LigB as a second ligase (unknown in structure) that has a different DNA, and therefore protein, sequence.
The overall aim of this work was first to clone, express, purify and test the kinetic and binding reactions of LigA and LigB as native proteins (not fusion-tagged versions, which most previous studies have used). This has not been reported before and the characterisation of the kinetics of LigA and LigB are very sparse in the literature. In this study we attempted to cover the lack of kinetic data on native LigA and LigB by studying how its functioning is affected by varying [DNA], [NAD+], reaction temperature, [NMN] and [NH4SO4]. The aim was to find the Vmax (maximal theoretical rate), Km (Michaelis equilibrium constant) and kcat (turnover number) for the nick-sealing reaction with each different variable.
We found that the Km values for LigA and LigB (how tightly they bound the nicked DNA) for singly-nicked DNA substrate were 12.22±6.42 and 3.50±1.34 nM, respectively. Therefore, native LigA was 6.4-fold tighter than native LigB. The Km values for varying NAD+ cofactor concentration were sharply different (1.55±0.33 vs. 0.16±0.03 µM), and show that LigB bound NAD+ 9.6 times tighter than LigA. However, the Vmax values showed that LigA was 4-fold faster than LigB. Both native enzymes had a similar optimum temperature for activity. For LigA this was ~20ºC, and for LigB approximately ~16ºC. The remaining two parameters, [NMN] and [NH4SO4], were only studied for LigA. The interesting result for NMN was that it inhibited the reaction of LigA with an IC50 of 31.34±1.58 µM. This shows that one of the ligation products (NMN) acts as a feedback inhibitor. The Km for the ammonium sulphate concentration experiment on LigA was 37.64±40.64 µM, and that [NH4SO4] stimulated the
LigA reaction when present about 1.6 fold.
The results from these characterisation studies allowed the development of a three-part molecular screening method (in silico to in vitro to in vivo) to build towards potential future studies that could then explore small molecule compounds that affect these basic kinetic parameters and find new ligase inhibitors. Therefore the second aim of the work was to explore the potential of DNA ligases (LigA in particular) as possible antibiotic targets. These involved the use of a molecular docking programme called Molecular Operating Environment (MOE).
This software (in silico) was used to identify eight chemical compounds from a large library of possible small molecular inhibitors. The key question in this work was to see if these compounds could inhibit the E.coli DNA LigA in vitro (by denaturing gel experiments) and in vivo (by Kirby-Bauer bacterial surface-inhibition experiments). We found that four out of the eight compounds (5-Azacytidine, Geneticin (G418), Chlorhexidine and Imidazolidinyl Urea) did inhibit the activity of LigA in vitro with IC50 values of 10.34±4.14, 45.31±13.81, 20.66±6.11 and 7.56±14.48 µM, respectively. Three of the eight compounds: Geneticin (G418), Chlorhexidine and Imidazolidinyl, did inhibit the growth of the bacteria (in vivo) with IC50 values of 0.63± 0.17, 0.17±0.03 and 233.25±143.35 mM, respectively. They had success in all three areas of study (in silico, in vitro and in vivo) and make them suitable candidates for future drug development studies as a promising chemical leads to target bacteria.
In conclusion, this thesis provides important kinetic and binding data on LigA and LigB that will help explain how they work in E.coli. This thesis also provides clear evidence for at least three new ligase inhibitors of E.coli.
|Date of Award||Jul 2018|
|Supervisor||Darren Gowers (Supervisor), Alan Thorne (Supervisor) & Andrew Pickford (Supervisor)|