DNA gyrase is an enzyme found in all bacteria that can, uniquely, introduce supercoils into DNA. Its topoisomerase reaction involves the generation of transient double-stranded breaks in DNA. Interruption of the DNA breakage-reunion step of this reaction can lead to a lethal lesion in bacteria. This mechanistic feature, plus its absence from humans and most other eukaryotes, has made it a highly-successful target for antibacterial agents. However, many questions remain concerning the strand-passage mechanism, particularly the dynamics of this process. The explosion of new structural information on gyrase and related enzymes from crystallography and cryo-EM now presents the possibility of establishing the dynamic aspects of this key enzyme. By using state-of-the-art all-atom simulations from such structures, we aim to address these questions with a view to developing novel ligands that can disrupt its catalytic activity.
Our objective is to map the strengths and dynamics of key interactions between gyrase, DNA and ligands. The preliminary simulations done in Agnes Noy’s lab have revealed protein gate opening/closing and novel DNA wrapping conformations, which are not apparent from experimental structures. This will enable revealing of new targeting approaches that will be instrumental in developing new strategies to combat antibiotic resistance and that will be tested in the labs of Inspiralis and Tony Maxwell.
The project will specifically aim to:
1) Model gyrase and its subunits bound/unbound to DNA to finding novel critical interactions and conformations.
2) Perform in silico screening for identifying potential ligands.
3) Test the candidates experimentally.
The project builds on a wealth of biochemical, structural and live-cell studies of DNA gyrase led by Tony Maxwell (John Innes Centre) (1) and Mark Leake (York) (2) (co-supervisors) and, thus, presents a timely opportunity to develop new mechanistic understanding into the mode of action of DNA gyrase, by using innovative all-atom simulation techniques with Agnes Noy (3,4) (principal supervisor) to extend our understanding of the mechanistic processes involved. This exciting interdisciplinary project includes the involvement of Inspiralis, a company supplying topoisomerase products to the pharmaceutical industries and academia to aid research in the anti-infectives and anti-cancer markets. The student will spend part of their studentship working in Inspiralis’ labs.
1. Feng L, Mundy JEA, Stevenson CEM, Mitchenall LA, Lawson DM, Mi K and Maxwell A. (2021). “The pentapeptide-repeat protein, MfpA, interacts with mycobacterial DNA gyrase as a DNA T-segment mimic”. Proc. Natl. Acad. Sci. USA 118, e2016705118
2. Stracy M, Wollman AJM, Kaja E, Gapinski J, Lee J, Leek VA, McKie SJ, Mitchenall LA, Maxwell A, Sherratt DJ, Leake MC and Zawadzki P. (2019). “Single-molecule imaging of DNA gyrase activity in living Escherichia coli”. Nucleic Acids Research, 47, 210–220
3. ALB Pyne, A Noy, K Main, V Velasco-Berrelleza, MM Piperakis, LA Mitchenall, FM Cugliandolo, JG Beton, CEM Stevenson, BW Hoogenboom, AD Bates, A Maxwell, SA Harris (2021). “Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides”. Nature Communications, 12, 1053
4. S Yoshua, G Watson, J Howard, V Velasco-Berrelleza, MC Leake, A Noy (2021). “Integration host factor bends and bridges DNA in multiplicity of binding modes with varying specificity ”. Nucleic Acids Research, 49,8684-8698
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