Single-molecule analysis of double-stranded DNA break repair in living bacteria

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Our objective is to study the in vivo mechanisms and kinetics of the repair of double-stranded DNA breaks (DSBs) inside living bacteria. In bacteria, DSBs are dealt with mainly via homologous recombination by proteins RecBCD and RecA: broken molecules are faithfully repaired by copying the missing information from another, intact, chromosome. RecBCD loads on a DSB, and starts unwinding and degrading it to help RecA form a filament that eventually finds homologous DNA, and catalyses strand exchange and DNA repair. RecBCD and RecA have been studied in vitro using biochemical and single-molecule approaches; however, such approaches cannot replicate the enormous complexity of the cellular cytoplasm, thus leaving severe gaps in our mechanistic understanding of DSB repair in cells.

To address the outstanding questions, we formed a collaborative three-site team that will analyse DSB repair mechanisms at the single-molecule level in live Escherichia coli. We will use in vivo single-molecule tracking of expressed fluorescent proteins and internalised fluorescent DNA substrates in single living bacterial cells, along with sophisticated data analysis. After validating the necessary proteins and DNA, we will characterize the RecBCD search determinants in cells with different degree and types of DNA breaks (e.g., ones generated using fluoroquinolones). We will monitor the process of DNA-end location in real-time using simultaneous tracking of RecBCD and target DNA molecules. We will use in vivo single-molecule FRET to characterise how DNA ends are processed by the endonuclease and helicase activities of RecBCD; these studies will be guided by complementary in vitro experiments. Finally, we will monitor the formation of RecA filaments on synthetic RecBCD substrates (to be formed controllably in cells) and monitor the kinetics of homology search; these studies will also examine the presence of 1-D search along DNA as a mechanism that accelerates this DNA-repair pathway.

The main proposal aim is to understand how bacteria repair their chromosomes; this aim has direct impact on understanding how bacteria become tolerant and resistant to clinically relevant antibiotics such as quinolones and trimethoprim. Our work will advance substantially our fundamental understanding of the mechanisms at play and develop cutting-edge techniques that elucidate the activity of these antibiotics in vivo. Moreover, our new technologies and assays will be applicable to identifying the mode-of-action of new potential antimicrobial lead compounds that target chromosomes and DNA-binding proteins in bacteria. Apart from the obvious academic impact, the project will have significant economic and social impact. Continuous engagement with our technology transfer offices and industrial partners will allow early identification of opportunities for translation of our scientific outputs.
Economic impact will be maximized through skills development and commercialisation options.
Skills development: The project will result in two highly trained PDRAs with a diverse and interdisciplinary set of skills, and experience of working at the interface between physical and biological sciences. Both PDRAs will gain skills in quantitative large-scale image processing (including machine-learning-based algorithms), an area with critical shortages of such skills in industry.
Commercialisation:
ANK is engaged in discussions with three clinicians at the Oxford John Radcliffe Hospital and industrial partner Oxford Nanoimaging to develop rapid antimicrobial susceptibility tests based on single-cell imaging. We expect that some mechanistic findings, electroporated DNA sensors, and imaging/data-analysis methods will be of interest for our clinical and industrial collaborators.
MeK is actively discussing with Sanofi Infectious Diseases applying single-molecule tracking assays to the mode-of-action characterisation of several new antimicrobials. Understanding the mode-of-action for existing and new antibiotics is important for their optimal use and for developing new antibiotics based on rational design.
Social impact. We will engage with the broader society and help stir enthusiasm for science via:
Workshops: the MeK team will deliver 2 workshops in local schools using the innovative and highly successful Science Art Workshop (SAW) Trust approach to teaching science to children using the power of words and art.
Videos: we will produce three 3-min videos for public engagement: one directed to the microscopy/instrumentation industry, one directed to microbiologists and clinicians, and one directed to high schools and general public.
Website: we will create a project website that will address the three communities targeted with the three videos (industry, clinicians, general public).
Science festivals: we will participate in our local Science festivals, with the Oxford Science Festival and the Edinburgh Science Festival being the main venues. The involvement will have sci-art entries, and/or practical demonstrations similar to the ones used in the SAW workshops.
Media: to enhance public understanding of science, the PI, co-Is and PDRAs will make every effort to communicate the importance and implications of our work to the media via interviews, press releases and radio/TV appearances, social media networks, as well as through public lectures, discussions, science Open days and outreach events.
Conference organisation. To maximise our impact, we will organise a conference to engage potential academic, clinical and commercial end-users. Specifically, we will organise "Single-Molecule Bacteriology 2020", a 3.5-day conference on single-molecule imaging in single bacteria. The workshop will familiarize clinicians and industrial attendees with the latest in single-molecule bacterial biology, and allow them to discuss clinical and industrial needs with academics, thus catalysing collaborations between academia, hospitals and industry.