Tackling tricky twists - how does DNA gyrase function inside living cells?

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DNA gyrase is a vital type II topoisomerase found in many kingdoms of life, in particular in bacteria, but absent from mammals. It performs a key role in bacteria of relaxing positive supercoils generated by DNA replication and transcription, however, despite extensive biochemical, structural and genetic data, we know little of how this is achieved in complex cellular environments. The role of gyrase in catalysing release of torsional stress from DNA has been studied extensively using ensemble average tools as well as single-molecule biophysics in vitro. Ensemble average methods mask heterogeneity and dynamic activity found in vivo whereas in vitro single-molecule approaches fail to reveal the complexity of gyrase's catalytic activity in the context of active DNA replication and transcription. In order to understand the mechanisms of gyrase's functional role we will study gyrase in live bacteria, exploiting the wealth of genetic and biochemical tools that are available for study with E. coli.

We will construct a range of fluorescent protein reporter E. coli cell strains in which different components of the gyrase complex, the replication fork and the transcription bubble, have been labelled, and track these in real time using millisecond single-molecule fluorescence microscopy in vivo in order to understand how gyrase acts to relax positive supercoils formed during DNA replication and transcription. We will track double-strand breaks in DNA formed via gyrase's catalytic activity and assess how these are affected by the presence of antibacterials which target gyrase. We will study how some cells can tolerate gyrase complexes which have been poisoned by antibacterials in order to understand how this can lead to antibacterial resistance.

Our analyses will address a fundamental question concerning the role of ATP hydrolysis in general topoisomerase activity. This will lead to a general and quantitative framework for understanding topoisomerases in all organisms.

Who will benefit from this research in addition to academic beneficiaries?

1. Non-academic workers. Specifically, there are unique interdisciplinary training opportunities in this project which have potential impact distinct from pure academic benefit.
2. Commercial sector. Specifically, pharmaceutical companies involved in antibiotic drug discovery, and SMEs involved in DNA gyrase and in the topoisomerase area in general (such as Inspiralis) and image analysis and microscopy control software applications.
3. General public.

How will they benefit from this research?

1. Non-academic workers.
a. Fostering of interdisciplinarity. Encouragement of collaborations across the life and physical sciences interface. For example, the production of a highly skilled 'scientifically-bilingual' postdoc and students in the applicants' lab groups.
b. Training development for novel technologies. For example, a new single molecule tool-kit, image analysis and microscopy control software: these have potential impact beyond pure academic research.
c. Training development in interdisciplinary dissemination via publications and presentations at national and international meetings. For example, increasing understanding of the way antibiotics that target DNA gyrase function.

2. Commercial sector.
a. New strategies for drug discovery. The annual financial value of antibiotics that act on topoisomerases is multi £M. Fluoroquinolones are highly successful antibiotics targeted to topoisomerases. These drugs are typically broad-spectrum and the risk associated with their loss is very high. Loss of this class of antibiotics due to the emergence of antimicrobial resistance is a clear and present threat as highlighted in the report and statements from Sally Davies the UK Chief Medical Officer (http://bsac.org.uk/news/antimicrobial-resistance-poses-catastrophic-threat-says-chief-medical-officer/).
Topoisomerases are regarded as a 'sweet-spot' by the pharmaceutical industry, since they are expressed in prokaryotic pathogens but not in mammalian (i.e. human) cells, and new compounds targeting these enzymes will be well-received. The research proposed may address un-met needs, for example by generating basic knowledge into the importance of double-strand break stability in the activities of DNA gyrase targeting antibacterials, and of how poisoned gyrase complexes can be tolerated by some cells. In particular, there is a possibility that the lifetime of existing antibiotics could be extended through reformulations which include new compounds that disrupt the cells ability to tolerate locked DNA gyrase
b. Better understanding of the roles of topoisomerases in general in bacterial cells. For example, working towards the development of new topoisomerase-targeting antibiotics and new anti-cancer drugs using new strategies.
c. Delivery of novel devices and technologies. For example, the of new microscopy systems, and technological developments which could facilitate new biosensing approaches, such as lab-on-a-chip, which would have far-reaching implications of diagnosis of disease.

3. General public.
a. Dissemination of new information through outreach activities. For example, in the use of new single-molecule methods to better understand how antibiotics work.
b. Increased public awareness of new devices and technologies - in particular those of relevance to new developments in personalized medicine through improved biosensing and more rapid diagnosis.
c. Engagement with local schools to enhance educational opportunities. School children in the UK are still required in general to make relatively early subject choices between the life and physical sciences - we will engage school children at pre-GCSE and pre-A-level subject choice stages to advertise the benefits of interdisciplinary approaches which capture both the life and physical sciences, in order to enhance their future educational opportunities.