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

DNA is the 'molecule of life' which provides the genetic code for most organisms. However, the action of DNA in living cells is not solely linked to the genetic code, but also to the dynamic shape, or 'topology', of the DNA. DNA gyrase is a type of molecular machine called a topoisomerase (or 'topo' for short), found in many types of organisms including bacteria, but not found in mammalian cells. It performs a vital function of relaxing excess twists known as 'supercoils' in the DNA that would otherwise prevent DNA from being replicated, or from its genetic code being read out. This essential role of DNA gyrase has resulted in the development of several antibiotics which kill the cell by specifically targeting gyrase and interfering with its function.

Although there has been extensive research performed on DNA gyrase using methods which can analyse its structure, genetics and biochemistry, very little is currently known about how it operates inside living cells. In this project we will use genetics techniques to attach specific single molecule tags called fluorescent proteins to the different subunits of gyrase and to other parts of the cellular molecular machinery which are involved in the activities of gyrase. We will apply advanced methods of bioimaging which allow us to observe these single fluorescent protein molecules and to track these different molecular components as they move in live bacterial cells at a speed which is faster than the molecules themselves can diffuse, enabling us to observe them unblurred and to determine their location very precisely. This will allow us to measure accurately where in the cell these molecules act and how many of them are involved in their cellular activities. Along with DNA gyrase we will also track the molecular machinery responsible for replicating the DNA, and also molecular machinery used in the process of 'transcription' in which the genetic code is read out and transcribed into different proteins in the cell. This will allow us to understand how DNA gyrase performs its essential role of relaxing supercoiled DNA during active DNA replication and transcription.

We will also study what happens to gyrase when antibiotics which are known to target gyrase are added to bacteria. Many of these antibiotics are believed to act by locking the DNA into a broken state at the point at which gyrase is bound to the DNA, and in order to study how this locked state occurs we will use a fluorescent label which binds specifically to the ends of broken DNA. However, some cells are known to be able to tolerate DNA gyrase molecules in this locked state. Such cells can survive the action of DNA gyrase targeting antibiotics, and this tolerance of antibiotics can subsequently lead to resistance against these drugs in a whole population of bacteria against these antibiotics, making them ineffective as a medicine to treat bacterial infections in humans. In order to study the mechanisms of cellular tolerance of such poisoned DNA gyrase molecules we will make modified bacteria which are particularly sensitive to a specific type of DNA gyrase targeting antibiotic, and compare how the DNA gyrase molecules and DNA replication machinery respond compared to normal cells.

Our single-molecule investigations of DNA gyrase using advanced light microscopy on live bacteria will allow us to explore a longstanding puzzle of how the molecules use the hydrolysis of ATP, the universal chemical energy currency in all living cells, in order to perform its vital role of relaxing torsional stress in DNA. In doing this it may help us, most importantly, to understand fundamental details about how ATP is used by this general class of molecular machine.

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.