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

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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.