Structure-function studies of the bacterial plasmid defence system Wadjet

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Microbes are found in many environments, and in almost all cases are under constant attack frombacteriophages and other selfish genetic elements. To avoid infection, bacteria have evolved a widerange of defence mechanisms. These different systems are found clustered on the genome in whatare called "defence islands". The defence systems include the well-know restriction enzymes andCRISPR-Cas9, which have been exploited for molecular biology and gene editing, respectively. Yetthere are many other uncharacterised systems within the defence islands that may provide yet moretools for scientists to exploit. This project aims to use biophysical analysis and structural biology toexplore the mechanism of one such system, named after the Egyptian goddess Wadjet.The Wadjet system prevents bacterial transformation by plasmids. It comprises four genes: jetA, jetB,JetC and JetD. jetABC encode gene products that are related at a sequence level to bacterialcondensins. Condensins are widespread across all organisms, and are ATP-dependent molecularmachines that help to regulate the process of genome segregation during cell division. Their exactmechanisms and roles are still debated. They form large protein complexes that use ATP hydrolysis todrive large-scale changes in DNA conformation, including tethering DNA together, or activelyextruding DNA loops as molecular motors. In this collaborative project between the Szczelkun andBerger-Schaffitzel labs, you will determine the overall structure of the Wadjet JetABCD complex, showhow it interacts with DNA, and determine the role for ATP hydrolysis. We propose that DNA captureor loop extrusion blocks plasmid replication. These activities will be directly distinguished andmeasured in real time using a single molecule magnetic tweezers assay.