New Antibiotic Targets in Bacterial DNA Double-strand Break Repair

Resistance to antibiotics is a major medical concern worldwide. There is an urgent need both to develop new antibacterial targets and to find ways to overcome bacterial antibiotic resistance mechanisms. Making better use of our existing range of antibiotics (e.g. increasing their efficacy) is also an important consideration. DNA replication and repair enzymes are different in humans and in bacteria so present opportunities for antibiotic targets. The repair of DNA breaks is already known to be essential for bacteria and there are already antibiotics (quinolones) that work by creating breaks in DNA. The main system that bacteria use to repair DNA breaks is called RecBCD or AddAB. These multi-protein complexes process the DNA ends to make them suitable for initiation of repair by a process called homologous recombination. If repair of the break is inhibited, then this will result in cell death when the cell next tries to divide. Previous work in my group has established molecular details of several steps in the enzyme-catalysed pathway for processing of the DNA ends by AddAB that might be targets to develop inhibitors of the enzyme that could be developed as antibiotics. This proposal aims to set up an assay system to allow us to exploit one such aspect of the AddAB mechanism that we have uncovered. An understanding of the molecular mechanism of the protein complex has uncovered an "Achilles heel" that we may be able to exploit to turn the bacterial repair system back upon itself and convert it from a repair system to one that will digest the bacterial genome to kill the organism. Antibiotics aimed at this unique feature of the enzyme should provide specific drugs with fewer side effects in humans. AddAB is present in many bacterial pathogens such as Listeria and MRSA but is replaced by the related RecBCD complex in bacteria such as E.coli and Salmonella. Consequently, we need to understand the mechanism of RecBCD to exploit similar weaknesses to develop equivalent antibiotics that will work on these bacteria. The established importance of the prevention of DNA break repair as a target for antibiotics such as quinolines suggests that there may be other targets to exploit for new drugs so I will examine some of these by looking to see which proteins provide additional sensitivity to quinolines to that provided by the established targets (DNA gyrase and topoisomerase IV). In particular, we will look to find proteins that act synergistically with RecBCD/AddAB inhibition as potential targets for combination therapies. Finally, two new proteins have recently been discovered that are involved in break repair in bacteria. We will determine the crystal structures of these proteins to understand how we might be able to design new antibiotics against them.