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.

DNA replication and repair is are essential processes in all organisms but are surprisingly different between eukaryotes and eubacteria. This proposal seeks to examine targets within the DNA double-strand break (DSB) repair for antibiotic development. A key component in bacterial DSB repair is the RecBCD/AddAB system that processes broken ends to prepare them for repair by homologous recombination. Using structural information gathered over the last 10 years we have identified a weakness in the AddAB mechanism that might be exploited. Extending our understanding of the mechanism and regulation of these complex systems will likely reveal additional opportunities for antibiotic development. In particular, understanding of specific aspects of regulation of RecBCD may provide opportunities for drug development. Quinolone antibiotics act upon DNA gyrase by stabilising a normally transient DSB during the enzyme mechanism. Mutations of a number of proteins other than DNA gyrase that are involved in DSB repair show sensitivity to quinolones and combinations will be sought that enhance sensitivity to quinolones in strains that carry mutant forms of RecBCD or AddAB that have lost regulation by Chi sequences. Finally, two new players in DSB repair in bacteria have recently been identified. I propose to determine the molecular mechanism of these proteins to seek opportunities for exploitation in drug development.

This research will provide information on molecular mechanisms which will underpin the design and interpretation of studies on biological function and antibiotic development. The proposed programme is basic research of potential value to the commercial private sector, specifically the biotechnology and pharmaceutical industries, to inform and guide the design of novel antibiotics. Antibiotic resistance has become a major medical concern and this proposal aims to evaluate the potential of inhibitors of DNA break repair in bacteria that would constitute a novel class of antibiotics and may also open up new opportunities for development of other targets in bacterial DNA repair.

Another area of impact for this research will be in the training and career development of young research scientists. I have been very fortunate in the high level of talent and enthusiasm shown by the young scientists who have worked with me and I believe my laboratory has provided them with an excellent training environment. Past pre- and post-doctoral members of my laboratory include three UK university professors, two pharmaceutical industry researchers, one research charity grant board coordinator, several group leaders in labs abroad, plus several post-doctoral research workers in posts both in the UK and abroad who are on career trajectories to run their own research groups in due course.