Molecular basis for repairing DNA double-strand breaks by non homologous end-joining

Our cells contain DNA, the so called "genetic blueprint of life" which encodes the information for our genes. DNA has a simple repeating structure composed of two complementary strands of DNA which form a double-helix structure. The integrity of DNA is constantly being challenged by various DNA-damaging agents. These agents include high energy UV and X-ray radiation from the sun, chemicals both man-made and environmental and even the oxygen we breathe can attack and damage DNA. In recent years it has been realised that our own cells produce a large number of proteins responsible for repairing this DNA damage which, if left unchecked, would lead to the development of conditions such as cancer. These protein "machines" can cut out and replace aberrant DNA mutations/structures and splice together broken DNA strands. One such protein is called DNA ligase, which interacts with its partner proteins: Ku , polymerase and nuclease to form a DNA break repair complex. This repair apparatus is able to detect physical breaks in the DNA helices, bring the ends back together, remodel them if required and, finally, reseal these breaks to restore the continuity of the double-strand DNA. The focus of this work is to understand how these repair complexes can bridge the break and make the DNA ends come back together prior to the sealing step.

DNA double-strand breaks (DSBs) are one of the most lethal forms of DNA damage encountered by cells and failure to repair such breaks can lead to genomic instability. DSBs are generated by both endogenous and exogenous agents, such as reactive oxygen species and ionizing radiation, and can arise following DNA replication fork collapse. DSBs are also produced during specialised cellular processes, such as meiosis and V(D)J recombination. In higher eukaryotes, the non homologous end-joining (NHEJ) pathway is critical for the repair of DSBs. A functionally homologous repair system has also been identified in many prokaryotes, where it is utilized to repair DSBs in stationary phase and sporulating cells.

The primary objectives of this proposal are to characterise the molecular architecture and modus operandi of prokaryotic and archaeal NHEJ complexes involved in DNA double-strand break repair. We will combine biochemical, biophysical and structural approaches to elucidate how the proteins (Ku, Lig, Pol and Nuc), that make up the prokaryotic NHEJ repair apparatus, co-operate to facilitate the complex task of break detection, synapsis, remodelling and end-ligation. In addition, we will screen for small molecule inhibitors of the NHEJ repair enzymes that will form the basis for the future development of potential antibiotics to target the NHEJ pathway in major pathogens, such as myobacteria.

This proposal focuses on the fundamental processes of DNA repair processes in prokaryotes and archaeal organisms. This research will have significant impact on our understanding of repair pathways associated with DNA breaks, as well as contributing to the knowledge of pathways related to its dysfunction e.g. cancer and also exploitation of this pathway to treat bacterial infections. In addition, characterisation of these basic DNA repair mechanisms will have significant impact on the development of novel biotechnology tools and compounds for drug development that could lead to the discovery of exciting lead compounds for new antibiotics.
The major areas of impact resulting directly from this research project:

1. Training impacts: This project provides ample opportunities to train researchers to use cutting-edge technologies, outlined in the application, to address fundamental scientific questions that have potentially important clinical and industrial applications These researchers will, in turn, supervise undergraduate and PhD students. This work will therefore significantly impact on the training of future young scientists, who will hopefully go on to set up their own research groups. Their newly acquired skills will also be readily transferable to other scientific areas, including clinical, biotechnology and industrial research, teaching and scientific writing. Training and maintaining a highly skilled scientific work force will significantly impact on the UK's ability to remain a world leader in academic and industrial research.

2. Clinical Impacts: NHEJ has been show to be an important cellular pathway required to maintain DNA stability, in both prokaryotes and eukaryotes, and its loss results in major genomic instability. Loss on NHEJ in mycobacteria makes them very prone to environmental stresses, such as desiccation, suggesting that this pathway could be targeted to treat a range of bacterial infections. We propose to exploit the molecular information obtained from these studies to develop potential lead compounds for future antibiotic development to treat a range of pathogenic bacterial infections e.g. TB.

3. Industrial impacts: We have previously patented and are currently developing a molecular biology system, based on this NHEJ DNA repair complex, into an application for the biotechnology sector. We will use this recently acquired experience to optimally exploit the newly discovered archaeal NHEJ enzymes, which also have excellent potential for commercial development for uses in the biotechnology sector e.g. uses in cloning and related molecular biology applications. Notably, this system can also aid our development of small molecular inhibitors of bacterial NHEJ pathways that could potentially act as lead compounds for a future drug development programme.