Elucidating the mechanism of non-canonical DNA mismatch repair in mycobacteria

Our cells contain DNA, the so called "genetic blueprint of life", which encodes the information for all our genes. DNA has a simple repeating structure composed of two complementary strands of DNA composed of bases, which form long, string-like, double-helix structures that make up the genome. Our genome is packaged away into chromosomes, contained within the nucleus of nearly every cell. This information must be faithfully copied as cells divide to produce daughter cells. Cells produce a large number of proteins responsible for "photocopying" this DNA blueprint. The proteins tasked with accurately copying the several billion letters of our genetic code are called DNA replication polymerases. During this copying process, the replication machinery can introduce mutations to the newly made DNA sequence that can, if left unrepaired, lead to the development of disease states, such as cancer.

Fortunately, our cells produce repair proteins whose role it is to remove the "mismatched" bases. We have recently discovered a novel bacterial repair gene called NucS and discovered that the protein it produces plays an important role in helping cells to excise mutations introduced during every round of cell division thus ensuring efficient genome replication. In this research programme, we are proposing to identify additional proteins that operate with NucS in the bacterial cell, determine how these repair machines are able to remove and correct DNA mutations, identify "when" and where" these complexes operate in cells, define the cellular consequences of deleting this repair pathway and, finally, determine if it co-operates with other repair pathways to ensure genome stability. This proposal will provide critical insights into a fundamental mutation avoidance pathway required to correct harmful genetic mismatch mutations that promote genetic instability.

Excessive accumulation of mutations can lead to uncontrolled cell growth that can result in the onset of diseases, such as cancer. However, in prokaryotes it can lead to the development of antibiotic resistance in major pathogenic bacteria. The rise of antibiotic resistance has been identified as one of the major threats facing global health. Therefore, understanding fundamental mechanisms and pathways that influence mutation rates in bacteria will uncover new strategies to predict and combat the development of antibiotic resistance.

Accurate replication of chromosomal DNA is vital to maintain genomic stability and preventing the accrual of genetic mutations. Cells ensure the maintenance of low DNA mutation rates using a combination of base selection, proofreading and mismatch repair mechanisms. Mismatch repair (MMR) is a sophisticated repair pathway that detects and removes incorrect mismatched bases. Loss of this pathway has important consequences, such as high rates of mutation and increased recombination between divergent DNA sequences. The MMR pathway is highly conserved among the three domains of life. However, most actinobacteria and archaea possess no identifiable MMR pathway but exhibit mutation rates similar to MMR-bearing species, suggesting the existence of unidentified mechanisms responsible for mismatch repair. We have identified a non-canonical MMR pathway in these organisms. Disruption of this repair pathway leads to hypermutation and the appearance of other genetic signatures associated with the loss of mismatch repair in other organisms. These results provide compelling evidence for the existence of an alternative MMR pathway in nature.

The major aims of this proposal are to define the cellular factors required to perform non-canonical mismatch repair and elucidate the molecular mechanisms of non-canonical mismatch repair in mycobacteria. We will employ molecular and cellular approaches to provide complementary mechanistic insights into MMR and delineate how the MMR pathway operates to facilitate mutation avoidance. Together, these holistic studies will significantly enhance our understanding of how this non-canonical mutation correction system excises and repairs DNA mismatches in prokaryotic cells. It is also likely to provide critical insights into how such repair processes are associated with the development of antibiotic resistance in microbial pathogens.

This proposal focuses on the fundamental processes of DNA mutation avoidance processes in prokaryotic cells. This research will have significant impact on our understanding of pathways associated with DNA replication and maintenance, as well as contributing to the knowledge of infectious diseases associated with antibiotic resistance. In addition, characterisation of basic DNA replication and repair mechanisms will open up exciting leads to a better understanding of why certain pathogens develop antibiotic resistance and may also have significant impact on the development of novel biotechnology reagents and novel antibiotics. Beyond academia and related research fields, the work in this project has the potential to impact, in the long-term, on the health sector and 'quality of life'/'Lifelong Health and Wellbeing'. This is because the programme will address the possible impact of DNA repair on the maintenance of normal cell function and on molecular pathologies associated with cell ageing and antibiotic resistance. For example, the project will investigate the link between mutation avoidance and the ability of pathogens to develop antibiotic resistance. A thorough understanding of the relationship between DNA damage, replication and pathologies associated with antibiotic resistance could thus help inform the health sector in respect to factors that promote infection. This research could thus, in the long-term, inform on environmental and life-style issues relating to 'healthy ageing across the life course' and 'Lifelong Health and Wellbeing'.

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, train 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: Mismatch repair (MMR) has been show to be a critical cellular pathway required to maintain DNA stability, in both prokaryotes and eukaryotes, and its loss results in major genomic instability. Loss on MMR in mycobacteria makes them very prone to increased mutation rates, suggesting that loss of this pathway is likely to be associated with the development of antibiotic resistance in a range of pathogens. We propose to exploit the molecular and cellular information obtained from these studies to help to develop a much better understanding of this phenomena and, in turn, guide the design of novel clinical strategies to treat a range of drug-resistance pathogenic bacterial infections e.g. TB.

3. Industrial impacts: We have previously developed a mycobacterial DNA repair system (Ku-Ligase D) into an application for the biotechnology sector. We will use this experience to optimally exploit this newly discovered enzyme (NucS), which also has potential for commercial development for uses in the biotechnology sector e.g. uses in molecular biology applications and in clinical diagnostics, e.g. SNPs analysis. Notably knowledge gained in this study will aid in the development of small molecular inhibitors that could potentially act as lead compounds for a future antibiotic development programme.