Molecular and cellular mechanisms utilized by Primase-Polymerase centric DNA repair pathways during stationary phase in mycobacteria

Our cells DNA, the so called "genetic blueprint of life", 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-helical 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 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. The integrity of DNA is also constantly being challenged by various 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 damage DNA.

Fortunately, our cells produce repair proteins whose role it is to remove the "damaged" bases. We have recently discovered a novel bacterial DNA repair "machine" that plays an important role in excising and repairing these "toxic" mutations thus ensuring genome stability and, ultimately, cell survival. In this research programme, we are proposing to determine how these repair machines detect, remove and correct DNA mutations, define the cellular consequences of deleting this repair pathway and, finally, determine if it co-operates with other related repair pathways to ensure genome stability. This proposal will provide critical insights into a fundamental DNA repair pathway required to correct harmful genetic mutations that promote genetic instability in bacterial pathogens.

Excessive accumulation of damage can lead to uncontrolled cell growth that can result in the onset of diseases, such as cancer. However, in bacteria 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.

The maintenance of genome stability requires a "Holy Trinity" of DNA repair pathways (excision repair, break repair and mismatch repair) that mend the majority of lesions arising in genomes. These repair mechanisms all rely on bespoke repair polymerases, which replace damaged DNA removed during repair pocesses. Cells also contain non-canonical polymerases called primase-polymerases (Prim-Pols), recently shown to play diverse roles in DNA metabolism including, replication, repair, damage tolerance and repriming. For example, Prim-Pol D (PPD) is required for the repair of DNA double-strand breaks formed during stationary phase in many prokaryotes. Although PPD's role in break repair is well characterised, particularly in mycobacteria, the biological functions of closely related Prim-Pol orthologues found in the same organisms has been unclear. We recently identified that a Prim-PolC (PPC)-dependent pathway is specifically involved in excision repair of lesions in mycobacteria, thus expanding the repertoire of pathways in which Prim-Pols operate. Furthermore, we also identified a PPD-dependent excision repair pathway that is non-epistatic with the PPC pathway.

The major aims of this proposal are to elucidate the molecular and cellular mechanisms employed by Prim-Pol dependent excision repair complexes in mycobacteria. We will use a variety of in vitro and in vivo approaches to provide complementary mechanistic insights into the modus operandi of these novel excision repair complexes and delineate how these pathways operate in unison to maintain cell survival in mycobacteria. Together, these holistic studies will significantly enhance our understanding of how Prim-Pol mediated repair pathways maintain genome stability during stationary phase in mycobacteria. These studies are also likely to provide critical insights into how these repair processes might be targeted by novel antibiotics and how these pathways may also contribute to antibiotic resistance.

This proposal focuses on the fundamental processes of genome repair processes in prokaryotic cells. This research will have significant impact on our understanding of pathways associated with DNA maintenance and repair, as well as contributing to the knowledge of pathways associated with antibiotic resistance. In addition, characterisation of basic DNA repair mechanisms will open up exciting leads to a better understanding of why certain pathogens are resistant to certain antibiotics and may also have significant impact on the development of novel antibiotics and biotechnology reagents. 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 research programme will address the impact of DNA repair mechanisms on the maintenance of bacterial cell function and define key molecular pathways associated with cell survival and antibiotic resistance. For example, the project will investigate how DNA repair pathways protect pathogens by enabling them to survive the toxicity of chemical agents. A thorough understanding of the relationship between DNA damage, repair and survival, associated with antibiotic resistance, will thus help inform the health sector in respect to factors that promote bacterial drug resistance. This research programme will inform on two strategic priority areas: 'Combatting antimicrobial resistance' and 'Healthy ageing across the life course'.

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: Base Excision Repair (BER) 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 BER in mycobacteria makes them very prone to oxidizing agents, suggesting that inhibition of this pathway could be a target for the development of novel antibiotics 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 these repair mechanisms 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 the newly characterised enzymes (PPC & LigC), which also have potential for commercial development for uses in the biotechnology sector e.g. uses in molecular biology applications and in clinical diagnostics. In addition, knowledge gained in this study will greatly aid in the development of small molecular inhibitors that could potentially act as lead compounds for a future antibiotic development programme.