Award details

Understanding the role of PrimPol in damage tolerance during genome replication in eukaryotic cells

Reference	BB/M008800/1
Principal Investigator / Supervisor	Professor Aidan Doherty
Co-Investigators / Co-Supervisors
Institution	University of Sussex
Department	Sch of Life Sciences
Funding type	Research
Value (£)	877,984
Status	Completed
Type	Research Grant
Start date	01/08/2015
End date	31/07/2019
Duration	48 months

Abstract

DNA damage tolerance pathways are critical for genome stability. Accurate replication of chromosomal DNA is vital to maintain genomic stability and prevent the accrual of oncogenic-promoting rearrangements. Replication forks are prone to stalling upon encountering obstacles on DNA, including secondary structures and lesions. Inability to restart stalled forks is a significant cause of replicative stress, which has been directly implicated in promoting oncogenous and accelerated ageing. Although numerous mechanisms exist to complete genome duplication in the absence of a pristine DNA template, identification of the pathways involved in these processes remains incomplete. The aim of this research programme is to address critical unanswered questions and new hypotheses concerning mechanisms of damage tolerance at mammalian replication forks. We will employ molecular and cellular approaches to pursue newly uncovered links between DNA replication and damage tolerance. This work will involve the use of bespoke gene targeting technologies in human cells, in conjunction with biochemical and structural biology approaches, to provide complementary mechanistic insights into lesion bypass processes involving a newly identified DNA polymerase called PrimPol, that functions at replication forks in mammalian cells. Loss of this damage tolerance protein in cells results in replication fork defects and genome instability. This programme of research will delineate how the PrimPol pathway functions, at both the molecular and cellular levels, to facilitate lesion bypass during replication. Together, these holistic studies will significantly enhance our understanding of how damage tolerance pathways assist replication forks to proceed through DNA lesions and structural barriers in mammalian cells. It is likely to also provide critical insights into disease-related pathologies associated with DNA damage sensitivity, replication stress and normal ageing processes in human cells.

Summary

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, which form long, string-like, double-helix structures that make up the genome. Our genome is packaged away into chromosomes, contained in the nucleus of nearly every cell, which must be 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 is frequently stopped by damaged DNA and this can lead to failure to replicate or the production of mutations to the sequence of the DNA that can, eventually, lead to the development of disease states, such as cancer. Fortunately, our cells produce damage tolerance proteins whose role it is to prevent this from occurring or restart replication when it stalls. We have recently discovered a novel human lesion bypass polymerase in human cells called PrimPol and shown that this protein, together with other cellular factors, plays an important role in helping the cell's replication machinery to bypass DNA damage it encounters during every round of cell division thus ensuring efficient genome replication. In particular, we have shown that loss of PrimPol leads to replication slowing, particularly past lesions produced by UV light, suggestive that it is required for efficient replication through these kinds of DNA damage. In this programme, we are proposing to identify how these molecular machines are able to bypass DNA damage, what are the cellular and organismal consequences of deletion or mutations in this gene (e.g. diseases associations) and, finally, how does PrimPol co-operate with other bypass proteins to ensure that replication proceeds in a faithful and completefashion. The accumulation of mutations can lead to uncontrolled cell growth that leads to the development of diseases, such as cancer. It is therefore important to understand how cells respond when the replication machinery stops at sites of damage. An "Achilles heel" of cancer cells is that they grow more rapidly than other cells in our body and thus they replicate their DNA more often. Because of this, many cancer treatments deliberately introduce damage to DNA replication in order to selectively slow/kill cancer cells. We hope that, by understanding how the replication machinery tolerates such damage, we may be able to increase the efficiency of cancer treatments by finding ways of making cancer cells even more likely to be killed by drugs that block DNA replication.

Impact Summary

This proposal focuses on the fundamental processes of DNA replication processes in human 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 diseases related to replication dysfunction and cancer. In addition, characterisation of basic DNA replication and repair mechanisms will have significant impact on the development of novel biotechnology reagents and compounds for drug development that could open up exciting leads for new therapies to treat cancer and related disorders. 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 damage tolerance on the maintenance of normal cell function and on molecular pathologies associated with normal ageing. For example, the project will investigate the link between aberrant damage tolerance and genome stability, a hallmark in the early stages of pre-cancerous cell growth. A thorough understanding of the relationship between DNA damage, replication and pathologies associated with proliferative diseases (cancer) and with ageing could thus help inform the health sector in respect to etiological factors that promote proliferative disease and 'unhealthy' ageing (e.g. environmental factors that induce excessive replication stress). 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: PrimPol has been show to be an enzyme required to maintain DNA replication under stress and loss of the gene results in genomic instability. Many common genetic disorders are associated with DNA instability and therefore PrimPol is an excellent candidate gene to screen human cell lines, from patients with such disorders, for mutations associated with this novel gene. This work could potentially pave the way for the development of simple diagnostic markers for particular cancers, provide insights into the role mutations of this gene may play in the progression of such diseases and exploit the molecular information gleaned from these studies to develop potential lead compounds for future drug development. 3. Industrial impacts: We have previously developed a DNA repair system (Ku-Ligase D) into an application for the biotechnology sector. We will use this recently acquired experience to optimally exploit this newly discovered enzyme (PrimPol), which also has potential for commercial development for uses in the biotechnology sector e.g. uses in molecular biology applications. Notably, this system may also be a potential drug target (see proposal) and the knowledge gained in this study will aid in the development of small molecular inhibitors that could potentially act as lead compounds for a future drug development programme.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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