Regulation of DNA repair pathway choice during development

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

The genetic code (DNA) contains a blueprint to produce all the machinery required for a cell to perform its various functions in a programmed manner. Preservation of this code is critical to maintain normal cellular functions. However, DNA is under continual attack from agents that cause changes in the genetic code. This can occur when cells copy their DNA (DNA replication), through changes in the sequence of DNA building blocks (mutation), or by creating breaks in the DNA molecule. Interestingly, DNA damage also occurs in a tightly regulated context during a number of normal cellular processes including development of the immune-system and transfer of genetic information as cells segregate their genomes in a process know as meiosis. Therefore, cells have developed a complex network of pathways to detect DNA damage when it occurs and correct these faults by DNA repair. The importance of DNA repair is underscored by the observations that defects in these pathways leads to a variety of debilitating clinical symptoms including premature ageing, immune-system failure, developmental abnormalities and increased cancer risk. Therefore, understanding DNA repair will provide insights into several fundamental biological processes in addition to clues regarding the molecular basis of a number of disease states. Breaks in both strands of the DNA double helix (double strand breaks; DSBs) are one of the most toxic varieties of DNA damage. These can be repaired by directly re-joining DNA breaks (non-homologous end-joining; NHEJ), or by using homologous DNA as a template to restore genome integrity (homology-directed repair; HDR). The components of these two pathways are well characterised. However, the factors that influence whether cells repair damage by NHEJ or HDR remains relatively ill defined. Defects in DNA repair lead to a variety of developmental abnormalities including defective antibody production, cranial-facial abnormalities and degeneration of the nervous system. Despite this, how these pathways are regulated as multi-cellular organisms develop into a variety of specialised tissues remains largely unexplored. DNA repair is achieved by similar mechanisms in all organisms from bacteria to humans. Therefore, studying these pathways in relatively simple model organisms has increased our understanding of how these processes work in humans. Unfortunately, certain commonly used model organisms lack key components of human DNA DSB repair pathways and do not undergo a developmental program into specialised cell types. In contrast, the soil dwelling amoeba Dictyostelium is an easily grown and manipulated organism that undergoes a relatively simple developmental program. As such, it is a commonly used model to study cell type specification and differentiation. In addition, our previous work has illustrated that DNA DSB repair pathways in this organism are more similar to humans than other commonly used model organisms. The aims of this research are to exploit these observations and use Dictyostelium as a model to study how DNA DSB repair pathway choice is regulated in the context of an organism's life cycle and developmental program. In addition to providing insights into how DNA repair pathways are regulated in Dictyostelium, these studies will likely be applicable to how these processes are achieved in other higher organisms including humans.

Technical Summary

DNA DSBs can be repaired by two pathways; homology directed repair (HDR), or non-homologous end-joining (NHEJ). Although the main components of these pathways are well characterised, how the decision is made to repair a break through HDR or NHEJ remains relatively ill defined. In particular, although the influence of the cell cycle on repair pathway choice has received much attention, the factors that influence this decision during the development of a multi-cellular organism remain largely unexplored. This application aims to exploit Dictyostelium to study DNA DSB repair pathway choice during development. Dictyostelium exists as single celled amoebae that under conditions of starvation aggregate and undergo a relatively simple developmental program. This, in addition to Dictyostelium being a genetically tractable organism, has made it a commonly used model to study the molecular basis of cell type fate and differentiation. Our recent work indicates Dictyostelium DNA DSB repair pathways are more similar to humans than other commonly used genetic models. In addition, our unpublished data illustrate a switch from HDR to NHEJ occurs at some stage during Dictyostelium development independently of the cell cycle. The experiments described here will build on this work and investigate the factors that regulate the choice between alternate DNA DSB repair pathways during development. We will use existing cell based assays to assess NHEJ and HDR efficiency through the Dictyostelium life cycle and analyse recruitment of NHEJ/HDR factors to DNA DSBs using cell imaging and chromatin immunoprecipitation technologies. Given that Dictyostelium is amenable to genetic analysis, we will assess the genetic requirements for these events and identify components required for regulating DNA DSB repair pathway choice either in Dictyostelium amoebae, or during the life cycle of this organism.

Planned Impact

Beneficiaries and how they will benefit: The immediate beneficiaries will primarily be the scientific community. Identifying mechanisms contributing to pathway choice during development in Dictyostelium will provide insights into more targeted strategies to assess these choices in other organisms. It will also provide data, reagents and methodologies of interest to Dictyostelium developmental biologists. In the longer term other beneficiaries will include:- i) Commercial sector - Commercial companies developing therapies targeted to either killing or protecting particular populations of differentiated or proliferating cells - Companies wishing to screen gene mutations that may contribute towards developmental abnormalities during embryogenesis. ii) Public sector - Policy decisions regarding effects of DNA damage during stem cell propagation and during embryonic development. - Policy makers by enabling them to develop policies to protect the general public from the long term effects of DNA damage on particular tissues. iii) Wider public in general - Facilitate the development of tailored, specific information regarding life style, diet etc. to protect from the effects of DNA damage which accumulate during the aging process or pregnancy. The realisation that particular differentiated tissues are reliant on certain DNA repair pathways will inform this. This is particularly important in light of the increasing numbers of elderly people and will allow the development of lifestyles at an early age which will increase the quality of life in later years. Management to increase impact: The impact of this project will be maximised through publicity and data management. The work will be published in peer-reviewed journals in compliance with open access policies, and presented at meetings, national and international, specialist and general. dictyBase (dictybase.org) is a central website and information portal for research on Dictyostelium and facilitates the publication of novel results as well as the reagents generated via the associated Stock Centre. Both investigators give seminars as invited speakers at other academic institutions. Within Oxford, the research will be presented at both the Chromosome Biology Club (an Oxford-wide club of researchers with an interest in chromosome biology that meets monthly) and within the Biochemistry Department at the weekly seminars run by the Laboratory of Chromosome and Cell Biology. This is a newly formed cluster of research groups working in a collaborative and synergistic way to advance our understanding of chromosome and cell biology using a variety of experimental systems and approaches. The labs of both investigators have web pages which are regularly up-dated and used to publicise research. The Biochemistry Department in Oxford has a dynamic web-site with current news items that is used to publicise exciting new discoveries. Blueprint is an Oxford-wide publication published monthly which is used to disseminate new discoveries throughout the University, to both scientific and non-scientific staff. Intermediary organisations will be engaged as appropriate to facilitate interaction with the commercial sector and the general public. These include the University media relations team for interaction with the general public, Research Services and Isis Innovation for interaction with the commercial sector. Science Oxford facilitates interaction with schools, the general public and the commercial sector. The investigators are committed to public understanding of science. They give presentations and help organise Open Days within the Biochemistry Department and Colleges of Oxford University which publicise their research to both prospective undergraduates and their parents, teachers etc. They speak in local primary schools to engage younger children in their research and to schools via Departmental and college based events.

Publications


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Couto AM (2013) Investigation of DNA repair pathway activity. in Methods in molecular biology (Clifton, N.J.)
Hsu DW (2011) DNA double-strand break repair pathway choice in Dictyostelium. in Journal of cell science
Pears CJ (2012) The role of ADP-ribosylation in regulating DNA double-strand break repair. in Cell cycle (Georgetown, Tex.)
Pears CJ (2012) The role of ADP-ribosylation in regulating DNA double-strand break repair. in Cell cycle (Georgetown, Tex.)
 
Description Repair of DNA double strand breaks (DSBs) is critical to maintain genome integrity. Furthermore, DSB repair plays a critical role in regulating a number of basic cellular processes including immunoglobulin gene maturation, site-specific recombination and meiosis. Therefore, in addition to providing insights into how genome stability is maintained, investigating these processes will increase our understanding of the several fundamental biological processes.



Previous BBSRC funding allowed us to pioneer Dictyostelium as a model to study DNA repair. These studies revealed a high degree of conservation between human and Dictyostelium DSB repair pathways and that this organism contains a number of DSB repair factors absent in more conventional model organisms used to study these pathways. The overall aim of this work was to extend these studies and gain a greater understanding of how the decision to repair a DSB by non-homologous end-joining (NHEJ) or homology directed repair (HDR) is achieved.



Our previous work illustrated a switch from HDR to NHEJ occurs when single celled vegetative Dictyostelium develop into a multi-cellular organism. During our current studies we developed an assay to assess NHEJ efficiency in Dictyostelium. This technology revealed that although vegetative Dictyostelium relay on HDR to tolerate DSBs, NHEJ is active during this stage of the life cycle. At the time these studies were performed, the prevailing view was that NHEJ repairs DSBs during G1 phase of the cell cycle, whilst HDR is active in S and G2. Given vegetative Dictyostelium have no discernible G1; these studies highlight a hitherto unrecognised role of NHEJ during S and G2. Our findings that loss of the NHEJ factor Ku increases HDR at the expense of NHEJ additionally illustrate NHEJ and HDR compete in vegetative Dictyostelium and that Ku is a critical factor in influencing DSB repair pathway choice. Our data illustrating disrupting genes that function downstream of Ku in the NHEJ pathway does not increase HDR, and that this can be supressed by disruption of Ku, additionally imply that Ku-dependent NHEJ is attempted first and if unsuccessful HDR is engaged. These studies were published in the Journal of Cell Science in 2011.



In a related study we analysed how ADP-ribosylation of proteins at DSBs by ADP-ribosyltransferases (ARTs) influences DSB repair pathway choice. Although the role of ADP-ribosylation by ARTs in single strand break (SSB) repair is well characterised, how these enzymes regulate DSB repair is less clear. We exploited Dictyostelium to uncover a novel ART that regulates NHEJ at the expense of HR. Three Dictyostelium ARTs (Adprt1a, Adprt1b and Adprt2) contain ART catalytic domains similar to PARP1, the principle DNA damage responsive ART in humans. We found that whilst adprt1a- cells are not sensitive to SSBs, adprt2 and adprt1b are required to tolerate this variety of damage. Thus, similar to humans, two ARTs are required for resolution of SSBs. Whilst Adprt1b is dispensable for PARylation at DSBs, adprt1a and to a lesser extent adprt2 are required for this event. Disruption of adprt1a and adprt2 in combination abrogates DSB-induced PARylation. NHEJ is compromised in adprt1a- cells with a concomitant increase in HR. Therefore, distinct ARTs respond to different varieties of DNA damage; whilst Adprt1b and Adprt2 signal SSBs, Adprt1a and Adprt2 respond to DSBs. These data identify a novel 3rd ART that responds to DSBs and that PARylation promotes NHEJ at the expense of HR.

Importantly, Ku enrichment at DSBs is compromised in adprt1a- cells, illustrating Adprt1a-induced PARylation promotes NHEJ by facilitating recruitment of NHEJ factors to DNA lesions. PAR polymers recruit repair factors to DNA lesions through PAR interaction domains. One such domain, the poly-ADP-ribose-binding zinc finger (PBZ) motif, is present in Ku70. We found that Ku70 binds PAR in vitro and this is dependent on the PBZ domain of the protein. This domain is also required for enrichment of Ku70 at DSBs and effective NHEJ. This represented the first illustration of how PARylation regulates classic NHEJ and provides a mechanistic explanation of these events. These studies were published in the Journal of Cell Biology in 2011 and resulted in an invited review in Cell Cycle in 2012.

We also uncovered redundancy between SSB- and DSB-responsive ARTs to maintain cell viability following DNA SSBs. Although adprt2- cells are sensitive to SSBs, robust PARylation is evident in these cells. Increased levels of H2AX phosphorylation and recruitment of Ku to DNA lesions in adprt2- cells exposed to agents that induce SSBs indicates DNA lesions are converted to DSBs. Disruption of the DSB-responsive adprt1a in combination with adprt2 abrogates MMS-induced PARylation, recruitment of Ku to DNA lesions, and further sensitises cells to agents that induce SSBs relative to adprt2 disruption strains. These data indicate that in the absence of Adprt2 and an effective SSB response, DNA lesions are channelled through Adprt1a and NHEJ to maintain cell viability in the face of genotoxic stress. In support of this hypothesis, disruption of the NHEJ gene dnapkcs in combination with adprt2 similarly increases sensitivity of cells to SSBs. Taken together, these data indicate overlapping functions between different ARTs in signalling DNA damage and a critical requirement for NHEJ in maintaining cell viability in the absence of an effective SSB response. This study was published in The Journal of Cell Science in 2013 and an invited review in DNA Repair in 2014.
Exploitation Route Immediate exploitation is primarily by the academic community. Identifying mechanisms that regulate DSB repair pathway choice in Dictyostelium will provide insights into how these pathways are regulated in other organisms, including humans. In this regard, our work prompted others to investigate how ADP-ribosylation regulates NHEJ in other organisms and led to the finding that human PARP3 is similarly required to promote DSB repair by accumulation of Ku at DSBs (Beck C, et al. Nucleic Acids Res. 42:5616-32).

Our data, reagents and methodologies have also provided technologies and knowledge to stimulate other Dictyostelium biologists to study DNA repair in the context of their own research interests. We were invited to write an article on technologies to study DNA repair in 'Dictyostelium Protocols II' (Couto, Lakin and Pears, 2013. Methods Mol Biol. 983:295-310) and have received many requests for reagents/advice from others interested in using our technologies in Dictyostelium.

In the longer term these studies can be exploited by medical care and pharmaceutical companies. Loss of DNA repair pathways results in a variety of debilitating clinical symptoms including developmental, neurological and immune system defects, premature ageing and increased cancer risk. Identifying and characterising factors that regulate DSB repair will provide potential therapeutic strategies to ameliorate these symptoms. In this regard, broad range inhibitors of ARTs are promising agents to treat ovarian and breast cancers. These studies have prompted us to exploit Dictyostelium as a model to assess the efficacy of ART inhibitors in these strategies, with a long term view of transferring these findings to humans and clinical settings.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology
 
Title Assays for DNA repair in Dictyostelium 
Description We developed several cell based assays to assess DNA repair efficiency in Dictyostelium cells. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact We were invited to write an overview of DNA repair assays in this organism (Couto, Lakin and Pears, 2013. Methods Mol Biol. 983:295-310) and have received requested for strains, reagents and advice on assays from other Dictyostelium researchers. 
 
Description Department of Biochemistry, University of Oxford Open Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact 2 Open days attended to publisise the Biochemistry course at Oxford, encourage students to apply to study this subject, an addition to stimulating a more general interest in science
Year(s) Of Engagement Activity 2013,2014
 
Description Invited seminar University of Zurich, Switzerland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Approximately 60 people attending the talk who are interested in aspects of cancer biology.

This event allowed me to publicise work funded by BBSRC and to network with other researchers.
Year(s) Of Engagement Activity 2012
 
Description Invited talk, International Dictyostelium Conference, Madrid, Spain 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact A seminar was presented to international researchers interested in all aspects of Dictyostelium biology.

This event allowed me to publicise work funded by BBSRC and to network with other researchers.
Year(s) Of Engagement Activity 2012
 
Description Invited talk, Society for General Microbiology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact A seminar presentation to researchers interested in various aspects of microbiology.

This event allowed me to publicise work funded by BBSRC and to network with other researchers.
Year(s) Of Engagement Activity 2012
 
Description Keynote address, CR-UK Leicester Centre, MRC Toxicology Unit Genome Sciences Theme Workshop, University of Leicester 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact An invited Keynote address at a workshop to open the CR-UK Leicester Centre, MRC Toxicology Unit & Genome Sciences Unit at the University of Leicester

To publicise our research funded by the RCUK and network with researchers.
Year(s) Of Engagement Activity 2014
 
Description PARP Conference, Cold Spring Harbor, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Data was presented to an audience of researchers interested in PARP biology.

An international collaboration was intiated during this activity.
Year(s) Of Engagement Activity 2014
 
Description PARP conference, Quebec, Canada 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact Data were presented to the scientific community interested in PARP biology.

Publicising research and forging contacts in a new area of research.
Year(s) Of Engagement Activity 2013
 
Description Research talk, University of Birmingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Participants in your research and patient groups
Results and Impact An invited seminar to researchers with a wide range of interests in cancer biology.

Invited seminar at the university of Birmingham allowed me to publicise work funded by BBSRC and to network with other researchers.
Year(s) Of Engagement Activity 2012
 
Description School Visit (Oxford) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Approximately 20 pupils attended a talk describing an overview of my research interests. This resulted in discussions regarding biomedical reserach, in addition to stimulating interest from pupils to embark on a university education/ research career.

This is difficult to assess, although the talk stimulated significant discussion and and invitation to talk again at the school.
Year(s) Of Engagement Activity 2014
 
Description Seminar, UK/EU Dictyostelium Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact A research senminar was presented to UK/EU researchers interested in various aspects of Dictyostelium biology.

This event allowed me to publicise work funded by BBSRC and to network with other researchers.
Year(s) Of Engagement Activity 2011