Regulation of DNA repair pathway choice during development

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.

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.

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.