The roles of DNA ligases in novel plant recombination pathways: from DNA repair to gene targeting.

DNA is essential for growth and reproduction. It contains the genetic information that is passed from one generation to the next, and encodes the factors needed for a cell to survive and divide. However, DNA in the cell is under constant attack from reactive molecules generated from within the cell or caused by environmental factors including carcinogens, UVB and soil pollutants such as heavy metals. DNA damage can have severe repercussions for the organism; a single DNA double strand break is sufficient to cause cell death if not accurately repaired. All organisms therefore require effective DNA repair mechanisms to counteract this damage. An essential step in nearly all DNA repair pathways is the re-joining of DNA ends, which is catalysed by a DNA ligase enzyme. Whilst yeast has two genes, higher organisms including mammals and plants have three DNA ligase genes, with specialised roles in maintaining the genome during DNA replication and repair. In this study we will determine the roles of the different DNA ligases in the higher plant Arabidopsis thaliana. Specifically, we determine the pathways in which each DNA ligase operates, and the importance for plant growth in adverse conditions. As part of this analysis we will characterise the role of each DNA ligase in recombination - the process whereby two DNA molecules are joined to make a new molecule. In DNA repair, recombination can occur by two processes; one method of repair uses an intact copy of the damaged DNA as a template for repair. This process is termed homologous recombination, and it involves the joining (recombining) of similar (homologous) sequences. The second process is largely independent of DNA sequence and is termed non-homologous end joining. It is important that we understand recombination processes in plants because DNA repair is required to allow growth of crop plants under conditions of adverse environmental stress, and failure of these pathways will both result in reduced crop yields and the accumulation of deleterious mutations in future generations. Given concerns over the impact of climate change on UK crop productivity, on it is now particularly important now that we understand how plants respond to environmental stresses. Understanding recombination in plants is also important for breeding new varieties of crop plants. Manipulation of DNA repair pathways will help develop crops that will tolerate altered climatic conditions and recent studies have also implicated homologous recombination in plant adaptation to give greater tolerances to pathogens. In addition, changing the activities of the plant recombination pathways will have effects on how we make transgenic plants. Transgenes usually integrate at random locations in the plant DNA. However, high levels of homologous recombination would allow us to target a transgene to a specific location in the genome, enabling more reliable expression and opening up the possibility of 'fine tuning' genes that are found naturally in the plant. This would lead to a second generation of transgenic plants that would address many of the criticisms of current methodologies.

DNA ligases play essential roles in all organisms, maintaining the integrity of the genome by repairing DNA breaks induced by DNA replication and DNA damage. Multiple DNA repair pathways has led to a diversification of DNA ligase function through evolution, such that mammals and plants both independently gained a third DNA ligase gene, in addition to conserved LIG1 and LIG4 orthologues found in all eukaryotes. In this study we will determine the roles of the three Arabidopsis DNA ligases in DNA repair. The novel DNA ligase unique to plants is termed LIG6, and recent studies in our lab have identified roles for this enzyme and AtLIG1 in the repair of DNA double strand breaks (DSBs), one of the most cytotoxic forms of DNA damage. The recombination mechanisms that repair DSBs are of great biotechnological significance as they mediate the integration of DNA into the plant genome during plant transformation. Recombination can either be sequence dependent homologous recombination, or can be largely independent of sequence, mediated by non-homologous end joining (NHEJ). Our studies indicate that plants have evolved multiple, highly active pathways of NHEJ, unlike mammals where backup pathways play a minor role. This study will determine the roles of DNA ligases in novel DSB repair pathways, and their requirement for plant growth in abiotic stress conditions. The recruitment of DNA ligases to repair complexes will be investigated by protein interaction studies, which will further delineate novel DNA repair pathways in higher plants. The roles of DNA ligases in plant transformation will be determined, including identification of DNA ligases required for gene targeting. These results will inform the development of improved gene targeting approaches in plants, which, together with increased knowledge of plant tolerance to abiotic stresses will help generate improved crop plants to meet the challenges of global food security and climate change facing us in the next decades.

Who will benefit from this research? Beneficiaries include The plant breeding industry Plant biotechnology companies Research scientists in the plant sciences Government sponsors of the biotechnology sector National and International regulatory bodies and biosafety organisations Developing world agriculture Seed conservation The plant breeding industry, biotechnologists and academic researchers This study will inform the development of improved gene targeting methods in the medium to long term, enabling the precise and directed manipulation of the genome. These represent valuable tools which would enable researchers to better understand the biological functions of genes in a wide range of species. They have a broad range of applications including in agriculture and biotechnology to maximise the biosynthetic potential of plants for production of food, biofuels and pharmaceuticals. In addition, improved tolerance of crop species to abiotic stresses (eg UVB, ozone, heavy metals) will help maintain yields under adverse climate conditions and growth of crops on poor soils, increasingly important in the face of continued population growth. Government and regulatory bodies Improved plant transformation technologies combined with the increasing need for improved yields should help reduce public opposition to genetically modified food crops as it allows the precise modification of endogenous genes rather than the introduction of new genetic material. This could benefit organisations such as DEFRA, the Food Standards Agency and the European Food Safety Agency which have the potential to inform public opinion on the potential benefit of crop improvement. Seed conservation There is a strong correlation between DNA repair capacity and seed viability, storability and vigour, which are important determinants of crop yield. DNA ligases and associated pathways potentially represent biomarkers for seed quality, which would be of potential importance to both agriculture and for seed conservation. Seed conservation is important for the preservation of plant germplasm in seedbanks. Staff Individual beneficiaries include the PDRA and technician employed on the project who will gain career training and development led both within in the Faculty of Biological Sciences and professional development courses run by a Central Staff Development Unit at the University of Leeds. Communication and exploitation The applicant has existing links with the Millennium Seed Bank (Kew) which will enable development and application of biomarkers for seed quality to seed conservation and has established links with the Department of Plant Science and Agricultural Resources, Khon Kaen (Thailand) with joint interests in plant resistance to abiotic stress. UOL CPS has strong links, programmes of collaboration and initiatives with Universities in India and China which could provide partners in developing countries who would be interested in any progress of this research. The UoL maintains specialist Knowledge Transfer and Media Relations expertise which will be used to publicise findings through Press releases and via media events organised by BBSRC, The Royal Society, and current national forums for publicising research developments including National Science Week, the Summer Science Exhibition, and the Science, Engineering and Technology for Britain initiatives. Both PI and PDRA will engage in public science communication events. Informing Beneficiaries: Publications in open access, peer reviewed journals will be available to the biotech industry. Additionally PI and Co-Applicant will attend and present work at appropriate UK and international conferences. Intellectual Property Intellectual Property generated from this programme will be protected, licensed to companies or made freely available to non-profit researchers as appropriate, through the IP mechanisms in place at the University Of Leeds.