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Understanding the mechanism of homologous recombination mediated gene targeting in Physcomitrella patens

Reference	BB/I006710/1
Principal Investigator / Supervisor	Dr Andrew Cuming
Co-Investigators / Co-Supervisors	Dr Yasuko Kamisugi
Institution	University of Leeds
Department	Ctr for Plant Sciences
Funding type	Research
Value (£)	461,659
Status	Completed
Type	Research Grant
Start date	09/04/2011
End date	08/10/2014
Duration	42 months

Abstract

Transgenes integrate when transforming DNA is captured and processed by host DNA repair machinery. In angiosperms, transgenes integrate randomly through a predominant non-homologous end-joining pathway. Only rarely does capture by the homologous recombination (HR) pathway result in targeted integration at homologous sites. Among plants, only Physcomitrella patens, a model species for comparative plant functional genomics, efficiently integrates DNA via the HR pathway, enabling precision genetic engineering by HR-mediated 'Gene Targeting' (HR-GT). GT is desirable because it (i) ensures predictable outcomes to transgenesis, (ii) permits the introduction of transgenic modifications without adventitious disturbances to genomes and (iii) allows 'cisgenic' modification of host genes by as little as a single base. To exploit GT in strategically important plants we need to understand the molecular mechanisms by which HR-GT occurs in plant cells. The analysis of this process in an HR-proficient model will deliver this understanding. This proposal will identify genes essential for high-efficiency HR-GT in Physcomitrella patens, focusing on chromatin remodeling helicases, identified through transcriptomic analysis of DNA repair. These genes will be functionally analysed by 'knock-out' and 'knock-in' mutagenesis. Protein-interaction studies will be used to search for and identify novel plant-specific members of molecular complexes that participate in HR-mediated DNA double-strand break repair and GT. The roles of these components and complexes will be analysed, in vivo, through analysis of targeted mutants. Identifying these components and understanding their interactions and activities is a necessary prerequisite for the development of knowledge-based strategies to extend the use of GT methodologies for crop breeding, to develop more environmentally robust plants and to enhance rates of meiotic recombination to accelerate genetic exchange in plant breeding programmes.

Summary

Land plants are static organisms, and their survival depends on their ability to withstand a variety of environmental stresses. The environmental impact of human activities is adversely affecting the severity of these stresses, and consequently limiting crop productivity. Ozone depletion leads to increased exposure to ionising radiation, and globally increasing temperatures and decreased water availability causing increased levels of drought stress. These stresses cause the generation of 'active oxygen species': highly reactive chemical agents that accumulate in cells and react with cellular components to inactivate or disrupt their functions. One key process that is highly susceptible to such damage is the maintenance of the genetic material. DNA, the molecule that encodes genetic information, is highly sensitive to damage by active oxygen species. The most severe forms of damage are breaks in the backbone of the DNA double-helix. If not repaired, these breaks result in irreversible and catastrophic loss of genetic material and subsequent cell death. To counter this, all organisms have evolved highly efficient mechanisms for the repair of such DNA double-strand breaks (DNA-DSBs). There are two principal mechanisms that are used for DNA-DSB repair. One is a 'quick and dirty' procedure called 'non-homologous end-joining' (NHEJ) that captures broken ends of DNA molecules and rejoins them. However, this process is inaccurate and incorporates DNA sequence errors at repair sites. The second mechanism captures broken ends and repairs them accurately by copying an homologous sequence. This process ('homologous recombination' - HR) is error-free, and is also used in the exchange of genetic material between maternal and paternal chromosomes when sperm or egg cells are produced by meiotic (reduction) division. This is process is responsible for the generation of genetic diversity within populations, and is exploited in plant breeding to introduce desirable traits into newcrop varieties. DNA repair mechanisms are also exploited by genetic engineers. Delivery of a foreign gene (a transgene) into a cell results in its being integrated into the host's genome when it is captured by the host cell's DNA repair machinery and integrated either randomly, by the NHEJ pathway, or at a specific site by the HR-mediated pathway. HR-mediated transgene integration occurs if (i) the transgene carries sequences identical to a target site in the genome and (ii) if the host cell displays a preference for HR-mediated repair over NHEJ-mediated repair. Very few organisms preferentially use the HR pathway for DNA repair and transgene integration. In those that do, it is possible to undertake precision engineering of genes by 'Gene Targeting' (GT). As little as a single base-pair of a host gene can be reliably altered by this means, without non-specific alteration of the genome. Because of its high degree of precision, the deployment of GT would be an attractive option for crop improvement strategies. Currently, the only plant in which efficient HR-mediated GT occurs is a moss, Physcomitrella patens, the first non-flowering land plant to have its genome completely sequenced and a model for studies of the evolution of plant gene function. Because GT in moss is routine and efficient, it provides an ideal model in which to identify the molecular mechanisms underlying this important DNA repair pathway. This research will identify and characterize key plant genes that direct efficient HR-mediated GT. This will provide the fundamental understanding necessary for (i) knowledge-based enhancement of GT rates in crop species, a prerequisite for 'clean' genetic engineering; (ii) identification of genes that can enhance resistance to DNA-damaging environmental stresses and (iii) identification of components of the HR machinery that could be modified to generate enhanced rates of meiotically-derived genetic variation for accelerated plant breeding.

Impact Summary

Beneficiaries include: Research scientists in the plant sciences The plant breeding industry Plant biotechnology companies Government sponsors of the biotechnology sector Research scientists in cancer & neurodegenerative disorders Understanding DNA-double-strand break (DNA-DSB) repair mechanisms in plants is important, both scientifically and strategically, because: (i) DNA-DSBs occur with high frequency as a consequence of environmental stress (ionising radiation and stress-related generation of active oxygen species) and collapse of DNA replication forks. (ii) DNA-DSBs are highly cytotoxic, and are a major cause of pathologies - including human cancers and neurodegenerative conditions. (iii) Transgene integration occurs through the recruitment of transforming DNA by DNA-DSB repair pathways. (iv) Repair of DNA-DSBs by homologous recombination (HR) is the underlying mechanism both for meiotic exchange and Gene Targeting (GT). Gene Targeting allows precise delivery of a transgene to a predetermined locus and enables custom modification of host genes. This level of sophistication is not available in either model flowering plants or in crop species. The ability to undertake GT in crop species would ensure 'clean' genetic manipulation and 'cis-genic' modification of genomes: a highly desirable outcome for plant breeders and policy makers who face difficulties in convincing the public of the benefits of transgenic modification of food crops. Understanding mechanisms underpinning plant HR will provide the fundamental knowledge necessary for developing knowledge-based strategies for developing wider applications of GT, enhancing the accuracy of DNA repair following stress, and for developing increased frequencies of meiotic recombination to accelerate plant breeding programmes. DNA repair defects are causes of many human pathologies, including cancers, neurodegenerative disorders and immunodeficiencies. Mutants in mammalian models suffer highfrequencies of early embryonic lethality. By contrast, plant development is highly plastic, and DNA repair mutants are viable, allowing an analysis of the DNA repair process. Analysis of HR in moss (like yeast) provides a platform for the role of this pathway in multicellular eukaryotes to analyse highly conserved components that are not amenable to study in mammals. Significantly, BBSRC's Strategic Plan 2010-2015 emphasises the importance of understanding the fundamental mechanisms that determine efficient DNA repair processes and gene targeting efficiency, by showcasing the research undertaken by Prof. Martin Evans (development of mammalian gene targeting technology) and Prof. Steve Jackson (determining pathways of DNA-double-strand break repair) as examples of how fundamental studies of underlying mechanisms lead to beneficial impacts for society. Ensuring benefits accrue: Experimental results will be published in high-impact international journals, ensuring timely access of peer-reviewed findings to the scientific community. Where appropriate, data will be deposited in publicly accessible databases (e.g. GenBank for sequence resources). Novel discoveries of strategic significance will be subject to intellectual property protection. Biological resources developed will be made generally available by deposition in the International Moss Stock Center at Freiburg University. Collaborations exist between the our laboratory and other groups studying DNA repair and gene targeting, including the German 'GABI-Precise' consortium, Dr. Fabien Nogué (INRA, Versailles) and Prof Karel Angelis (Czech Academy of Science) with whom we have submitted joint grant proposals and are co-authoring publications. We have a collaboration directed toward the use of P. patens for the analysis of genes associated in human pathologies, focused on the role of aprataxin in excision repair, with Dr. Steve West (Cancer Research UK) and Dr. Ivan Ahel (Paterson Institute).

Committee	Research Committee B (Plants, microbes, food & sustainability)
Research Topics	Plant Science
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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