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

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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 new crop 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.