DNA damage response and metals

Copper is an important trace metal ion in biology as some essential enzymes use it as a cofactor. Copper, however, is potentially toxic if it binds to the wrong components of a cell. The amount of copper taken into cells is therefore carefully regulated so as not to cause damage. Central to this regulation is the ability of a cell to sense the amount of copper it needs and how much it has. Presently, we have little understanding of how this is achieved in complex organisms. We use bakers yeast as a model to analyse how cells sense metal ions as it has been shown that some aspects of this are conserved throughout biology. Our work has established that yeast treated with chemicals that cause DNA damage switch on genes that are involved in copper uptake. We want to understand why copper is needed under these conditions and identify the mechanisms that are used by yeast to activate copper uptake. We will compare these processes with those of another yeast, Cryptococcus neoformans, which causes a potentially fatal human disease.

Copper is an essential enzyme cofactor that is potentially toxic. Cells therefore coordinate the need for copper with its uptake and distribution. Unlike bacterial systems, little is known of how copper is sensed by eukaryotic cells. The yeast Saccharomyces cerevisiae is an established model for studying eukaryotic metallobiology. One aspect of metal regulation in yeast involves the control of metal responsive transcription. Although the factors that mediate this in yeast have been identified, the molecular mechanisms determining how these discern different metals are not known. Gene induction under copper limiting conditions is dependent on the Mac1 factor. Copper binds to Mac1 in vitro, which has supported a model whereby copper binding to the factor is the signal of copper availability. Recently however, superoxide dismutase activity has been shown to be necessary for Mac1 function suggesting a role for redox sensing in Mac1 function. Here, we demonstrate the novel finding that Mac1 activates copper uptake genes in response to known DNA damaging agents and that copper is required for yeast to grow in the presence of these agents. Molecular analysis will determine why copper is needed during genomic stress and identify the regulatory pathways that link copper and the response to DNA damage. This experimental system allows us the opportunity to distinguish between the copper and the redox sensing mechanisms of Mac1. In addition, we will address the hypothesis that in yeast, the signal transduction pathway that mediates the cell's response to DNA damage also directly regulates copper uptake. Analysis of copper regulation in the human pathogenic yeast Cryptococcus neoformans will also determine to what extent the link between copper and the DNA damage response is conserved. Together, these studies will address a novel aspect of eukaryotic copper homeostasis and provide further understanding of how eukaryotic cells sense metals.

This research is based on the question: how do eukaryotic cells sense the need and availability of copper? Copper is an essential element yet potentially toxic so that there are complex mechanisms to ensure copper homeostasis. There are many diverse aspects of eukaryotic copper biology that have health and economic implications. The misregulation of human copper homeostasis can impact on human health and has been linked with Alzheimer's disease. Fungal copper laccases oxidise many substrates and are used for a variety of industrial purposes. Some fungal plant and human pathogens require melanin as a virulence factor, which they produce via the action of copper enzymes. Lastly, copper based fungicides have been used for over a century to protect crops. Central to the treatment of copper related disease, the development of copper enzymes for industry, the potential targeting of copper pathways to treat fungal pathogens, and the development of more efficient copper fungicides is an understanding of the copper sensing mechanisms within eukaryotic cells. Who will benefit from this research? Potential beneficiaries of this research are: (1) patients who suffer from copper related disease and companies developing relevant treatments; (2) companies producing copper enzymes for industrial purposes and the end users of those enzymes; (3) patients who suffer from fungal infections and companies developing antifungal drugs and (4) those within agriculture that lose crops to fungal disease and companies that are developing copper based antifungal agents. How will they benefit from this research? The development of treatments for copper related disease and human fungal infections have the potential to significantly improve the quality of a patient's life. Companies that are able to produce and sell treatments for copper related disease, copper enzymes and antifungal agents will benefit financially with a consequent impact on the economy of the nation. As an example, the fungus Mycosphaerella graminicola is the cause of wheat blotch, which has a serious impact throughout the world. It is estimated that $275 million is lost annually to wheat growers in the United States and that £280 million is spent in Europe on antifungal agents to protect cereals [1]. There is a clear economic market for efficient and effective agrochemicals. The proposed research can be described as 'basic science' so that any potential impact from it may be long term, particularly with regard to the treatment of human disease. It is worth noting, however, that the study of metal homeostasis in yeast has resulted in an understanding of the molecular mechanisms involved in Wilson and Menkes disease and Friedreich's ataxia. More immediate practical applications may arise with regard to the development of copper fungicides. Aspects of copper sensing that will be studied may be conserved with plant fungal pathogens allowing a better understanding of the mechanism of action of copper fungicides. What will be done to ensure that they benefit from this research? The 'Metals in Cells' group at Newcastle University plans to foster contacts between itself and UK companies that may benefit from its research. Some preliminary contacts have already been made. Discussions are underway relating to the holding of a yearly meeting at which the Metals in Cells group at Newcastle will present their work to other academics and potential industrial partners. [1] http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html