Modelling genomic instability in Drosophila melanogaster: genetic and molecular analysis of Dm WRN exonuclease

Some people age well while others succumb to many and various diseases associated with old age including cancer, diabetes and heart disease, but we still don't know why some people age more healthily than others. By studying a human premature ageing disease called Werner's Syndrome, which essentially mimics normal ageing but much more quickly, scientists hope to understand the causes of many of the age-associated diseases. It is now known that most, if not all, of the many premature ageing aspects of Werner's syndrome result from the loss of a single protein known as WRN. We have shown that cells taken from patients with Werner's syndrome have problems copying their genetic blueprint, the DNA, and that such problems result in their DNA becoming very unstable. Such 'genomic instability' is known to be important in the development of many cancers, and it may also make people more prone to develop other serious diseases. To find out how WRN has such wide-ranging effects, we need to study the protein on its own in the test tube and to test the effect of making changes in it (mutations) in cells and whole organisms. Although some progress has been made using cells taken from patients, each batch of cells is different and we cannot precisely ascribe any changes to the WRN protein itself. In addition, we need to work out which parts of the WRN molecule are particularly important in keeping the DNA intact, and whether changes to these regions can affect how long a cell and even an organism lives. Obviously this is both impractical and unethical in mammalian systems, so we have chosen to examine the fruit fly, Drosophila. The fly is very well characterised, with the sequence of all the 'letters' of its DNA code already known, and many different genetic procedures are possible to 'mix and match' different combinations of genes. To date, we are the only researchers to have identified a WRN-like function in flies and we have very recently, and excitingly, shown that flies lacking this activity show a big increase in abnormal changes to their DNA, just like in patients with Werner's syndrome. By providing a full molecular and genetic analysis of the action of WRN in flies, we hope to answer significant questions as to what the protein is doing to protect our DNA. We are therefore planning to make molecular probes to look at when and where the fly WRN protein is made, what it does to DNA, what other proteins it works with and what aspects of the DNA copying process are affected when the protein is altered. Finally, by replacing the fly version of the WRN gene with the human WRN gene, we hope to develop a model system for future use in testing drugs that either block human WRN activity (for example to make otherwise long-lived cancer cells age quickly), or to promote longevity of normal cells, with the hope of avoiding many of the problems associated with ageing of vitally important cells in old age. The long term aim of such ageing research, and in particular, the development of model ageing systems, is to help achieve a healthy old age and prevent many of the debilitating illnesses associated with getting old.

Werner's Syndrome is very useful model of ageing. Loss of function of the unique WRN exonuclease/helicase leads to pleiotropic effects including increased genomic instability, which may arise from illegitimate recombination at stalled replication forks. We have identified a homologue of the WRN exonuclease in Drosophila, and shown that mutation of this gene results in a large increase in genomic instability. We propose to identify the processes of DNA metabolism for which DmWRNexo is required through parallel genetic, cell biological and biochemical studies of DNA replication, recombination and repair. DmWRNexo mutation effects will be analysed phenotypically through development and in the post-mitotic adult. Using RT-PCR, novel antibodies and tagged gene variants, we shall assess DmWRNexo spatial and temporal expression through development and the cell cycle. Sensitivity of WRNexo mutant flies to DNA damaging agents will be examined. We shall characterise the biochemical activities of the DmWRNexo in terms of DNA substrate preferences, its impact on stalled replication fork resolution, and roles in recombination and DNA repair. We propose to identify protein partners of DmWRNexo, in particular examining the known RecQ helicases in Drosophila. Complementation of the genetic instability phenotype by human WRN transgenes will be tested, providing the possibility of a 'humanised' model system as a powerful tool for the analysis of agents that impact on ageing through the WRN pathway, whilst avoiding use of mammalian models. The role of Holliday junction persistence in genomic instability in DmWRNexo null flies will be tested by introducing RusA, a bacterial HJ resolvase, under the control of the WRNexo promoter. The data generated should provide exciting insights into the roles of the exonuclease activity of WRN distinct from its helicase activity. The contribution of genomic instability to accelerated ageing may also be made possible by this DmWRNexo mutant fly.