) A breakdown in the maintenance of genome stability is an invariant feature of tumor cells and one of the defining features of the transformed phenotype. Much of the work on genome stability has focussed on the role of 'mutator' genes, which when themselves mutated, allow high overall mutation rates in cells, precipitating the rapid disruption of oncogenes and tumor suppressor genes in cancer. Examples of such mutator genes are the mismatch repair genes such as MSH2 which are frequently mutated in HNPCC. Less work has focussed on identifying the key players responsible for abnormal recombination and missegregation of chromosomes in cancer cells. Part of the problem has been the lack of systems for rapidly screening defects in chromosome segregation. If such screening tools were available, it would be possible to identify new genes involved in maintaining the fidelity of chromosome segregation. It would be expected that such genes may be mutated in cancer cells, potentially constituting new tumor suppressor genes. Some key guardians of such genome stability have already been identified from a family of DNA helicases, named after the prototypic member RecQ, a bacterial helicase which is required both for initiating the RecF pathway of homologous recombination and for the suppression of illegitimate replication. Members of this RecQ gene family are associated with the intriguing cancer susceptibility syndromes, Bloom's syndrome-BLM, Werner's syndrome-WRN and Rothmund-Thompson syndrome. The overall goal of the proposed application is to establish and validate robust vertebrate models to identify defects in chromosome segregation. The proposed models will allow a sufficiently high throughput that they could be used for genome-wide mutational screens and large-scale screening of candidate peptide blockers of complexes indicated in the maintenance of genome stability The first goal of this project is devoted to the isolation and testing of such peptide blockers isolated using a yeast screen. We suggest that the peptide blockers themselves will prove to be very useful dominant negative reagents to unravel the pathways involved in genome stability, since they allow the uncoupling of different activities of multifunctional proteins. In this way they will simulate the effects of dominant mutations and may even allow 'epistasis' analyses to determine the relationship between different gene products. The goal of the second phase is the establishment and validation of zebrafish and mouse models to detect defects in chromosome segregation during mitosin and meiosis. Since these models employ robust fluorescent screening technology they will be suitable for high throughput industrial application.
