The role of CyclinG for the genome stability in Drosophila

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It is vital to the organism to sustain genomic stability. Accordingly the repair of damaged DNA and the consequent elimination of irreversibly damaged cells is of central importance. This process is monitored and controlled by so-called checkpoints that prevent cell cycle progression to gain time for the necessary repair steps. In a coordinated interplay, a dense network of multiple factors controls checkpoint activity and the induced repair and apoptotic processes. In the model system Drosophila, we identified the gene cyclin G (cycG) as an essential factor for the sensing and the repair of DNA-double strand breaks during meiosis. Our preliminary work indicates that CycG is also engaged in the DNA-repair of somatic cells. The aim of this proposal is hence to investigate the mechanism of action of CycG during mitotic DNA-repair using molecular and genetic tools and to compare it with our findings on the role of CycG during meiotic DNA-repair.The first set of experiments addresses the role of CycG in the irradiation induced stress response. We will irradiate larval tissue that contains cells with normal CycG content in a clearly defined region, whereas the neighbouring cells are mutant for cycG. Using immuno-histology, the exact timeline of the apopotic cell death induction and the cell cycle response is recorded. Moreover, examination of the cytology shall reveal spontaneous and X-ray induced chromosomal aberrations in cycG mutants. The second set of experiments focuses on a possible specific role of CycG in the various repair pathways. On one hand the sensitivity towards additional genotoxic agents will be examined. On the other hand we will use a genetic test system, which allows upon targeted induction of double strand breaks to determine the utilized repair pathway by simple phenotypic inspection of the offspring. Subsequent PCR- and sequence analyses help to accurately unravel the accumulated errors. The third set of experiments addresses the molecular and genetic interrelation of CycG with the tumour suppressor P53, a known principal player in the context of DNA-repair. We have observed a direct interaction between the two proteins in vitro the relevance of which we want to verify by molecular and genetic approaches. CycG's mode of action may involve the protein phosphatase PP2A known to counteract multiple checkpoint players. A direct protein-protein interaction between CycG and the regulatory B' subunits of PP2A has been observed. Hence we want to address genetically, whether CycG influences DNA-repair via PP2A. These results not only extend our image of the vital control of genomic stability in Drosophila, but moreover enhance our understanding on the respective control mechanisms in vertebrates.