The role of CyclinG for the genome stability in Drosophila

维持基因组的稳定性对生物体至关重要。因此，修复受损的DNA和随后的不可逆受损细胞的消除是至关重要的。这个过程由所谓的检查点监测和控制，这些检查点阻止细胞周期的进展，为必要的修复步骤赢得时间。在一个协调的相互作用中，一个密集的多因素网络控制着检查点活动和诱导修复和凋亡过程。在果蝇模型系统中，我们发现基因周期蛋白G （cycG）是减数分裂过程中感知和修复dna双链断裂的重要因子。我们的初步工作表明，CycG也参与体细胞的dna修复。因此，本文的目的是利用分子和遗传工具研究CycG在有丝分裂dna修复过程中的作用机制，并将其与我们在减数分裂dna修复过程中CycG的作用进行比较。第一组实验解决了CycG在辐照诱导应激反应中的作用。我们将在一个明确界定的区域照射含有正常CycG含量的细胞的幼虫组织，而邻近的细胞则是CycG的突变体。利用免疫组织学，记录凋亡细胞死亡诱导和细胞周期反应的确切时间线。此外，细胞学检查应揭示自发和x射线诱导的cycG突变体染色体畸变。第二组实验集中于CycG在各种修复途径中可能的特定作用。一方面，将检查对其他遗传毒性物质的敏感性。另一方面，我们将使用基因检测系统，该系统允许通过对后代进行简单的表型检查来靶向诱导双链断裂以确定所利用的修复途径。随后的PCR和序列分析有助于准确地解开累积的错误。第三组实验解决了CycG与肿瘤抑制因子P53的分子和遗传相互关系，P53是dna修复中已知的主要参与者。我们在体外观察到两种蛋白质之间的直接相互作用，我们希望通过分子和遗传方法验证其相关性。CycG的作用模式可能涉及蛋白磷酸酶PP2A，已知可抵消多个检查点参与者。已经观察到CycG与PP2A的调节B'亚基之间的直接蛋白-蛋白相互作用。因此，我们想从遗传学上解决CycG是否通过PP2A影响dna修复的问题。这些结果不仅扩展了我们对果蝇基因组稳定性的重要控制的认识，而且增强了我们对脊椎动物各自控制机制的理解。

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.