The role of CyclinG for the genome stability in Drosophila

CyclinG 对果蝇基因组稳定性的作用

• 批准号: 247944451
• 负责人: Dr. Anja Christina Nagel
• 金额: --
• 依托单位: Institut für Genetik (240) (aufgelöst)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/247944451?language=en
• 关键词: role; CyclinG; genome; stability; Drosophila

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.

维持基因组稳定性对生物体至关重要。因此，修复受损的 DNA 以及随后消除不可逆受损的细胞至关重要。这一过程由所谓的检查点进行监测和控制，这些检查点阻止细胞周期进展，为必要的修复步骤赢得时间。在协调的相互作用中，多种因素的密集网络控制着检查点活动以及诱导的修复和凋亡过程。在果蝇模型系统中，我们确定了基因周期蛋白 G (cycG) 是减数分裂期间 DNA 双链断裂感知和修复的重要因子。我们的初步工作表明 CycG 也参与体细胞的 DNA 修复。因此，本提案的目的是使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 的作用的发现进行比较。第一组实验探讨了 CycG 在辐射诱导的应激反应中的作用。我们将照射在明确定义的区域中包含具有正常 CycG 含量的细胞的幼虫组织，而邻近的细胞是 cycG 突变的。使用免疫组织学，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，细胞学检查将揭示 cycG 突变体中自发的和 X 射线诱导的染色体畸变。第二组实验侧重于 CycG 在各种修复途径中可能的特定作用。一方面，将检查对其他基因毒性剂的敏感性。另一方面，我们将使用基因测试系统，该系统允许有针对性地诱导双链断裂，从而通过对后代的简单表型检查来确定所使用的修复途径。随后的 PCR 和序列分析有助于准确地揭示累积的错误。第三组实验探讨了 CycG 与肿瘤抑制因子 P53 的分子和遗传相互关系，肿瘤抑制因子 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学方法验证其相关性。 CycG 的作用方式可能涉及已知可对抗多个检查点参与者的蛋白磷酸酶 PP2A。已观察到 CycG 和 PP2A 的调节 B' 亚基之间存在直接的蛋白质-蛋白质相互作用。因此，我们想要从基因角度解决 CycG 是否通过 PP2A 影响 DNA 修复。这些结果不仅扩展了我们对果蝇基因组稳定性重要控制的认识，而且增强了我们对脊椎动物各自控制机制的理解。