Genetic and Molecular Analysis of DNA Damage induced Apoptosis and Cell Cycle Arrest in C. elegans

响应于遗传毒性损伤，细胞激活DNA损伤检查点。检查点信号传导导致有效修复DNA损伤所需的短暂细胞周期停滞，或受影响细胞的程序性细胞死亡（凋亡），这可能对整个生物体有害。我们仍然对DNA损伤的感知和程序性细胞死亡机制的激活之间的分子联系知之甚少。为了阐明DNA损伤诱导细胞凋亡的分子基础，我们建议使用C。elegans作为一个模型系统来追求向前的遗传策略。我们发现，在DNA损伤C。线虫生殖细胞可以经历细胞周期停滞以及凋亡。我们已经隔离了三个C区。elegans突变体在DNA损伤反应中有缺陷，其中之一mrt 2影响C. elegans同源的S.酿酒酵母外切核酸酶rad 17。为了鉴定和表征参与检查点信号传导的新基因，我们将（任务-1）克隆和表征C。线虫rad-5检查点基因。此外（任务2），我们将进行大规模的遗传筛选，以筛选对辐射诱导的细胞死亡具有抗性的突变体。利用这些突变体，我们建议克隆两个新的检查点基因。此外（任务-3），我们将分类C。elegans检查点突变体，并计划通过上位性实验将其置于遗传途径中。为了支持这些遗传上位性实验，我们计划开发指示DNA损伤依赖性检查点信号传导的细胞学和生化标记物。

In response to genotoxic insults cells activate DNA damage checkpoints. Checkpoint signaling leads to a transient cell-cycle arrest needed to efficiently repair DNA lesions, or to programmed cell death (apoptosis) of affected cells that might potentially become harmful for the entire organism. We still know relatively little about the molecular connection between the sensing of DNA damage and the activation of the programmed cell death machinery. To address the molecular basis of DNA damage induced apoptosis we propose to use C. elegans as a model system to pursue a forward genetic strategy. We found that upon DNA damage C. elegans germ cells can undergo cell cycle arrest as well as apoptosis. We already isolated three C. elegans mutants defective in DNA damage responses one of which, mrt2, affects the C. elegans homolog of the S. cerevisiae exonuclease rad17. To identify and characterize novel genes involved in checkpoint signaling we will (task-1) clone and characterize the C. elegans rad-5 checkpoint gene. Furthermore (task-2), we will perform a large-scale genetic screen for mutants that are resistant to radiation-induced cell death. Taking advantage of those mutants, we propose to clone two novel checkpoint genes. In addition (task-3), we will classify C. elegans checkpoint mutants according to their phenotypes and plan to place them into genetic pathways by epistasis experiments. To support these genetic epistasis experiments we are planning to develop cytological and biochemical markers indicative of DNA damage-dependent checkpoint signaling.