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

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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.