Investigation of DNA Repair Pathway Activity

DNA is constantly being damaged from endogenous and exogenous sources and efficient repair of different types of DNA lesions is essential for the survival of the organism. Dictyostelium is highly resistant to DNA damage and its genome sequence has revealed the presence of multiple repair pathways conserved with vertebrates but lost in other genetically tractable invertebrate models. As such, Dictyostelium is a powerful model organism to study selected human DNA repair pathways and may provide insights into the molecular basis of how cells become resistant to DNA damage. Here we describe a range of assays used to study DNA repair in Dictyostelium . Genes required for repair of DNA damage can be identified and analyzed by comparing the ability of control or mutant cells to survive exposure to genotoxic agents that induce different types of DNA lesion. We also describe assays that assess the presence of markers for DNA repair within chromatin either in the form of posttranslational modification of proteins at sites of damage or the recruitment of repair factors to DNA lesions. Finally, we also describe more direct assays to assess repair of DNA double-strand breaks by either homologous recombination or non-homologous end joining.