Host: The Japan Radiation Research Society
Co-host: Asian Association for Radiation Research
The Analysis of double strand break repair by chromatin immunoprecipitation in mammalian cellsMikio Shimada, Reiko Ohba and Kenshi KomatsuDepartment of Genome Repair Dynamics, Radiation Biology Center, Kyoto-University, Kyoto 606-8501, JapanRestriction enzyme, similar to ionizing radiation, induces DNA double strand breaks (DSBs) in living cells. I-Sce1 endonuclease recognizes highly specific 18-bp sequence, which is not present in mammalian cells. Since DR-GFP repoter gene contains the I-Sce1 recognition sequence, DSBs can be generated at a specific site of the reporter gene-integrated chromosome by introduction of I-Sce1 endonuclease. Althoug DSBs are rejoined by both homologous recombination (HR) and non homologous end joining (NHEJ), the latter is dominant in mammalian cells. Here we report Chromatin-immunoprecipitation (ChIP) analysis to investigate the repair process from I-Sce1-induced DSBs in mammalian cells. When Ku70 null mouse cell line, NHEJ deficient, was analyzed by ChIP, phosphorylation of H2AX was detected at distances of ~ 1kbp and ~ 4kbp from the I-Sce1- induced DSB. This ChIP analysis totally depends on rejoining rate of DSBs, since this phosphorylation was not detected in mKu70-complemented cell lines. ChIP assay has been successfully applied for analysis of the interaction between chromosome and DNA repair factors in yeast. Present results suggest that ChIP is also a useful tool for analysis of mammalian repair process when DSBs are generated by I-Sce1.
