Abstract
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.
