Analyzing cell cycle checkpoints in response to ionizing radiation in mammalian cells

Exposure of cells to DNA-damaging agents, such as ionizing radiation (IR), results in perturbation of cell cycle progression. IR activates cell cycle checkpoints that arrest the cell cycle at the G1/S, S, and G2/M phases. The DNA damage-signaling network involves a number of important DNA damage response factors that are required for maintaining genome stability and prevention of cancer. These factors are involved in the regulation of cell cycle checkpoints and include ATM, NBS1, BRCA1, Chk2, and p 53. Here we describe a series of assays that are often used to analyze cell cycle checkpoints after IR. These assays include a G1/S checkpoint assay that measures 5-bromodeoxyuridine incorporation into DNA, an S-phase checkpoint assay that measures DNA synthesis at a very early time point after IR, and a G2/M checkpoint assay that quantitates histone H3 phosphorylation. This collection of assays allows us to investigate the specifi c functions of proteins involved in regulating different cell cycle checkpoints in mammalian cells as a response to IR.