Nonradiolabeled Radioresistant DNA Synthesis and S-Phase Checkpoint Analysis.

DNA damage activates cell cycle checkpoints and repairs damages during cell cycle arrest. Radiation-induced S-phase arrest occurs in normal cells, but cells from ataxia telangiectasia (AT) patients present radioresistant DNA synthesis (RDS). AT-derived cells proceed DNA synthesis with DNA damage. Upon detection of radiation-induced DSBs, ATM becomes activated and initiates a signaling cascade that triggers multiple cellular responses aimed to repair the damaged DNA. One of them is the S-phase checkpoint to stop DNA synthesis. Originally, RDS was detected using pulse chase of two radiolabeled thymidines. This chapter introduces modified nonradiolabeled RDS assay using dual labeling of EdU and BrdU and detection of thymidine analogous by specific antibody and Click reaction.