Assaying for Radioresistant DNA Synthesis, the Hallmark Feature of the Intra-S-Phase Checkpoint Using a DNA Fiber Technique.

During S-phase the cell replicates its DNA which is critical to maintaining the integrity of the genome and cell survival amidst damaging events. The cell is equipped with a series of checkpoints to slow progress throughout the cycle and facilitate DNA repair. Ataxia telangiectasia mutated (ATM), defective in the human genetic disorder ataxia-telangiectasia (A-T), is the key to initiating a signaling cascade activating the intra-S-phase checkpoint. This was first identified in A-T cells as radioresistant DNA synthesis using 14 C thymidine and 3 H thymidine to pulse label replicating cells before and after damage. This technique has been superseded now by direct labeling that distinguishes DNA replication initiations from ongoing sites of replication which are the target for the intra-S-phase checkpoint. Here, we outline how sites of replication are pulse labeled with two different thymidine analogs before and after damage. The DNA is then stretched out as fibers for immunolabeling to enable visual distinction and counting of ongoing replication forks from new initiations. It is this extent of new initiations that is used to detect the intra-S-phase checkpoint after DNA damage.