Analysis of RB Action in DNA Damage Checkpoint Response

Cell cycle checkpoints play a key role in maintaining genome stability by monitoring the order and integrity of cell division events. Checkpoints induced by DNA damage function to limit the propagation of potentially deleterious mutations. The retinoblastoma tumor suppressor (RB) is a critical effector of DNA damage checkpoint function by eliciting G1-phase cell cycle arrest following genotoxic stress. Here, we describe methodologies for evaluation of three facets of RB action in the DNA damage checkpoint response: (1) transcriptional repression of E2F-regulated genes (cyclin A reporter assay); (2) induction of cell cycle arrest (Brd-U incorporation assay); and (3) inhibition of DNA double-strand break accumulation (phosphorylated-histone H2A.X detection). Together, this combination of techniques allows the evaluation of RB action in the coordinated checkpoint response to DNA damage.