Reconstitution of an ATM-dependent checkpoint that inhibits chromosomal DNA replication following DNA damage

Cell cycle checkpoints lead to the inhibition of cell cycle progression fol lowing DNA damage. A cell-free system derived from Xenopus eggs has been es tablished that reconstitutes the checkpoint pathway inhibiting DNA replicat ion initiation. DNA containing double-strand breaks inhibits replication in itiation in a dose-dependent manner. Upon checkpoint activation, a prerepli cative complex is assembled that contains ORC, Cdc6, Cdc7, and MCM proteins but lacks Cdc45. The checkpoint is ATM dependent. Cdk2/CyclinE acts downst ream of ATM and is downregulated by Cdk2 phosphorylation on tyrosine 15. Cd k2AF/CyclinE is refractory to checkpoint signaling, and Cdc25A overrides th e checkpoint and restores DNA replication. This report provides the descrip tion of a DNA damage checkpoint pathway that prevents the onset of S phase independently of the transcriptional function of p53 in a vertebrate organi sm.