Absence of an immediate G1/S checkpoint in primary MEFs following γ-irradiation identifies a novel checkpoint switch

Abstract

DNA double-strand breaks caused by ionizing radiation have been shown to induce G1/S, intra-S-phase, and G2/M cell cycle checkpoints. However, analysis of the immediate induction of G1/S checkpoint at a cellular level has been hampered by the inability to distinguish cells that were already replicating DNA at the time of damage from cells that entered S phase following the DNA damage. We have developed a novel strategy for assessing the initiation of the G1/S checkpoint following γ-irradiation within asynchronous, low passage, primary mouse embryonic fibroblast cultures (MEFs) using a staggered CldU/IdU double-labeling protocol. Contrary to the current model of the G1/S checkpoint, we found that 65% of late- G1 primary MEFs still proceeded into S phase after a g-irradiation dose of 5 Gy. The delayed p53-dependent G1/S checkpoint was intact in these cells, and a G2/M checkpoint that was over 90% effective was induced within one hour and maintained through six hours post-irradiation. Furthermore, these cells also exhibited an intra-S-phase replication slow-down, as there was a decrease in the S/ G2 transition frequency of primary MEFs following g-irradiation. The absence of an immediate G1/S checkpoint in primary MEFs suggests that in late G1 these cells may predominantly respond to DNA damage at the level of individual replication origins, rather than by inducing a complete shut-down of S-phase entry. ©2006 Landes Bioscience.