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Absence of an Immediate G1/S Checkpoint in Primary MEFs Following γ-irradiation Identifies a Novel Checkpoint Switch

Kendra L. Cann and Geoffrey G. Hicks

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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-labelling protocol. Contrary to the current model of the G1/S checkpoint, we found that 65% of late-G1 primary MEFs still proceed into S phase after a γ-irradiation dose of 5 Gy. The delayed p53-dependent G1/S checkpoint is intact in these cells, and a G2/M checkpoint that over 90% effective was induced within 1 h and maintained through 6 h post-irradiation. Furthermore, these cells also exhibited an intra-S-phase replication slow-down, as there is a decrease in the S/G2 transition frequency of primary MEFs following ?-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 Sphase entry.

This is an open-access article

 Download PDF

If the document does not open, please right-click on the link (control-click on a Macintosh) and select the option to save the file to disk.