Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

Jacob Peter Matson; Amy M. House; Gavin D. Grant; Huaitong Wu; Joanna Perez; Jeanette Gowen Cook

Cells typically load extra MCM in G1 to license dormant origins so they can tolerate replication stress in S phase (Woodward et al., 2006; Ge et al., 2007; Ibarra et al., 2008) . Given our observations that the first S phase is underlicensed relative to the subsequent S phase, we hypothesized that cells re-entering the cell cycle from G0 would be hypersensitive to replication stress in the first S phase. To test that idea directly, we treated cells in the first or second cell cycle after G0 with low-dose gemcitabine, a drug that depletes nucleotides to cause replication stress ( Fig. 2A) . We used flow cytometry to measure the expression of γH2AX, a common replication stress marker (Ewald et al., 2007) (Fig. 2B, and Fig. S2A ). We specifically analyzed mid-S phase cells to account for differences in cell cycle distribution, and we scored the number of γH2AX-positive S phase cells with expression equal to or greater than the top 5% of untreated cells (Fig. 2C , dashed line). By this measurement, cells in the first S phase after G0 are significantly more sensitive to replication stress than cells in the second S phase (Fig. 2D) . Moreover, gemcitabine-treated first S phase cells expressed double the amount of γH2AX per cell than cells in the second S phase, suggesting that not only were more total cells exhibiting a replication stress response but also that there was more replication stress per cell (Fig. 2E, F) . This hypersensitivity to replication stress in the underlicensed first cell cycle suggests that cell cycle re-entry is an inherently higher-risk cycle with respect to genome stability compared to subsequent cell cycles.