Figure 4.

Cells abruptly lose the capacity for HDR of DSBs and begin to exhibit a requirement for mutagenic TLS when they pass the G2/M checkpoint. (A) Rev1 and Polζ are required for survival when damage is incurred between the G2/M checkpoint and mitotic spindle disassembly. The mean and s.d. of the experiments from Figure 3I are plotted. The median times established in Figure 2B are shown for the indicated landmarks. (B) Populations of 300 cells were imaged after exposure to 6.3 Gy of X-rays, a dose chosen to produce 1 DSB/cell on average (0.5 DSB/genome).⁶³ The probability that a genome received one or more DSBs was 0.39 at this level of exposure assuming a Poisson distribution, so 39% loss of viability was expected if each DSB were lethal. Viability of wild-type cells, calculated as in Figure 3, is plotted as a function of cell cycle stage. Two separate experiments are shown. (C) Wild-type cells exhibit the same X-ray sensitivity as cells that lack HDR when irradiated after the G2/M checkpoint. Loss of viability due to X-ray exposure was calculated by subtracting the fraction of viable cells in an X-irradiated population from that of a mock-irradiated population. Error bars show 95% c.i. The pre-G2/M checkpoint subset represents cells that underwent cleavage 1 hour or more after irradiation, and the post-G2/M checkpoint subset is cells that cleaved less than 1 h after irradiation.