FIG. 5.
Influence of DNA-PKcs on checkpoint induction. (A) The intra-S-phase checkpoint defect of Nbs1ΔB/ΔB mutants was indistinguishable from that of Nbs1ΔB/ΔB Prkdcscid/scid double mutants. Prkdcscid/scid MEFs showed significantly stronger arrest than WT MEFs (Wilcoxon rank sum test; at 10 Gy, P = 3.18e−2 [two sided], and at 20 Gy, P = 2.98e−5 [two sided]). Atm−/− cultures are shown for comparison. (B) G1/S checkpoint responses in early-passage MEFs. Cultures were irradiated with 5 or 10 Gy, and DNA synthesis levels were normalized to those in untreated cultures. Both Prkdcscid/scid and Nbs1ΔB/ΔB Prkdcscid/scid MEFs showed stronger G1/S checkpoint arrest than WT MEFs. Representative flow cytometry data are included in Fig. S5 in the supplemental material. (C) Deficient G2/M checkpoint arrest in Nbs1ΔB/ΔB mutants was rescued by the Prkdcscid/scid mutation. MEFs of the indicated genotypes were mock treated, incubated with 10 mM caffeine, exposed to 10 Gy of IR, or exposed to 10 Gy IR and treated with 10 mM caffeine, and their mitotic indices were determined 1 h posttreatment. Treatment with caffeine abolished the checkpoint response in all backgrounds. Representative flow cytometry data are included in Fig. S6 in the supplemental material.