Figure 1. Loss of Chk2 does not exacerbate the checkpoint defects, damage sensitivity, or chromosome instability conferred by Mre11 complex hypomorphism.

(A) Diagram of the hypomorphic Mre11^ATLD1 and Nbs1^ΔB alleles (reviewed in Stracker et al., 2004).

(B) Intra-S phase checkpoint response of WT (◇), Chk2^−/− (◆), Nbs1^ΔB/ΔB (□), Nbs1^ΔB/ΔBChk2^−/− (■), Mre11^ATLD1/ATLD1 (○), Mre11^ATLD1/ATLD1Chk2^−/− (●) and Atm^−/− (△) fibroblasts. The DNA synthesis ratios (untreated and IR treated cultures, normalized to untreated samples) are plotted. Wedge denotes increasing IR dose (0, 10, or 20 Gy) and horizontal bars indicate the average value of replicate experiments. Chk2^−/− cultures are not significantly different than WT (P=0.33 at 10 and 0.82 at 20 Gy, WT vs. Chk2^−/−) and loss of Chk2 does not exacerbate the defect of Mre11^ATLD1/ATLD1 (P=0.88 at 10 and 0.55 at 20 Gy, Mre11^ATLD1/ATLD1 vs. Mre11^ATLD1/ATLD1Chk2^−/−). However, Nbs1^ΔB/ΔBChk2^−/− cultures showed increased RDS compared to Nbs1^ΔB/ΔB (P=1.5×10⁻³ at 10 and 2.5×10⁻³ at 20 Gy, Nbs1^ΔB/ΔB vs. Nbs1^ΔB/ΔBChk2^−/−). Wilcoxon rank sum test, 2-sided.

(C) G2/M checkpoint responses of exponentially growing MEFs. The mitotic ratios (mock or IR treated normalized to mock treated) of mock (white) or 10 Gy IR (black) treated cells at 1 hr post treatment are presented. Results are the average of 3–6 experiments performed in triplicate for each genotype. Error bars denote standard deviation.

(D) G1/S checkpoint in early passage MEF cultures. The S-phase ratios (%BrdU positive of irradiated or unirradiated/ average %BrdU positive unirradiated) are plotted. Results are the average of 2–4 experiments performed in triplicate for each genotype. Error bars denote standard deviation.

(E) IR sensitivity determined by clonogenic survival assay. WT (◇), Chk2−/− (▲), Mre11^ATLD1/ATLD1 (○), and Mre11^ATLD1/ATLD1Chk2^−/− (●) SV40 transformed MEF cultures were exposed to IR at the indicated doses and plated for colony formation. Results from 2 representative experiments performed in triplicate are shown. Error bars denote standard deviation.

(F) Genomic instability in mock and irradiated primary proliferating splenocytes. Examples of metaphases from IR treated Mre11^ATLD1/ATLD1 Chk2^−/− spreads are shown: 2 chromatid breaks (top) and a rearrangement (bottom) are indicated. The number of spreads analyzed and aberrations identified are described in Supplementary Table S1.