Generation of a conditional RAD17 allele in human somatic cells. (A) Targeting strategy for establishing a conditional RAD17 allele. Construct 1 contains a duplicated exon 5 as well as an exon 5 disrupted in-frame with the complete coding sequence of the neomycin resistance (Neo) gene and polyadenylation sequence (black squares). Three lox sites (triangles) were positioned as indicated. Following targeting with Construct 1, exon 5 with the Neo insertion was removed by selective Cre incision between the first two lox sites to generate the Conditional allele and allow recycle of the Neo marker. A second round of targeting with Construct 2 generated the Inactivated allele. (B) Southern blot analysis of genomic DNA isolated from the indicated cell lines after digestion with SpeI. +/+, RAD17 wild-type HCT116 cells by the presence of wild-type alleles (8.1 kb); flox/+, cells that had been successfully targeted with Construct 1, showing a wild-type band and band 1 (9.6 kb); flox/−, cells with the Inactivated allele (band 3, 8.5 kb) and the Conditional allele (band 3, 7.3 kb). A SpeI site immediately downstream of the last lox site in both Construct 1 and 2 reduced the length of the conditional allele band (band 3) by 0.8 kb, compared with the wild-type band. (C) Immunoblot analysis of RAD17^flox/− cells 3 d after Ad–Cre and Ad–GFP infection, respectively. The loss of the Rad17 protein was nearly complete whereas Rad9 levels remained constant in both cells.

Defects in DNA damage-induced checkpoint activation in RAD17^flox/− mutant cells. Wild-type (RAD17^+/+) and mutant (RAD17^flox/−) cells were exposed to γ-radiation (20 G) or UV (50 J/m²) 3.5 d after Ad–Cre infection. Cells were harvested for protein extract preparation 2 h after treatment. The numbers 43 and 10 designate two independent RAD17^flox/− clones—KO43 and KO10, respectively. (A) Rad17 levels in each mutant at the time of protein harvest. (B) Immunoblot with a phospho-Ser 345-specific Chk1 antibody. (C) Immunoblot analysis with a phospho-Thr 68-specific Chk2 antibody. (D) Immunoblot with a phospho-Ser 966-specific Smc1 antibody. (E) Immunoblot with an anti-Nbs1 antibody, showing unaltered Nbs1 phosphorylation pattern on DNA damage. (F) β-actin as loading control.

DNA damage-induced mitotic entry delay is impaired in RAD17^flox/− mutant cells. (A) Mitotic entry checkpoint in RAD17^flox/− cells. RAD17^+/+, RAD17^flox/−, and ATR^flox/− cells were infected with Ad–Cre for 2.5 d and exposed to IR (10 G). Nocodazole (1 μg/mL) was added to the medium at the time of IR treatment to retain cells that have entered into mitosis. Sixteen hours later, cells were harvested for propidium iodide staining and immunostaining with an antibody to phospho-histone H3, a mitotic indicator, followed by FITC-conjugated secondary antibody. Flow cytometry was performed to determine DNA contents as well as the percentage of phospho-histone H3-positive cells. (B) Comparison of cell cycle distributions on DNA damage (10 Gy) in RAD17^+/+, RAD17^flox/−, and ATR^flox/− cells. Cell cycle distribution was derived from flow cytometry analysis in A. The percentage of G2 cells was derived by subtracting the percentage of cells in mitosis from G2–M as determined by flow cytometry. (C) Samples prepared similarly as in A were analyzed by a laser scanning cytometer to determine phospho-histone H3-positive cells. Decrease in the mitotic cell fraction, induced by IR treatment, was expressed by the ratio of phospho-histone H3-positive cells between IR-treated and mock treated cells with each genotype. At least 5000 cells were scanned from each sample slide. (D) Mitotic index analysis by Giemsa staining after the indicated cells were treated with γ-radiation (10 G). Mitotic population is indicated as a percentage of total cell number. Each experiment was performed multiple times and representative results are shown.

Chromosomal aberrations in RAD17^flox/− cells. Metaphase chromosome spread of cells with indicated genotype 5 d after Ad–Cre infection. (A) Normal diploid metaphase spread found in RAD17^+/+ cells. (B) Normal diploid metaphase spread found in RAD17^flox/+ cells. (C) Chromosomal abnormalities in RAD17^flox/− cells. Arrows point to gapped or broken chromosomes. (D) Metaphase chromosome spread from RAD17^flox/− cells with partially endoreduplicated chromosomes. (E) Flow cytometry analysis of RAD17^+/+ and RAD17^flox/− cells. Cells were infected with Ad–Cre for 3 d and subsequently subjected to 48 h of nocodazole block (200 ng/mL). The DNA contents of cells were determined by propidium iodide staining. The percentage of cells with hyperdiploid DNA content in the RAD17^flox/− mutant was consistently higher than that of the RAD17^+/+ cells. (F) Accumulation of chromosome breaks in RAD17 and ATR mutants. Cells with indicated genotypes were infected by Ad–Cre (day 0) and metaphase chromosome spreads were prepared at 3, 4, and 5 d post infection. Breaks per cell were derived from analysis of 100 metaphase spreads for each sample point.