ATR^Δ/− cells divide and then exit the cell cycle. (A) Asychronously expanding ATR^flox/− MEFs expressing EGFP–Cre are selectively lost from culture. The cells that continue to express EGFP–Cre over the course of 8 d of growth were quantitated as a percentage of their initial representation 1 d after infection with lenti-EGFP–Cre. ▪, wild type; ♦, ATR^+/−; ●, ATR^flox/+; ▴, ATR^flox/−. (B) Western blot quantitation of ATR protein levels in ATR^flox/− and ATR^+/− MEFs 60 h after NLS-Cre expression (lenti-Cre). (C) Cell cycle analysis of lenti-Cre-infected wild-type and ATR^flox/− MEFs. DNA content was determined by propidium iodide staining and flow cytometric analysis. (D) Exit of ATR^Δ/− cells from the cell cycle. Cells treated as in C are shown after 1 and 6 d of FBS stimulation. Cells were split 1:4 at day 3.

ATR loss does not eliminate the DNA replication checkpoint. (A) Both wild-type and ATR^Δ/− cells are prevented from entering mitosis by aphidicolin (Aph) treatment. Cre-expressing wild-type and ATR^flox/− cells were treated with aphidicolin upon S-phase entry following FBS stimulation (15 h post-FBS), and the percentage of cells entering mitosis was quantitated by phosphohistone H3 staining. ▪, wild-type; ♦, ATR^Δ/−; ●, wild-type + Aph; ▴, ATR^Δ/− + Aph. (B) Cyclin B1-associated H1 kinase activity in wild-type and ATR^Δ/− cells. After 15 h of FBS stimulation, Cre-expressing wild-type and ATR^flox/− cells were treated with aphidicolin (Aph), exposed to 20 Gy of γ-radiation (IR), or were left untreated (nt). Cells were then harvested 3, 6, and 9 h later, and immunoprecipitated cyclin B1-associated H1 kinase activity was assayed. Autoradiographs and PhosphorImager-quantitation (graph) of ³²P-labeled H1 following SDS-PAGE are shown. Immunoprecipitates were also Western-blotted and detected for cyclin B1 to control for equivalent cyclin B1 recovery (Western: cyclin B1). ▪, wild-type; ♦, ATR^Δ/−; ✖, wild-type + IR; ✚, ATR^Δ/− + IR; ●, wild-type + Aph; ▴, ATR^Δ/− + Aph. (C) Chromosome condensation is prevented by DNA replication inhibitors in lenti-Cre-infected wild-type and ATR^flox/− cells. Aphidicolin (5 μM) or hydroxyurea (0.5 mM) was added 16 h after FBS stimulation. Following a 2-h incubation to allow cells already in G2 and M phase at the time of treatment to pass through to G1, cells were treated with nocodazole (0.5 μM) for 5 h (18–23 and 23–28 h of FBS stimulation). Mitotic cells were quantitated by chromosome condensation (panel 1) and histone H3 phosphorylation (panel 2). Note that quantitation by chromosome condensation includes mitotic cells with normal or fragmented (PCC) chromosome structure. (D) The DNA replication checkpoint is intact in ATR^Δ/− ATM^−/− and ATR^Δ/− p53^−/− cells. Mitotic entry was analyzed by phosphohistone H3 staining cells as described in C with nocodazole treatment for 5 h, from 18 to 23 h of FBS stimulation. Error bars represent the standard deviation (S.D.) from the mean of values obtained in these experiments.

Cdc2 phosphorylation on T14 and Y15. (A) Cdc2 is phosphorylated normally as cells enter S phase. After 18 h of FBS stimulation, lysates from lenti-Cre-infected ATR^flox/− cells were generated and subjected to SDS-PAGE and Western blot detection of Cdc2. The unphosphorylated (fastest-migrating), singly phosphorylated (phospho-T14 or phospho-Y15), and doubly phosphorylated (slowest-migrating, phospho-T14 and phospho-Y15) forms of Cdc2 are indicated. (B) Accumulation of phospho-T14/Y15 Cdc2 following aphidicolin or IR does not occur in ATR^Δ/− cells. After 15 h of FBS stimulation, Cre-expressing wild-type and ATR^flox/− cells were either left untreated (nt) or treated with either aphidicolin (Aph) or 20 Gy of γ-radiation (IR). Cells were then harvested 9 and 12 h after treatment, and lysates were Western-blotted and detected for Cdc2 as in A.

Generation of a floxed conditional allele of ATR (ATR^flox). (A) Schematic representations of the targeting vector, wild-type ATR locus, recombined locus, and the final conditional allele of ATR (ATR^flox) are shown. Following confirmation by Southern blot, the ES cells with a recombined ATR allele were transfected with the pCAGGS-FLPe expression vector (Buchholz et al. 1998) to remove the selection cassette. Removal of the selection cassette results in the lox-conditional (ATR^flox) allele shown. (B) Confirmation of the ATR^flox allele by Southern blot. DNA prepared from ES cell clones was digested with BamHI + KpnI or HindIII alone and Southern-blotted for hybridization with the 5′ and 3′ probes indicated in A, respectively. DNA fragments representing wild-type, internally recombined, recombined, and ATR^flox alleles are discussed in the text and indicated in A.

ATR and ATM cooperate in checkpoint responses to γ-irradiation. (A) Serum-deprived and lenti-Cre-infected wild-type, ATR^flox/−, and ATM^−/− MEFs were γ-irradiated (20 Gy) after 17 h of FBS stimulation and then treated with 0.5 μM nocodazole for 5 h (corresponding to 18–23 h of FBS stimulation). The percentage of cells exhibiting chromosome condensation (panel 1) and phosphorylated histone H3 (panel 2) were quantitated by chromosome spread preparation and immunostaining, respectively. Differences between the total percent of cells with condensed chromatin (panel 1) versus phosphohistone H3 staining (panel 2) is consistent with the phosphorylation of histone H3 occurring prior to condensation and remaining phosphorylated throughout M phase. (B) ATR is required for late-phase mitotic delay in response to IR. Entry into mitosis (without nocodazole treatment) was quantitated by phosphohistone H3 staining before and after treatment with 20 Gy of γ-radiation at 17 h of FBS stimulation. ▪, wild-type; ♦, ATR^Δ/−; ●, wild-type + IR; ▴, ATR^Δ/− + IR. (C) ATR and ATM cooperate in the cell cycle delay elicited by IR. Mitotic entry was assessed after IR exposure as in B to compare mitotic entry of ATR^flox/− (no lenti-Cre infection), ATM^−/−, and lenti-Cre-infected ATR^flox/− and ATR^flox/− ATM^−/− (ATR^Δ/− and ATR^Δ/− ATM^−/−) MEFs. ATR^flox/− and ATR^flox/− ATM^−/− MEFs were derived from embryonic littermates. (D) ATR and ATM cooperate in p53 S18 phosphorylation. Lysates generated from lenti-Cre-infected wild-type, ATR^flox/−, and ATM^−/− cells at various times after IR treatment were Western-blotted and detected with a phospho-specific antibody to phospho-S18 p53. (E) Cells treated as described in C were analyzed for p53 S18 phosphorylation by Western blot detection as described in D. Error bars represent the standard deviation (S.D.) from the mean of values obtained in these experiments. nt, not treated with IR.

Regulation of Chk1 and Chk2 by ATR and ATM, respectively. (A) Chk1 phosphorylation in response to either aphidicolin or IR requires ATR. After 15 h of FBS stimulation, Cre-expressing wild-type and ATR^flox/− MEFs were treated with aphidicolin or IR. Lysates were prepared at various times posttreatment, and Western blots were detected for phospho-S345 Chk1 (upper panel) or total Chk1 (lower panel). (B) Chk2 phosphorylation in response to aphidicolin and IR is not dependent on ATR. Samples generated as in A were Western-blotted and detected for Chk2. The slower-migrating phosphorylated and faster-migrating unphosphorylated forms are indicated.

Aphidicolin causes DSBs in ATR-depleted cells, but DSBs do not prevent mitotic entry upon aphidicolin release. (A) Aphidicolin causes phosphorylation of H2AX in ATR^Δ/− cells. At 15 h of FBS stimulation, lenti-Cre-infected wild-type and ATR^flox/− cells were left untreated or treated with aphidicolin. Cells were harvested 1, 2, and 4 h later. Lysates were Western-blotted and detected for phosphohistone H2AX. (B) Schematic representation of an assay to quantitate the effect of DSBs on preventing mitotic entry and detect increased chromosome breakage upon aphidicolin treatment. (C) Mitotic entry of aphidicolin-treated wild-type and ATR^Δ/− cells after aphidicolin release. After a 2-h aphidicolin treatment, Cre-expressing wild-type and ATR^flox/− MEFs were washed four times with 10% FBS DMEM to remove aphidicolin. Two and a half hours later, cells were collected in mitosis by nocodazole treatment (0.5 μM) for 1.5 h. Chromosome spreads were prepared to quantitate the percentage of cells entering mitosis and to assess chromosome breakage as described in D and E below. Cells were released from or maintained in aphidicolin as indicated. (D) Mitotic spreads with differing degrees of chromosome breakage were observed and quantitated using the assay described in B and C. Examples of spreads with 1–10 breaks and >10 breaks are shown. (E) Aphidicolin induces breaks specifically in ATR^Δ/− cells. Cre-expressing wild-type and ATR^flox/− MEFs released from aphidicolin treatment as described in B and C (+Aph & Release) were analyzed for the frequency of each category of mitotic chromosome spread shown in D. The chromosome breakage observed in cells left untreated with aphidicolin is also shown (−Aph). N, normal chromosome spread. (F) Aphidicolin treatment does not cause apoptosis in ATR^Δ/− cells. Cre-expressing wild-type and ATR^flox/− MEFs left untreated, treated with aphidicolin, or treated with and released from aphidicolin were stained for Annexin V and quantitated by flow cytometry.