(A) Schematic of the TRF2^ΔRap1 mutant. H, predicted helix. (B) Co-IP of Myc-TRF2 or Myc-TRF2^ΔRap1 with FLAG-tagged Rap1 or Apollo from co-transfected 293T cells. In, 2.5% of input. (C) Immunoblots for TRF2 and Rap1 from TRF2^F/-p53^-/- MEFs expressing TRF2, TRF2^ΔRap1, or vector control, 72 and 96 hours after H&R-Cre. (D) IF-FISH to monitor TRF2, Rap1, and TIN2 at telomeres in TRF2^F/-p53^-/- MEFs expressing TRF2^ΔRap1 or vector control at day 4 after Cre. (E) Telomeric ChIP of TRF2^F/-p53^-/- MEFs expressing TRF2 or TRF2^ΔRap1 at day 7 post Cre. Duplicate dot blots were probed for telomeric DNA or the dispersed BamHI repeats. ChIP ratios represent the % telomeric DNA recovered in TRF2^ΔRap1 vs. TRF2 expressing cells calculated as in Fig. 1.

(A) Rap1 and TRF2 from TRF2^F/-Ku70^-/- MEFs expressing TRF2, TRF2^ΔRap1, or vector control analyzed 4 days after Cre. (B) CO-FISH analysis on cells as in (A). Arrows: T-SCEs. (C) Enlarged T-SCE events in Cre-treated TRF2^F/-Ku70^-/- MEFs expressing TRF2^ΔRap1. (D) Quantification of T-SCEs as assessed in (B). Bars represent averages from three independent experiments (n>1100 chromosome ends each) and SDs. P values based on Student’s two-tailed t-test. (E) Quantification of T-SCEs as assessed in (B) in cells of the indicated Rap1 and Ku70 status. Method as in (D). Errors bars: SEMs except for SDs for Rap1^F/FKu70^-/-. (F) The functions of shelterin components. See text for details.

(A) Schematic of Rap1 (top), and maps of the mouse Rap1 (Terf2ip) locus, the targeting construct, the floxed allele, and the Δex2 allele. N, NdeI; B, BamHI; F1, F2, and R, PCR primers. Rap1 shRNAs are indicated at the bottom. At right, the Rap1^_Δex2 encoded protein. (B) Genomic blot of NdeI-digested DNA from wild type and targeted ES cells. Probe as in (A). (C) PCR genotyping of tail DNAs. Primers in (A). (D) Immunoblots for Rap1 (Ab1252), TRF2 (Ab1254), and TRF1 (Ab1449) from Rap1^F/F and Rap1^F/+ MEFs five days after Cre, First and second +Cre lanes, H&R-Cre and pWZL-Cre, respectively. (E) Loss of Rap1 IF signal from Rap1^F/F MEFs at day 5 after Cre. Red, Rap1; green, telomeric FISH; blue, DNA (DAPI). (F) Telomeric ChIPs on Rap1^F/F MEFs at day 5 after Cre. Numbers represent ratios of % telomeric DNA in the ChIPs (corrected for pre-immune (PI) signal) on cells + and -Cre. (G) Proliferation of SV40LT-immortalized Rap1^F/F and Rap1^F/+ MEFs infected with pWZL-Cre or vector. (H) Offspring from Rap1^Δex2/+ and Rap1 ^Δex2/Δex2 intercrosses.

(A) TIF assay on Rap1^F/F MEFs treated with Cre and the indicated shRNA. Red, IF for 53BP1; green, telomeric FISH; blue, DNA (DAPI). (B) TIF assay quantification. Averages of two independent experiments (n≥100 nuclei each) and SEMs. (C) Chk2-P in Rap1-deficient MEFs. TRF2 null cells and IR-treated cells (1 hr post 2 Gy,) serve as positive controls. (D) Quantification of TIF assays on TRF2^F/-p53^-/- cells expressing TRF2, TRF2^ΔRap1 or vector control at day 4 post Cre. Mean of three independent experiments (n≥100 nuclei each) and SDs. (E) Chk1 and Chk2 phosphorylation in TRF2^F/-p53^-/- MEFs expressing TRF2, TRF2^ΔRap1 or vector control. UV (1 hr post 25 J/m2) and IR (1 hr post 2 Gy) treated cells serve as positive controls. (F) Metaphase chromosomes from Rap1^F/F cells 5 days post Cre. Red, DNA (DAPI); green, telomeric FISH. (G) Quantification of telomere fusions, detected as in (F) in Rap1^F/F MEFs with the indicated Cre and shRNA treatments. Average (av) % of telomeres fused is given. (H) Quantification of telomere fusions in TRF2^F/-p53^-/- MEFs (+ or − Cre, day 4) complemented with TRF2 or TRF2^ΔRap1 or vector control and treated with TPP1 shRNA as indicated.