Fig 2: CPD and EXO1 Are Recruited by TRF2 to Dysfunctional Telomeres That Resemble Stalled Replication Forks(A) SMARCAL1 co-localizes with telomeres in U2OS cells expressing the indicated cDNAs. Telomere PNA-FISH (red), antibody staining (green), and DAPI (blue). Scale bar for all panels: 5 μm.(B) Co-localization of GFP-DONSON and HA-Claspin with endogenous EXO1 at telomeres. Data represent the mean of 2 independent experiments ± SD; n > 150 nuclei analyzed per experiment. *p = 0.01, **p = 0.001 by 1-way ANOVA. ns, non-significant.(C) WT GFP-DONSON or mutant DONSON interactions with V5-EXO1. Inputs represent 5% of the total cell lysate used for the IP. γ-tubulin, loading control.(D) (Top) diagram illustrating that the TRF2TRFH domain interacts with the ClaspinTBM domain in its C terminus. (Bottom) representative proteins that contain the TBM (F/Y/H-X-L-X-P) are indicated.(E) ITC measurements of the interaction between TRF2TRFH and ClaspinTBM peptides.(F) Chromosome and chromatid fusions in Apollo/SNM1B−/− MEFs treated with indicated shRNAs for 120 h. TelG-FAM, TelC-Cy3, and DAPI were used to visualize fused chromosomes (arrowheads). Representative G1/G2 chromosome fusions (white arrows), G2 chromosome fusions (orange arrows), leading-leading strand (green arrows), sister-sister telomere fusion (pink arrows), and interstitial telomeres (red arrows) are indicated. Scale bar for all panels: 5 μm.(G) Quantification of G1/G2 and G2 chromosome-type fusions in (F). Data represent the mean of 2 independent experiments ± SD from a minimum of 50 metaphases analyzed per experiment.(H) Quantification of the chromatid-type and sister fusions in (F). Data represent the mean of 2 independent experiments ± SD from a minimum of 50 metaphases analyzed per experiment.

Fig 3: Recruitment of CPD to Telomeres Lacking TRF2 and POT1a/b-TPP1(A) Co-localization of p-RPA32, RAD51, and Flag-ATRIP with telomeres (arrowheads) in Nbs1+/+, Nbs1−/− MEFs, or NBS-ILB1 cells. Telomeres were visualized by peptide nucleic acid-fluorescent in situ hybridization (PNA-FISH) (red), indicated proteins (green), and nuclei (blue). Scale bar for all panels: 5 μm.(B) Percentage of cells containing ≥5 TIFs in (A). Data are the mean of 2 independent experiments ± SD; n > 200 nuclei analyzed per experiment. ****p < 0.0001 by 1-way ANOVA. ns, non-significant.(C) Co-IP of HA-Claspin with GFP-DONSON or Myc-PCNA in 293T cells. Input, 5% of the total cell lysate. γ-Tubulin, loading control.(D–F) Localization of Claspin (D), PCNA (E), and DONSON (F) in Nbs1−/− MEFs expressing indicated DNAs. Endogenous DONSON TIFs were detectable only in HA-claspin-expressing cells. Telomeres were visualized by PNA-FISH (red), and endogenous CPD was visualized with the indicated antibodies (green). Scale bar for all panels: 5 μm.(G) Percentage of cells containing ≥5 CPD+ foci co-localizing with telomeres, detected in (D)–(F).(H) Co-expression of HA-Claspin with GFP-DONSON (top panel) or HA-Claspin, Myc-PCNA, and GFP-DONSON (bottom panel) in U2OS cells. Top panel: GFP-DONSON (green) and HA-Claspin(red). Bottom panel: Myc-PCNA (red) and GFP-DONSON (green). For bothpanels, anti-TRF2 antibody visualized telomeres (white).(I) HA-Claspin and GFP-DONSON co-localized with BrdU+ foci in U2OS cells. GFP-DONSON (green), BrdU (red), and DAPI (blue). Scale bar for all panels: 5 μm.