Figure 3.

Effect of CTC1 disruption on G-overhang structure and telomere length. CTC1 conditional cells were treated with/without tamoxifen (TAM) for the indicated times. (A and B) Analysis of G-overhang abundance by in-gel hybridization. (A) Gel showing hybridization of TAA(C₃TA₂)₃ probe to genomic DNA under native and denaturing conditions. (B) Quantification of relative G-overhang abundance. Overhang abundance in CTC1^−/− cells was normalized to that of CTC1^F/F cells (i.e. t = 0). Mean ± S.E.M., n = 3 independent experiments. (C) Analysis of telomere length by FISH. Metaphase spreads were hybridized with (C₃TA₂)₃ telomere G-strand probe and the signal intensity was quantified. Histograms show the distribution of relative telomere lengths expressed as fluorescence intensity (TFU, telomere fluorescence unit), av.; median value, >2000 telomeres were quantified for each sample. A minimum intensity of 100 TFU was set as the cut-off. (D) Cartoon illustrating how loss of C-strand fill-in leads to shortening of lagging strand telomeres following each round of replication. (E) Analysis of telomere length by FISH as in (C) except probe was (G₃T₂A)₃. (F) Southern blot showing length of telomere restriction fragments. Exo1+, DNA was treated with Exo1 prior to restriction digestion. Probe was TAA(C₃TA₂)₃. Brackets indicate telomere that appear to have undergone elongation or shortening. Mean telomere length is indicated below each lane.