Functional Human Telomeres Are Recognized as DNA Damage in G2 of the Cell Cycle  Ramiro E. Verdun, Laure Crabbe, Candy Haggblom, Jan Karlseder  Molecular Cell  Volume 20, Issue 4, Pages 551-561 (November 2005) DOI: 10.1016/j.molcel.2005.09.024 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Proteins Involved in the DNA Damage Response Are Recruited to the Telomeres during G2 (A) Protein extracts from synchronized IMR90 cells were subjected to chromatin immunoprecipitation (ChIP) experiments with the indicated antibodies. Indicated amounts of total input DNA were subjected to Southern blot analysis by using telomeric or ALU repeat-specific probes. The signals obtained were quantified by densitometry, and the percentage of precipitated DNA was calculated as a ratio of input signals and plotted. Five independent experiments were evaluated. (B) The same extracts were used for Western analysis, and each lane represents the protein of 1 × 105 cells. The graphs for ChIP (A) and Western analysis (B) represent a minimum of three independent experiments. The cell cycle profile of the time points was determined by FACS analysis, and cell cycle phase as well as hours after release are indicated. Error bars indicate the SD. Molecular Cell 2005 20, 551-561DOI: (10.1016/j.molcel.2005.09.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Chromosome Ends Are Accessible in G2 (A) Schematic representation of the products visualized by the TdT-FITC assay. (B) Simultaneous visualization of TTAGGG repeats (red channel) with TdT-FITC signal (green channel) in synchronized IMR90 cells 10 hr after release from a G1/S block. The images for both channels were deconvoluted and merged to evaluate colocalization. (C) Quantification of the colocalization of TTAGGG repeats with TdT-FITC signals during the cell cycle. (D) Quantification of TdT-FITC-positive cells during the cell cycle. Cell cycle distribution and hours after release are indicated. (E) Quantification of TdT-positive telomeres in individual cells. Cell cycle distribution and hours after release are indicated. TdT positivity is given as a percentage of the total number of telomeres detected by TTAGGG in situ hybridization. In (C)–(E), 100 nuclei per time point were evaluated, and error bars indicate the SD of three independent experiments. Molecular Cell 2005 20, 551-561DOI: (10.1016/j.molcel.2005.09.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Telomeres Are Recognized as DSBs during G2 Phase (A) Protein extracts from synchronized IMR90 cells were subjected to ChIP assays by using antibodies against the indicated proteins. IgG antibodies were used as negative control. Precipitates were analyzed for the presence of TTAGGG repeats and ALU repeats by Southern analysis. The signals obtained were quantified by densitometry, and the percentage of precipitated DNA was calculated by comparison to input signals. The same extracts were used for Western analysis of the proteins indicated, and protein from 1 × 105 cells was loaded per lane. (B) Quantification of ChIP and Western analysis. Cell cycle phases and hours after release are indicated. Caffeine was added 3 hr after release from the aphidicolin block where indicated. Error bars indicate the SD between three independent experiments. (C) IMR90 cells in G2 were stained for S1981-ATM (red channel) and TdT-FITC or TTAGGG repeats (green channel), respectively. The signals were merged where indicated, and colocalization of the merged signals with DAPI stained DNA (blue channel) is shown. (D) Western analysis of protein extracts during the cell cycle with antibodies against indicated proteins. Protein from 1 × 105 cells was harvested at the indicated time points after G1/S release. Cells were exposed to 25 J/m2 of UVC where indicated. Molecular Cell 2005 20, 551-561DOI: (10.1016/j.molcel.2005.09.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 DNA Damage Proteins Activities Are Required for Telomere Function (A) Western and FACS analysis of control IMR90 cells and cells expressing adenovirus proteins E1b55k, E4orf6, or both with anti Mre11 antibodies. Equal cell numbers were loaded in each lane, and hours after infection are indicated. (B) Quantification of TdT-FITC-positive cells in unsynchronized IMR90 cells infected with adenoviral proteins as in (A). The immunofluorescence of an IMR90 cell with the telomeric DNA (red channel) and TdT-FITC signal (green channel) 24 hr after the infection with E1b55k and E4orf6 expressing adenovirus is shown. Total DNA was stained with DAPI. The bar graphs represent the TTAGGG and TdT-FITC colocalization results from the analysis of 50 nuclei per assay in three independent experiments involving IMR90 cells expressing E1b55k/E4orf6 or transfected with siRNAs targeting MRE11. (C) The abundance of total P53 protein was analyzed by Western analysis in IMR90 cells infected with a control retrovirus or a virus expressing HPV16 E6-E7 oncoproteins. Cells were exposed to UV (25 J/cm2), and p53 was detected with an anti p53 antibody 24 hr after damage. (D) Telomeric FISH on metaphase spreads of IMR90 cells infected with HPV16 E6-E7. Cells were exposed to 10 mM caffeine for 16 hr where indicated. The white arrows show a chromosome fusion with telomeric DNA at the fusion site. The bar graph represents the quantification of cells with telomeric fusions, and the number of telomere fusions per cell, observed after the analysis of 50 nuclei in three independent experiments. (E) Bar graph of the quantification of ATM-deficient cells with telomeric fusions. Caffeine was added, and E6 and E7 were expressed in the cells where indicated. Fifty metaphases were analyzed for each set of cells. Fifty metaphases were analyzed for each set of cells in three independent experiments. In (B) and (E), error bars indicate the SD. Molecular Cell 2005 20, 551-561DOI: (10.1016/j.molcel.2005.09.024) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Telomeres Are Processed Similar to DSBs Step-by-step model of a comparison of DSB repair and telomere processing. Molecular Cell 2005 20, 551-561DOI: (10.1016/j.molcel.2005.09.024) Copyright © 2005 Elsevier Inc. Terms and Conditions