Cdc13 and Telomerase Bind through Different Mechanisms at the Lagging- and Leading-Strand Telomeres  Virginie Faure, Stéphane Coulon, Julien Hardy, Vincent Géli  Molecular Cell  Volume 38, Issue 6, Pages 842-852 (June 2010) DOI: 10.1016/j.molcel.2010.05.016 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 38, 842-852DOI: (10.1016/j.molcel.2010.05.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 An Assay to Analyze the Association of a Telomere-End-Binding Protein to the Two Daughter Telomeres Scheme of the strategy to differentiate the leading-strand telomere from the lagging-strand telomere. Cells are grown in the presence of BrdU, an analog of thymidine for one division. Since only the parental strand for the leading-strand telomere synthesis contains adenine (C1-3A), the leading-strand telomere will be labeled with BrdU during the first cell cycle while the lagging-strand telomere will be not. Then BrdU is chased and cells are allowed to go through an additional cell cycle. The leading-strand telomere becomes the parental strand for the lagging-strand telomere synthesis. During the second cell cycle, only the lagging-strand telomere will be labeled with BrdU. Analysis of the BrdU associated with the ChIP performed with antibodies directed against a tagged telomere-end-binding protein at the two cell cycles allows for discrimination between the binding of the protein to leading or lagging telomere. In this figure, telomere-end binding proteins that bind only to the leading- or lagging-strand telomeres are represented in pale green and blue, respectively. Molecular Cell 2010 38, 842-852DOI: (10.1016/j.molcel.2010.05.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Telomeric DNA Enrichment in the Cdc13-ChIP (A) Schematic representation of the Y′ and TG1-3 probes used for direct hybridization of the unampified DNA associated to the Cdc13-ChIP. (B) FACS analysis of the first and the second cell cycle of the pp529b-CDC13-MYC strain. (C) Telomere binding of Cdc13 analyzed by ChIP and qPCR. The positions of the primers are indicated. The relative enrichment of bound telomeric DNA (TeloXI) over background (GAL2A) is represented. (D) Input DNA and Cdc13-ChIP associated DNAs were hybridized with a radioactive telomeric (TG1-3) probe (left panel) or a subtelomeric (Y′) probe (right panel). (E) Quantitation of telomeric enrichment in the Cdc13-ChIP DNA are represented. Molecular Cell 2010 38, 842-852DOI: (10.1016/j.molcel.2010.05.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Cdc13 Binds to the Leading- and Lagging-Strand Telomere (A) FACS analysis of the first and second cell cycle. (B) Telomeric DNA binding of Cdc13-myc was monitored by ChIP performed with anti-Myc antibodies on cells harvested at the indicated times during the first and second cell cycle. The relative enrichment of bound telomeric DNA (Tel XI) over background (GAL2A) is represented. Error bars are standard deviations from three independent synchronies. (C) DNAs associated to the Cdc13-ChIPs that were performed at the time points indicated by arrows in Figure 3B were used as templates to perform LM-PCR. The amplified DNAs were analyzed by ethidium bromide (EtBr) stained agarose gel and Southern blot probed, either with a TG1-3 telomeric probe or with a Y′ subtelomeric probe. (D) The DNAs associated to the Cdc13-ChIPs performed at each time point were spotted onto a nitrocellulose membrane and crosslinked by UV treatment. Immunodetection of BrdU was performed using a monoclonal antibody against BrdU as indicated in the Experimental Procedures. The signal of the dots was quantified by scanning the optic density of each dot. In the left panel, input DNAs from which the ChIPs were performed were spotted to analyze total BrdU incorporation. In the right panel, ChIPs were spotted. The graph represents the ratio ChIP/input. Molecular Cell 2010 38, 842-852DOI: (10.1016/j.molcel.2010.05.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Deleting MRE11 Reduces the Binding of Cdc13 to the Leading-Strand Telomere, but Not to the Lagging-Strand Telomere (A) Binding of Cdc13 to telomeres in mre11Δ cells was analyzed by ChIP as in Figure 3B. Error bars are standard deviations from three independent synchronies. (B) Subtelomeric contamination of the DNA associated to the Cdc13-ChIPs. The ChIP samples that were analyzed are indicated by arrows in Figure 4A. (C) Left panel, BrdU total incorporation; right panel, BrDU associated to the Cdc13-ChIP in mre11Δ cells (ChIP/input). The FACS and the spot assay are shown in Figures S2E and S2F. Molecular Cell 2010 38, 842-852DOI: (10.1016/j.molcel.2010.05.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Est1 and Est2 Telomerase Subunits Bind to Both Daughter Telomeres (A) Binding of Est1 (left panel) and Est2 (right panel) to telomeres analyzed by ChIP. Error bars are standard deviations from three independent synchronies. (B) Subtelomeric contamination of the DNA associated to the Est1- and Est2-ChIPs. (C) Left panel, BrdU total incorporation and BrdU associated to the Est1-ChIP (ChIP/input); right panel, BrdU total incorporation and BrdU associated to the Est2-ChIP (ChIP/input). The FACS and the spot assay are shown in Figure S3. Molecular Cell 2010 38, 842-852DOI: (10.1016/j.molcel.2010.05.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Deleting MRE11 Reduces the Binding of Est1 and Est2 to the Leading-Strand Telomere, but Not to the Lagging-Strand Telomere (A) Binding of Est1 (left panel) and Est2 (right panel) to telomeres analyzed by ChIP in mre11Δ cells. Error bars are standard deviations from three independent synchronies. (B) Subtelomeric contamination of the DNA associated to the Est1- and Est2-ChIPs in mre11Δ cells. (C) Left panel, BrdU total incorporation and BrdU associated to the Est1-ChIP in mre11Δ cells; right panel, BrdU total incorporation and BrdU associated to the Est2-ChIP in mre11Δ cells (ChIP/input). The FACS and the spot assay are shown in Figure S4. Molecular Cell 2010 38, 842-852DOI: (10.1016/j.molcel.2010.05.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Mre11 Binds Mainly to the Leading-Strand Telomere (A) Binding of Mre11 to telomeres analyzed by ChIP. (B) Subtelomeric contamination of the DNA associated to the Mre11-ChIPs. (C) Left panel, BrdU total incorporation; right panel, BrdU associated to the Mre11-ChIP (ChIP/input). The FACS and the spot assay are shown in Figure S5. Molecular Cell 2010 38, 842-852DOI: (10.1016/j.molcel.2010.05.016) Copyright © 2010 Elsevier Inc. Terms and Conditions