1. The DNA Damage Machinery and Homologous Recombination Pathway Act Consecutively to Protect Human Telomeres 
Ramiro E. Verdun, Jan Karlseder 
Cell 
Volume 127, Issue 4, Pages (November 2006)
DOI: /j.cell
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 Telomeres Incorporate BrdU in a Two-Peak Pattern
(A) FACS analysis of IMR90 cells released from a G1/S block. Time after release and approximate cell-cycle phases are indicated.
(B) Schematic of BrdU-ChIP experiments. Synchronized IMR90 fibroblasts were released, and BrdU was added to the medium 1 hr prior to harvest.
(C) ChIP experiments on synchronized IMR90 cells incubated with BrdU as described in (A). Precipitations were performed with antibodies against TRF1 and TRF2. The precipitated DNA was analyzed with anti-BrdU antibodies. The graph represents the densitometric evaluation of three independent experiments, with error bars indicating the standard deviation. The total DNA represents 0.1% of genomic DNA.
(D) Telomeric ChIP of the precipitated DNA.
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3. Figure 2
Proteins Involved in DNA Replication and Repair Are Recruited to the Telomeres
(A) Protein extracts from synchronized IMR90 cells were subjected to ChIP experiments using indicated antibodies. IgG antibodies were used as negative control. An indicated amount of total DNA (input) was subjected to Southern blot analysis 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. Three independent experiments were evaluated, and error bars indicate the standard deviation.
(B) As in (A), using antibodies against proteins in the replication machinery. Three independent experiments were evaluated, and error bars indicate the standard deviation.
Cell  , DOI: ( /j.cell )
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4. Figure 3 DNA Damage Proteins Are Recruited to the Telomeres
(A) Protein extracts from synchronized IMR90 cells were subjected to ChIP experiments using the indicated antibodies. IgG antibodies were used as negative control. An indicated amount of total input DNA was subjected to Southern blot analysis 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. Three independent experiments were evaluated and error bars indicate the standard deviation.
(B) As in (A), using antibodies against proteins involved in homologous recombination. Three independent experiments were evaluated, and error bars indicate the standard deviation.
Cell  , DOI: ( /j.cell )
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5. Figure 4
Homologous Recombination Can Generate a D Loop Structure with Telomeric Sequences In Vitro
(A) Schematics suggesting structural similarities between Holliday junction intermediates and telomeric D loops.
(B) Schematics showing the steps of the D loop in vitro assay. The length of the double- and single-stranded telomeric probe is indicated. Only the telomeric probe was radioactively labeled (marked with a star).
(C) The D loop in vitro assay was performed with different probes and reaction conditions as indicated. 3′, telomeric probe with a unique 3′ G-rich telomeric overhang. 5′, telomeric probe with a 5′ C-rich telomeric overhang. B, telomeric probe with both ends blunt. 3′nt, probe with double-stranded telomeric region as in (3′), but with a 3′ nontelomeric overhang. 70°C, nuclear extract preheated to 70°C for 5 min before reaction. The signals corresponding to the D loop formation or free probe are indicated on the left. The EtBr panel shows the upper region of the gel prior to exposure to evaluate plasmid loading and integrity. The asterisks mark the new plasmid form observed after D loop formation.
(D, E, and F) D loop assays were performed with nuclear extracts (IMR90 cells) previously depleted of the indicated proteins. The signals corresponding to the loop or free probe are indicated at the left.
Cell  , DOI: ( /j.cell )
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6. Figure 5
D Loop Formation Is HR Dependent in Cells with or without Telomerase
(A) Three independent assays performed with nuclear extracts from IMR90 cells at the indicated cell-cycle phases. The quantification is provided, and error bars indicate the standard deviation.
(B) D loop assays with nuclear extracts from the indicated cell lines. The nuclear extracts were depleted of the indicated proteins using specific antibodies prior to the reaction. IgG antibodies were used as a negative control. The signals corresponding to the D loop or free probe are indicated on the left. The quantification represents three independent experiments, and the error bars indicate the standard deviation.
Cell  , DOI: ( /j.cell )
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7. Figure 6
Proposed Model for Telomere Processing during and after Replication in Human Primary Cells
Step-by-step model of telomere end processing during and after replication. Phase I represents the restart of replication after stalling at the telomere. Phase II represent a DSB-like repair/processing by HR after replication of telomere ends.
Cell  , DOI: ( /j.cell )
Copyright © 2006 Elsevier Inc. Terms and Conditions