Journal: Oncotarget

Article Title: Optogenetic regulation of site-specific subtelomeric DNA-methylation

doi: 10.18632/oncotarget.10394

Figure Lengend Snippet: a. Schematic of the HALO tagged TCON and ECON fusion proteins, b. Western blot analysis demonstrates production of intact TCON (~149 kD; lane-1), ECON (~223 kD; lane-2), and TCON plus ECON (lane-3) fusion proteins in HeLa cells. Representative images showing the nuclear localization of TCON c. ECON d. their co-localization e. and their overlap with DAPI stained nucleic acids f. g. Schematic showing the binding of TCON to the telomeric repeat sequences through TRF1. FCS measurements showed the distinct distribution and diffusion pattern of TCON tagged EGFP j, k. in comparison to the control EGFP h, i. Co-localization of TCON and TRF2 is revealed using the overlapping fluorescence of EGFP of TCON and Alexa-647 TRF2 (m) antibody bound to TRF2 l, m and n. in HeLa cells. Because TRF2 is telomere specific binding protein, the co-localization of TCON to the same locus reveals the association of TCON with telomeres.

Article Snippet: Blocking was performed with PBS solution containing 5% goat serum and 0.3% Triton X-100 for 1 h. Mouse monoclonal anti-TRF2 antibody (ab13579, Abcam) diluted 1:500 was incubated overnight with the cells at 4°C.

Techniques: Western Blot, Staining, Binding Assay, Diffusion-based Assay, Comparison, Control, Fluorescence

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 1. Physical interactions between TRF2 and BER proteins. A, increasing concentrations of various proteins were spotted in replicate (0.6, 0.8, 0.9, and 1 ng) on a grid of the Discover-Light Protein Array membrane and then hybridized with TRF2 protein (10 ng/mL). After washing, bound TRF2 protein was detected by Western blotting with an anti-TRF2 antibody. B, coimmunoprecipitation of TRF2 and Pol h. HeLa whole-cell extracts (500 ng) were immunoprecipitated with either rabbit anti-TRF2 (lane 3) or control IgG (lane 4) antibodies. The immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis with anti–Pol h or anti-TRF2 antibodies as indicated. TRF2 (lane 1) and Pol h (lane 6) were loaded as markers and positive controls. Input, 10% loaded (lane 2). C, coimmunoprecipitation of TRF2 and FEN-1. HeLa whole-cell extracts (500 ng) were immunoprecipitated with either rabbit anti-TRF2 (lane 3) or control IgG antibodies (lane 1). The immunoprecipitates were probed with anti-FEN-1 or anti-TRF2 antibodies as indicated. Input, 10% loaded (lane 2).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Protein Array, Membrane, Western Blot, Immunoprecipitation, Control, SDS Page

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 2. Mapping the sites of Pol h and FEN-1 interactions with TRF2. A, schematic of the known domains and structural motifs of TRF2 and the borders of the various GST-tagged fragments. B, basic NH2 terminus; TRFH, dimerization domain; Myb, Myb-like telomere DNA binding domain. Numbers indicate the amino acid sequence. B, Coomasie staining of the recombinant GST-tagged TRF2 fragments (2 Ag each) used in the binding assay after single-step purification and SDS-PAGE. C, TRF2 domains that interacted with Pol h and FEN-1. HeLa nuclear extracts (400 AL) were incubated with either GST alone (lanes 2 and 9) or GST-tagged TRF2 fragments (lanes 3-6 and 8) that were prebound to glutathione beads. Eluted proteins were separated by SDS-PAGE, transferred to a membrane, and stained with amido black to ensure equal loading of the various TRF2 fragments. The membrane was probed with mouse anti–Pol h or rabbit anti-FEN-1 antibodies.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Binding Assay, Sequencing, Staining, Recombinant, Purification, SDS Page, Incubation, Membrane

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 4. FEN-1 incision activity in the presence of TRF2. A, FEN-1 incision of a 10-nt flap substrate. Reactions contained 120 pmol/L FEN-1 incubated with a 10-nt flap substrate (100 nmol/L) either alone (lane 2) or together with increasing TRF2 concentrations (9, 18, 90, 180, or 900 pmol/L; lanes 3-7, respectively) at 37jC for 10 minutes. The relative percent incision activity was calculated as described in Materials and Methods and normalized to the FEN-1 alone control (lane 2). Values represent the average and SD of at least three independent experiments. B, FEN-1 incision of a telomeric flap substrate. Reactions contained 10 pmol/L FEN-1 incubated with a 15-nt flap substrate harboring telomeric sequence 5V to the flap (10 nmol/L). FEN-1 was incubated alone (lane 2) or together with increasing TRF2 concentrations (100, 300, and 1,000 pmol/L; lanes 3-5) at 37jC for 10 minutes. The relative percent incision activity was calculated as in (A).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Activity Assay, Incubation, Control, Sequencing

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 5. TRF2 specifically stimulates primer extension by Pol h. A, TRF2 effects on Pol h activity. Pol h (0.5 nmol/L) was preincubated with increasing TRF2 amounts (0, 0.5, 1.5, or 3.0 nmol/L; lanes 2-5, respectively) for 5 minutes on ice. Reactions were initiated by adding the nontelomeric mix15/mix34 substrate (25 nmol/L) and were incubated for 15 minutes at 37jC. Reaction products were run on a 20% denaturing polyacrylamide gel and visualized by a Phosphorimager. Lane 1, substrate alone. E, 0.5 nmol/L (lane 6) and 3.0 nmol/L (lane 7) of heat-denatured TRF2 protein. Lanes 8 and 9, TRF2 (0.5 or 3.0 nmol/L, respectively) in the absence of Pol h. B, quantitation of Pol h primer-extension. Percent of total products with the indicated number of nucleotides incorporated was calculated as described in Materials and Methods. Columns, mean from three independent experiments; bars, SD. C, Klenow activity. Increasing Klenow concentrations (lanes 2-7) were incubated with the mix15/mix34 substrate (25 nmol/L) for 15 minutes at 37jC. Lane 1, substrate alone. Products were analyzed as in (A). D, TRF2 affects on Klenow primer extension. Klenow (0.32 nmol/L) was preincubated with increasing TRF2 amounts (0, 0.32, 0.96, 1.92, or 3.84 nmol/L; lanes 2-6, respectively) and Pol h (0.3 nmol/L) was preincubated with 1.8 nmol/L TRF2 (lane 8) for 5 minutes on ice. Reactions were initiated by adding mix15/mix34 substrate (25 nmol/L) and were incubated for 15 minutes at 37jC. Products were analyzed as in (A).

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Activity Assay, Incubation, Quantitation Assay

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 6. Comparison of TRF2 stimulation of Pol h on telomeric and nontelomeric template substrates. A, reactions contained Pol h (0.25 nmol/L) alone or together with increasing TRF2 concentrations (0.125, 0.25, 0.75, and 1.5 nmol/L). Reactions were initiated by adding 25 nmol/L substrate with either nontelomeric template sequence (mix15/mix34; lanes 1-6) or telomeric template sequence (mix15/tel34; lanes 7-12) and were incubated for 15 minutes at 37jC, followed by analysis on a 20% denaturing gel. Quantitation and calculation of primer extension products for the nontelomeric (B) or telomeric (C) template substrates was as described in Materials and Methods. Columns, mean from three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Comparison, Sequencing, Incubation, Quantitation Assay

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 7. TRF2 promotion of Pol h primer extension on substrates with TRF2 binding sites. A, reactions contained Pol h (0.25 nmol/L) alone or together with increasing TRF2 concentrations (0.125, 0.25, 0.75, and 1.5 nmol/L). The reactions were initiated by adding 25 nmol/L telomeric substrate (tel21/tel40) and were incubated for 15 minutes at 37jC, followed by analysis on a 20% denaturing gel. E, 1.5 nmol/L (lane 7) heat-denatured TRF2 protein. B, the percent of products with the indicated number of nucleotides incorporated was calculated as described in Materials and Methods. Columns, mean from at least three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Binding Assay, Incubation

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 8. TRF2 stimulates Pol h strand displacement DNA synthesis on a nontelomeric BER substrate. A, schematic of the 34-bp DNA substrate containing an 8-oxo-guanine at position 17 is shown both before and after treatment with OGG1 and APE1. OGG1 removes the 8-oxo-guanine base and APE1 incises the DNA strand 5V to the resulting apurinic/apyrimidinic site. B, the substrate was pretreated with OGG1 (128 nmol/L) for 20 minutes at 37jC. The pretreated DNA (100 nmol/L) was incubated with 3.4 ng/AL APE1 for 25 minutes at 37jC together with increasing Pol h concentrations (0.6, 1.2, 2.5, and 5 nmol/L; lanes 2-5 and 6-9, respectively). The reactions in lanes 6 to 9 also contained increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L, respectively). C, the OGG1-pretreated DNA substrate was incubated with 3.4 ng/AL APE1 in the absence () or presence (+) of Pol h (5 nmol/L; lane 1) and with increasing concentrations of TRF2 (0, 5, 15, and 30 nmol/L; lanes 2-5, respectively). D, quantitation of unreacted substrate (0), short-patch (1), and long-patch (2-6) BER intermediates. E, quantitation of individual long-patch BER intermediates. Reaction products were calculated as a function of total radioactivity as described in Materials and Methods for reactions containing 5 nmol/L Pol h alone (solid columns) or together with 30 nmol/L TRF2 (hatched columns). Columns, mean of two independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: DNA Synthesis, Incubation, Quantitation Assay, Radioactivity

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 9. TRF2 stimulation of Pol h on telomeric BER substrates. A, a schematic of the 39-bp DNA substrate containing an 8-oxo-guanine at position 17 within two tandem telomeric repeats is shown both before and after treatment with OGG1 and APE1. B, the substrate was pretreated with OGG1 (128 nmol/L) for 20 minutes at 37jC. The pretreated DNA (100 nmol/L) was incubated with 3.4 ng/AL APE1 for 25 minutes at 37jC together with increasing Pol h concentrations (0.62, 1.2, 2.5, and 5 nmol/L; lanes 2-5 and 6-9, respectively). The reactions in lanes 6 to 9 also contained increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L, respectively). C, the OGG1-pretreated DNA substrate was incubated with 3.4 ng/AL APE1 in the presence (+) or absence () of Pol h (1.2 nmol/L), TRF2 (7.5 nmol/L), or FEN-1 (30 nmol/L) as indicated. E, heat-inactivated control. D, quantitation of unreacted substrate (0), short-patch (1), and long-patch (2-7) BER intermediates. E, quantitation of individual long-patch BER intermediates. Reaction products were calculated as a function of total radioactivity as described in Materials and Methods for reactions containing 1.2 nmol/L Pol h alone (solid columns) or together with 7.5 nmol/L TRF2 (hatched columns). Columns, mean of three independent experiments; bars, SD.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Incubation, Control, Quantitation Assay, Radioactivity

Journal: Cancer Research

Article Title: Telomere Repeat Binding Factor 2 Interacts with Base Excision Repair Proteins and Stimulates DNA Synthesis by DNA Polymerase β

doi: 10.1158/0008-5472.can-05-2742

Figure Lengend Snippet: Figure 10. TRF2 enhances Pol h extension of the 3V tail of a telomeric D-loop. The telomeric D-loop substrate (25 nmol/L) was incubated with increasing Pol h concentrations alone (0.62, 1.2, 2.5, and 5 nmol/L; lanes 2-5) or together with increasing TRF2 concentrations (3.7, 7.5, 15, and 30 nmol/L; lanes 6-9). The reactions were initiated by adding substrate and were incubated for 15 minutes at 37jC. The reaction products were run on a 20% denaturing and were visualized by a Phosphorimager.

Article Snippet: After washing with PBS-T, the membranes were probed with monoclonal mouse anti-TRF2 antibody (1:500, Imgenex, Sorrento Valley, CA) overnight.

Techniques: Incubation

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Elevated levels of TRF2 protein in a number of breast cancer and melanoma cells. Immunoblotting was performed to detect TRF2 in whole-cell extracts of the following human cell lines: Primary fibroblasts: IMR90, BJ and WI38; Breast cancer cells: MDA-MB-231, MDA-MB-453, MDA-MB-468, ZR-75-1, MCF-7 and SK-BR-3; Melanoma cells: Lox, CaCL 73-36, WM115, WM278, WM983A, WM983B and WM1158. Fibrosarcoma cell: HT1080. Tubulin was used as a loading control. ( b ) Assessing TRF2 overexpression levels. Parallel cultures of HT1080 clone A6 (a subclone of HT1080 cells that maintain stable telomere length) cells infected with lentiviruses expressing GFP or TRF2 were examined by immunoblotting (top panel) or immunostaining (bottom panel). Fold of TRF2 expression was quantified by the ImageJ software and normalized to tubulin levels. ( c ) Terminal Restriction Fragment analysis of HT1080 A6 cells infected with lentiviruses expressing GFP or TRF2. Cells were continuously passaged and collected at the indicated population doublings (PD). ( d ) Schematic diagram of STELA analysis. ( e ) Individual telomere lengths measured by STELA analysis in HT1080 A6 cells overexpressing GFP or TRF2 at PD6. Each lane represents a single PCR reaction performed with 100 pg of genomic DNA, followed by Southern blotting detection of XpYp telomeres using an XpYp subtelomeric probe.

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: Western Blot, Over Expression, Infection, Expressing, Immunostaining, Software, Southern Blot

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Representative metaphase spread image of HeLa1.2.11 cells infected with lentivirus expressing GFP or TRF2. Infected cells were passaged and collected at PD7 for metaphase spread followed by FISH analysis. Chromosomes (blue) were hybridized with PNA probes for telomeric sequences (green) or centromeric sequences (red). Regions in white boxes are enlarged to the bottom of the corresponding image for better visualization. Yellow arrows indicate signal-free telomeres; arrowhead indicates chromosome end-to-end fusions. For b and c , 50 metaphases (∼3,360 chromosomes) each of GFP- or TRF2-overexpressing cells were examined for telomeric abnormality. All quantifications were carried out blindly. Each point on the scatter plot represents a single metaphase. Mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( b ) Quantification of signal-free telomeres in HeLa1.2.11 cells overexpressing GFP or TRF2. ( c ) Quantification of chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing GFP or TRF2. ( d ) Schematic diagram of Fusion PCR analysis. ( e ) Individual chromosome end-to-end fusions assessed by Fusion PCR. HeLa1.2.11 cells overexpressing GFP or TRF2 were harvested at PD6. Multiple aliquots of 100 ng of genomic DNA were independently subjected to fusion PCR using a mix of XpYp, 17p and 21q subtelomeric primers. PCR products were resolved on 1% agarose-TBE gel and detected by Southern hybridization with an XpYp-specific subtelomeric probe. ( f ) Representative sequence of fusion molecules between XpYp, 17p and 21q. The fusion points, size of deletion, and microhomology (in red) are indicated.

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: Infection, Expressing, Two Tailed Test, Hybridization, Sequencing

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Formation of thinly stretched telomere bridges between anaphase chromosomes in HeLa1.2.11 cells overexpressing TRF2. Telomeric DNAs were detected by in situ hybridization with a PNA telomeric probe (red). Chromosomes were stained with DAPI (blue). ( b ) Quantification of telomeric anaphase bridges and chromosome end-to-end fusions in HeLa1.2.11 cells overexpressing TRF2 at PD3 and PD7. HeLa1.2.11 cells were infected with lentiviruses expressing TRF2. Parallel cultures were collected at PD3 or PD7 for PNA telomere-FISH to examine telomeric anaphase bridges or for metaphase spreading followed by PNA telomere-FISH to examine chromosome end-to-end fusions. ( c ) Quantification of fragile telomeres in HeLa1.2.11 cells overexpressing GFP control or TRF2 at PD3. Cells were collected for metaphase spreading followed by PNA telomere-FISH to examine fragile telomeres. Representative fragile telomeres are labelled by yellow arrows on images at the left panel. All quantifications were carried out blindly. For b and c , mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( d ) TRF2 overexpression stalled replication at telomeres. Representative chromatin fibre FISH images showed the incorporation of IdU (blue) or CldU (green) at telomeric (red) and adjacent subtelomeric regions in LOX cells infected with lentiviruses expressing luciferase control or TRF2. At PD3, cells in logarithmic growth were labelled sequentially with 30 μM of IdU and then CldU for 4 h each before Chromatin fibre-FISH analysis was carried out. Telomeres were identified by FISH with a telomeric repeat probe. IdU and CldU were identified by immunostaining with analogue-specific antibodies. Dotted line represents the start of telomeric sequences. We did not see dual IdU and CldU labelling at replicating telomeres due to the long labelling time (4 h) used for each halogenated nucleotide. ( e ) Quantification of fraction of telomeric fragments that was labelled with CldU and/or IdU. For control of stalled replication, cells were treated with 1 μg ml −1 aphidicolin for 16 h before they were labelled with IdU and CldU in the presence of aphidicolin (see for representative images).

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: In Situ Hybridization, Staining, Infection, Expressing, Two Tailed Test, Over Expression, Luciferase, Immunostaining

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Representative anaphase images showing the staining of PICH, telomeres, and centromeres in HeLa1.2.11 cells overexpressing TRF2. Cells were infected with lentivirus expressing TRF2 and collected at PD2 after infection for immunostaining-FISH analysis. Telomeres (magenta) and centromeres (red) were identified by PNA FISH. PICH (green) were identified by immunostaining with an anti-PICH antibody. Chromosomes were stained with DAPI (blue). PICH-aligned telomeric anaphase bridges were marked by white arrows. Note that the image represents a single section on the z axis. ( b ) Quantification of PICH bridges, as well as centromere-associated PICH bridges, in HeLa1.2.11 cells overexpressing GFP control or TRF2. Bars represent mean values and s.e.m. (>150 anaphases from three independent experiments examined for each line). Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. ( c ) Quantification of fraction of telomeric UFBs that associate with the PICH protein. 75 telomeric UFB-containing anaphases from three independent experiments were examined for the association between PICH and telomeric UFB. ( d ) Representative anaphase images showing the staining of PICH and centromeres in HeLa1.2.11 cells treated with 20 μM DNA topoisomerase II inhibitor ICRF-159.

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: Staining, Infection, Expressing, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Elevated levels of TRF2 induce telomeric ultrafine anaphase bridges and rapid telomere deletions

doi: 10.1038/ncomms10132

Figure Lengend Snippet: ( a ) Long telomere length exacerbates TRF2-induced telomeric UFBs. Left panel: quantification of telomeric UFBs in cells with different mean telomere lengths. Approximately 50 anaphases from each cell line were analysed for telomeric UFBs. Mean values are indicated in red. Two-tailed Student's t -tests were performed to make pairwise comparison for statistical significance. No telomeric UFBs were detected in HeLa1.2.11 and HT1080 cells infected with lentivirus overexpressing GFP. Telomeres in UM-UC-3 cells (∼3 kb) were pre-extended by expression of the telomerase RNA subunit (hTR) for 11 days (to ∼8 kb) and 20 days (to ∼15 kb). Cells were infected with lentivirus expressing TRF2, and then fixed for telomeric FISH 48 h after infection. TRF2 overexpression did not induce any telomeric UFBs in control UM-UC-3 cells that were infected with an empty lentiviral vector. Right panel: terminal restriction fragment analysis in UM-UC-3 cells expressing vector control or hTR using a telomeric repeat probe. ( b ) TRF2 overexpression fails to induce telomere shortening in cells containing very short telomere length.

Article Snippet: Immunoblots were incubated with a mouse monoclonal anti-TRF2 (BD Transduction Laboratories, 1:500), followed by a horseradish peroxidase-conjugated donkey anti-mouse IgG (Jackson ImmunResearch).

Techniques: Two Tailed Test, Infection, Expressing, Over Expression, Plasmid Preparation

Journal: The Journal of biological chemistry

Article Title: Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.

doi: 10.1074/jbc.M205396200

Figure Lengend Snippet: FIG. 1. TRF2 associates with WRN in vivo. A, HeLa NE (400 l) were incubated with either GST protein (lane 1) or the GST-WRN fragment 949–1432 fusion protein (lane 2) that were pre-bound to glutathione beads. Eluted proteins were separated by SDS-PAGE and analyzed by Western blot with anti-TRF2 antibodies. Input, 3% loaded (see lane 3); reactions, 25% loaded (see lanes 1 and 2). B, Amido Black staining of the membrane indicates the relative amounts of GST and GST-WRN fragment 949–1432 that were loaded. C and D, HeLa nuclear extracts (400 l) were immunoprecipitated with either anti-TRF2 antibodies (lane 2) or control IgG (lane 3). The immunoprecipitates were analyzed by SDS-PAGE and Western blot with anti-WRN (panel C) and anti-TRF2 antibodies (panel D). Purified WRN and TRF2 proteins were loaded as markers and positive controls (lane 4). Input, 2.5% loaded (see lane 1); reactions, 35% loaded (see lanes 2 and 3).

Article Snippet: Bound TRF2 from HeLa NE were detected by Western blot with mouse monoclonal anti-TRF2 antibodies (1:250 dilution; Imgenex).

Techniques: In Vivo, Incubation, SDS Page, Western Blot, Staining, Membrane, Immunoprecipitation, Control, Purification

Journal: The Journal of biological chemistry

Article Title: Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.

doi: 10.1074/jbc.M205396200

Figure Lengend Snippet: FIG. 2. TRF2 and EGFP-WRN co-localize in human cell lines. Exponentially growing cells expressing EGFP-WRN were fixed and stained with mouse anti-TRF2 antibody followed by addition of anti-mouse secondary antibody conjugated to Texas Red (red fluorescence). A, U-2 OS (telomerase-negative) nucleus showing EGFP-WRN localized in nuclear foci. B, TRF2 localization (red) in the U-2 OS nucleus. C, co-localization of EGFP-WRN and TRF2 in nuclear foci (yellow) in the U-2 OS nucleus. D, transmitted image of the U-2 OS nucleus. E, HeLa nucleus with EGFP-WRN localized to the nucleus and nuclear foci. F, TRF2 (red) localizes outside the nucleolus in the HeLa nucleus. G, co-localization of TRF2 with EGFP-WRN containing foci in the HeLa nucleus (white arrows). H, transmitted image of HeLa nucleus. I, HeLa nucleus showing nucleolar exclusion of EGFP-WRN. J, TRF2 staining in the HeLa nucleus. K, co-localization of TRF2 with EGFP-WRN containing nuclear foci (yellow foci, highlighted by white arrows). L, transmitted image of HeLa nucleus.

Article Snippet: Bound TRF2 from HeLa NE were detected by Western blot with mouse monoclonal anti-TRF2 antibodies (1:250 dilution; Imgenex).

Techniques: Expressing, Staining, Fluorescence

Journal: The Journal of biological chemistry

Article Title: Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.

doi: 10.1074/jbc.M205396200

Figure Lengend Snippet: FIG. 3. Mapping of the WRN and TRF2 interaction sites. A, known struc- tural motifs and domains of the WRN pro- tein. The exonuclease domain, conserved RecQ ATPase/helicase domain, RecQ C- terminal region (RQC), helicase-related domain (HRDC), and the nuclear localiza- tion sequence (NLS) are shown. B, WRN domains that interact with TRF2. A se- ries of recombinant GST-WRN fragment fusion proteins, shown schematically, and GST alone were bound to glutathione beads and incubated with in vitro-trans- lated TRF2 (35S-TRF2). Total protein was eluted, and equal amounts were run on 10% SDS gels. Bound 35S-TRF2 was de- tected by autoradiography. Ten percent of the input was loaded. C, fine mapping of the TRF2 interaction sites in the WRN C terminus.

Article Snippet: Bound TRF2 from HeLa NE were detected by Western blot with mouse monoclonal anti-TRF2 antibodies (1:250 dilution; Imgenex).

Techniques: Sequencing, Recombinant, Incubation, In Vitro, Autoradiography

Journal: The Journal of biological chemistry

Article Title: Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.

doi: 10.1074/jbc.M205396200

Figure Lengend Snippet: FIG. 4. Direct interaction between TRF2 and two RecQ helicases. A, purified recombinant proteins TRF2, WRN, and BLM. Proteins (1–2 g) were separated on 10% SDS-PAGE gels and were detected by Coomassie staining. TRF2 protein migrated similar to the 66-kDa marker, consistent with previous reports (18). Bands migrating below TRF2 represent degradation products as determined by Western blot using anti-TRF2 antibody (Santa Cruz Biotechnology). The lower band in the WRN lane (about 66 kDa) is BSA (see “Experimental Procedures”). B, detection of WRN and BLM binding with TRF2 by ELISA. Wells coated with BSA (control), WRN (75 ng), BLM (75 ng), or TRF2 (75 ng) were incubated with either 0 or 75 ng of TRF2 for 2 h at 37 °C. After washing bound TRF2 was detected using rabbit polyclonal anti-TRF2 antibody (Santa Cruz Biotechnology) by ELISA. DNaseI (5 g/ml) was included were indicated. C and D, affinity of WRN and BLM for TRF2. Wells were coated with either 9 nM WRN (C) or 9 nM BLM (D) and incubated with increasing concentrations of TRF2. Bound TRF2 was determined by ELISA as above. Absorbance values were corrected for background and plotted against TRF2 concentration (see “Experimental Procedures”). Values and error bars represent the mean and standard deviation from three independent experiments.

Article Snippet: Bound TRF2 from HeLa NE were detected by Western blot with mouse monoclonal anti-TRF2 antibodies (1:250 dilution; Imgenex).

Techniques: Purification, Recombinant, SDS Page, Staining, Marker, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Control, Incubation, Concentration Assay, Standard Deviation

Journal: The Journal of biological chemistry

Article Title: Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.

doi: 10.1074/jbc.M205396200

Figure Lengend Snippet: FIG. 5. TRF2 binding to DNA heli- case substrates depends on the te- lomere repeat number. Binding reac- tions were analyzed by native gel electrophoresis. A, binding reactions con- tained 5 nM of the (TTAGGG)13 radiola- beled duplex, 0.5 g of competitor calf thymus DNA, and 0, 5, or 50 nM recombi- nant TRF2. B, reactions contained 0.5 nM (TTAGGG)4 forked duplex and 0, 20, or 50 nM recombinant TRF2. C, reactions in- cluded 0.5 nM non-telomeric forked du- plex and 0, 20, or 50 nM recombinant TRF2. D, reactions contained 0.5 nM (TTAGGG)2 forked duplex and 0, 20, or 50 nM purified TRF2. In all panels an arrow indicates the position of the well where the TRF2DNA complexes were retained. The percent DNA bound and retained in the well (%B) is shown at the bottom of each lane.

Article Snippet: Bound TRF2 from HeLa NE were detected by Western blot with mouse monoclonal anti-TRF2 antibodies (1:250 dilution; Imgenex).

Techniques: Binding Assay, Nucleic Acid Electrophoresis, Recombinant, Purification

Journal: The Journal of biological chemistry

Article Title: Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.

doi: 10.1074/jbc.M205396200

Figure Lengend Snippet: FIG. 6. TRF2 interaction with WRN or BLM stimulates their helicase ac- tivity but does not alter WRN exonu- clease activity. 0.5 nM either WRN (A) or BLM (B) was pre-incubated with in- creasing amounts of TRF2 (0 to 4 nM; monomer) for 5 min at 25 °C. Reactions were initiated by the addition of the (TTAGGG)2 fork (0.5 nM) and incubated for 15 min at 37 °C, followed by product analysis on a 12% native polyacrylamide gel. Œs, heat-denatured substrate; ŒE, heat-denatured TRF2 protein (4 nM; mon- omer). The percent displacement was cal- culated as described under “Experimental Procedures” and plotted against TRF2 concentration (nM; monomer). Values rep- resent the mean and standard deviation from three independent reactions. C, WRN exonuclease in the presence of TRF2. WRN (95 nM) was pre-incubated with increasing amounts of TRF2 (0, 25, 50, 100, 200, 300, and 400 nM; monomer (see lanes 2–8)) for 5 min at 25 °C. Reac- tions (10 l) were initiated by the addition of the 53/74-mer substrate (3 fmol) and were incubated for 1 h at 37 °C. Products were analyzed on a 15% denaturing poly- acrylamide gel. ŒE, heat-denatured TRF2 protein (400 nM; monomer (see lane 10)).

Article Snippet: Bound TRF2 from HeLa NE were detected by Western blot with mouse monoclonal anti-TRF2 antibodies (1:250 dilution; Imgenex).

Techniques: Activity Assay, Incubation, Concentration Assay, Standard Deviation, Acrylamide Gel Assay

Journal: The Journal of biological chemistry

Article Title: Telomere-binding protein TRF2 binds to and stimulates the Werner and Bloom syndrome helicases.

doi: 10.1074/jbc.M205396200

Figure Lengend Snippet: FIG. 7. WRN and BLM are active on telomeric substrates pre-bound by TRF2. The (TTAGGG)4 forked duplex (0.5 nM) was pre-incubated with or with- out TRF2 for 5 min at 25 °C. Reactions were initiated by adding either the heli- case alone or together with RPA and were incubated for 15 min at 37 °C. Œs, heat- denatured substrate. A, analysis of WRN helicase activity on TRF2-bound sub- strates. Where indicated, the reactions contained 50 nM TRF2 (monomer), 7.5 nM WRN (monomer), and 8.5 nM RPA (het- erotrimer) (lanes 1–8). The products were analyzed on a 12% native denaturing gel. The percent displacement (%D) values represent the mean and standard devia- tion from three independent reactions. B, analysis of WRN exonuclease activity on TRF2-bound substrates. The reactions contained 50 nM TRF2, 7.5 nM WRN, and 8.5 nM RPA. The products were analyzed on a 14% denaturing gel. C, analysis of BLM helicase activity on TRF2-bound substrates. The reactions contained 50 nM TRF2, 7.5 nM BLM (monomer), and 8.5 nM RPA, where indicated. Products were ana- lyzed on a 12% native gel. The %D values represent the mean and standard devia- tion from three independent experiments.

Article Snippet: Bound TRF2 from HeLa NE were detected by Western blot with mouse monoclonal anti-TRF2 antibodies (1:250 dilution; Imgenex).

Techniques: Incubation, Activity Assay