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: Metallomics : integrated biometal science

Article Title: Pt-ttpy, a G-quadruplex binding platinum complex, induces telomere dysfunction and G-rich regions DNA damage.

doi: 10.1093/mtomcs/mfab029

Figure Lengend Snippet: Fig. 4 TRF2 foci quantification detected by immunofluorescence on A2780 and A2780cis cells treated with cisplatin, Pt-ttpy, and Pt-tpy for 96 h at their respective IC80 concentration. (A) A2780 cells were processed for immunofluorescence using antibodies against TRF2. (B) % of TRF2 foci after A2780 cell treatments with cisplatin, Pt-ttpy, and Pt-tpy. (C) % of TRF2 foci after A2780cis cell treatments with cisplatin, Pt-ttpy, and Pt-tpy (mean of at least three experiments). ** Indicates a Mann and Withney test P-value < 0.01 and * P < 0.05 (GraphPad PRISM software).

Article Snippet: Briefly, 20 μg proteins were electrophoresed in SDS-PAGE (SDSPolyacrylamide 10%) under denaturing conditions, then transferred to a PVDFmembrane (Polyvinylidin Difluoride) (Amersham HybondTM P+, GE Healthcare) or nitrocellulose, which were hybridized with mouse monoclonal anti-TRF2 antibody (4A794, Upstate), the Anti-TRF2 Thr-188P polyclonal antibody, and antiactin HRP (SC1616-HRP, Santa-Cruz).

Techniques: Immunofluorescence, Concentration Assay, Software

Journal: Metallomics : integrated biometal science

Article Title: Pt-ttpy, a G-quadruplex binding platinum complex, induces telomere dysfunction and G-rich regions DNA damage.

doi: 10.1093/mtomcs/mfab029

Figure Lengend Snippet: Fig. 5 Proteins bound to telomeres quantified from dot blots. Dot blot membranes of ChIP of TRF2, TRF1, POT1, and H3 on A2780 cells treated by doses of Pt-ttpy (A), Pt-tpy (B), and cisplatin (C) for 96 h at their respective IC80 concentration. (D) % of proteins bound to telomeres of A2780 cells. (E) % of proteins bound to telomeres of A2780cis cells. Telomeric sequences were evidenced in a DNA fraction immunoprecipitated by an anti-TRF1, anti-TRF2, anti-H3 antibody using a 32P radio labelled 800pb telomeric probe and normalized with α 32P radio labelled Alu sequences in untreated, cisplatin, Pt-ttpy, and Pt-tpy treated cells. 200 ng of DNA were blotted for each sample. The % represents the quantitative values of telomeric DNA signals in the samples originating from cells with treatment compared to the cells without any treatment. Quantitative values of telomeric DNA signals are calculated as the ratio between telomeric DNA signal precipitation and telomeric DNA signals in the INPUT for the same amount of blotted DNA. These values have been normalized by the amount of blotted DNA for each sample quantified by the non-specific Alu probe, following the formula: (telomere IP/telomere INPUT)/(Alu IP/Alu INPUT). (Means of at least three experiments) ** Indicates a Mann and Withney test P-value < 0.01 and * P < 0.05 (GraphPad PRISM software). Statistical analysis was made by comparing the amount of protein bound to telomeres for each treatment.

Article Snippet: Briefly, 20 μg proteins were electrophoresed in SDS-PAGE (SDSPolyacrylamide 10%) under denaturing conditions, then transferred to a PVDFmembrane (Polyvinylidin Difluoride) (Amersham HybondTM P+, GE Healthcare) or nitrocellulose, which were hybridized with mouse monoclonal anti-TRF2 antibody (4A794, Upstate), the Anti-TRF2 Thr-188P polyclonal antibody, and antiactin HRP (SC1616-HRP, Santa-Cruz).

Techniques: Dot Blot, Concentration Assay, Immunoprecipitation, Software

Journal: Metallomics : integrated biometal science

Article Title: Pt-ttpy, a G-quadruplex binding platinum complex, induces telomere dysfunction and G-rich regions DNA damage.

doi: 10.1093/mtomcs/mfab029

Figure Lengend Snippet: Fig. 6 Western blot of TRF2, pTRF2, and telomere length from A2780 treated cells with cisplatin, Pt-ttpy, and Pt-tpy for 96 h. (A) Western blot of TRF2 membranes, (B) TRF2 relative protein level normalized with actin (Mean of three experiments), (C) Western blot of pTRF2 membranes for 50 μM 8 h treatment, and 85 mM etoposide as positive control (D) Southern blot or (E) Relative telomere length measured by qPCR. The values were normalized using 36B4u gene (mean of three experiments).

Article Snippet: Briefly, 20 μg proteins were electrophoresed in SDS-PAGE (SDSPolyacrylamide 10%) under denaturing conditions, then transferred to a PVDFmembrane (Polyvinylidin Difluoride) (Amersham HybondTM P+, GE Healthcare) or nitrocellulose, which were hybridized with mouse monoclonal anti-TRF2 antibody (4A794, Upstate), the Anti-TRF2 Thr-188P polyclonal antibody, and antiactin HRP (SC1616-HRP, Santa-Cruz).

Techniques: Western Blot, Positive Control, Southern Blot