Journal: The Journal of Biological Chemistry

Article Title: c-Myc Quadruplex-forming Sequence Pu-27 Induces Extensive Damage in Both Telomeric and Nontelomeric Regions of DNA *

doi: 10.1074/jbc.M113.505073

Figure Lengend Snippet: Pu-27 induces phosphorylation of H2AX (γH2AX). U937 cells and ΔB-U937 cells treated with Pu-27 and were subjected to Western blot and FACS analysis. A, 3-day treatment of Pu-27 showed profound up-regulation of γH2AX expression in U937 cells but almost no changes in dB-U937 cells. Relative expressions of TRF2 in U937 and ΔB-U937 cells were also shown. TRF2 expression is down-regulated in U937 cells and no changes in ΔB-U937 cells when treated with Pu-27 (consistent with RT-PCR data in Fig. 6). Bottom panel, relative expression presented in bar graphs. *, p < 0.05. B, by FACS analysis γH2AX has shown continued to be highly expressed from days 1–3, and again there was no significant change in ΔB-U937 cells. Bottom panels, relative expression presented in bar graphs. *, p < 0.05.

Article Snippet: Aliquots of 0.4 ml of cells were transfected with 2 μg of the plasmid pLPC TRF2 ΔB (Addgene), which contains the TRF2 gene in which the basic domain of amino acids 1–44 is deleted.

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: c-Myc Quadruplex-forming Sequence Pu-27 Induces Extensive Damage in Both Telomeric and Nontelomeric Regions of DNA *

doi: 10.1074/jbc.M113.505073

Figure Lengend Snippet: Pu-27 inhibits the molecules related to DNA damage repair machinery in U937. A, RT-PCR of U937 and ΔB-U937 cells treated with Pu-27 for 3 days. Pu-27 inhibits ATM, RAD17, RAD50, CHK14, and CHK2 but not H2AX, BRCA1, and telomerase reverse transcriptase in U937 cells. B, ΔB-U937 cells showed no changes of TRF2, TRF1, POT1, TIN2, RAD17, RAD50, and 53BP1; down-regulation of ATM; up-regulation of CHK1 and CHK2; and H2AX and BRCA1.

Article Snippet: Aliquots of 0.4 ml of cells were transfected with 2 μg of the plasmid pLPC TRF2 ΔB (Addgene), which contains the TRF2 gene in which the basic domain of amino acids 1–44 is deleted.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Composition and DNA binding affinity of the Flag–TIN2 complexes. ( A ) Experimental plan for characterization of Flag–TIN2 containing Shelterin complexes. Extracts made from HeLa cells transfected with Flag–TIN2 are incubated with the [ 32 P]-labeled probe, after which Flag–TIN2 containing complexes are immunoprecipitated with magnetic beads coated with the anti-Flag M2 antibody. Amount of probe recovered can then be used as a measure of the affinity of each probe for Shelterin complexes (Left arm). Alternatively, the isolated complexes can be released by incubation with an excess of 3XFLAG peptide, after which the eluted complexes are analyzed by native electrophoresis on composite gel containing TAM buffer (Right arm). Exposing the complexes to supershifting antibodies prior to the electrophoresis allows determination of the composition of the complexes. ( B ) Graphical representations of the probes D4 and D4-nTel. Shaded areas represent telomeric DNA. The probes carry four binding sites (ds-TTAGGGTTA motifs) for the Myb domain of TRF2 followed by a telomeric (D4) or non-telomeric (D4-nTel) 3′-overhang. [ 32 P]-phosphate was at the 5′-end of the bottom strand. ( C and D ) Subunit composition of the isolated Flag–TIN2 complexes. Extracts of HeLa cells transfected with (+) or without (–) Flag–TIN2 or Flag-TRF2 were incubated with [ 32 P]-probe D4. Protein/DNA complexes containing the Flag-tagged proteins were captured with beads coated with the M2 antibody and released by incubation with an excess of 3XFLAG peptide. After exposure to the indicated antibodies, the eluted complexes were resolved by native electrophoresis in a composite gel containing TAM buffer. Short arrow indicates positions of the supershifted Shelterin/DNA complex. ( E ) Saturation binding curve of the interaction of Flag–TIN2 complexes with probe D4. Extracts of HeLa cells transfected with Flag–TIN2 were incubated with increasing concentrations of probes D4 or D4-nTel, after which point Flag–TIN2 complexes were recovered with beads coated with either the anti-Flag antibody (M2 Ab) or normal mouse IgG (normal IgG). Scattered plot shows the amount of probe recovered (in fmoles) as a function of the initial probe concentration (pM). Dotted line shows fitting of the data to either a linear curve (Black, D4-nTel) or a one site saturation binding curve (Gray, D4). Nonlinear regression of the D4 data allowed determination of the dissociation constant ( K d = 1.5 ± 0.3 nM) and maximum number of binding complexes ( B max = 2.3 ± 0.2 fmol/reaction).

Article Snippet: Plasmids pcDNA3.1-Flag-TRF2 ΔB and pcDNA3.1-Flag-TRF2 were made from their pCMV1 equivalents, by transfer of their TRF2 cassettes to vector pcDNA3.1(–) (Invitrogen). pCMV1-Flag-TRF2 ΔB was made by the transfer of a SacII–BamHI fragment from plasmid pTetFLAGhTRF2 45-501 (a gift from Titia de Lange, Rockefeller University, NY, USA) ( ) to pCMV1.

Techniques: Binding Assay, Transfection, Incubation, Labeling, Immunoprecipitation, Magnetic Beads, Isolation, Electrophoresis, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Characterization of the DNA binding specificity of complex T2. ( A and C ) Graphical representations of the structures of all probes tested. Probes were designed to harbor different end structures and to contain different number of ds-TTAGGGTTA motifs. Shaded areas represent telomeric DNA. ( B ) DNA binding by complex T2 requires a functional POT1 binding site. Probes described in A were incubated with no extracts (lane 4) or with nuclear extracts of HT1080-Vector (lane 5) or HT1080-TRF2 cells (lanes 1–3 and 6–13), after which point the protein/DNA complexes were resolved by native electrophoresis in composite gels containing TAM buffer. Five minutes prior to loading, antibodies were added to samples in lanes 7 (anti-Flag M2 antibody) and 8 (Normal mouse IgG). Lanes 1–3 were overexposed to allow detection of the signal of probe A2. Short arrow indicates positions of the supershifted complex T2. ( D ) DNA binding by complex T2 requires at least one Myb binding site. Probes described in C were incubated with no extracts (lane 1) or with nuclear extracts of HT1080-Vector (lane 2) or HT1080-TRF2 cells (lanes 3–8), after which point the protein/DNA complexes were resolved by native electrophoresis in composite gels containing TAM buffer. Five min prior to loading, antibodies were added to samples in lanes 4 (anti-Flag M2 antibody) and 5 (Normal mouse IgG). Short arrow indicates positions of the supershifted complex T2. ( E ) Densitometric quantification of the T2/DNA complexes detected in (D).

Article Snippet: Plasmids pcDNA3.1-Flag-TRF2 ΔB and pcDNA3.1-Flag-TRF2 were made from their pCMV1 equivalents, by transfer of their TRF2 cassettes to vector pcDNA3.1(–) (Invitrogen). pCMV1-Flag-TRF2 ΔB was made by the transfer of a SacII–BamHI fragment from plasmid pTetFLAGhTRF2 45-501 (a gift from Titia de Lange, Rockefeller University, NY, USA) ( ) to pCMV1.

Techniques: Binding Assay, Functional Assay, Incubation, Plasmid Preparation, Electrophoresis

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Characterization of the DNA binding specificity of Flag–TIN2 complexes. ( A ) Graphical representations of the structures of all tested probes. Probes were designed to harbor different end structures and to contain different number of ds-TTAGGGTTA motifs. Shaded areas represent telomeric DNA. ( B ) DNA binding specificity of Flag–TIN2 Complexes. Extracts of HeLa cells transfected with no DNA (Mock) or with Flag–TIN2 (+ Flag–TIN2) were incubated with the [ 32 P]-labeled probes described in A, after which Flag-tagged protein/DNA complexes were captured with the M2 antibody and the amount of radioactivity recovered was counted. Results in triplicates show the percent of each probe recovered by the M2 antibody (mean ± standard deviation; n = 3). The percent of probe C2 recovered from the Mock-transfected cell extract defines the value of the background (<0.1%). ( C ) DNA binding specificity of Flag–TIN2/TRF2 ΔB complexes. Extracts of HeLa cells transfected with TRF2 ΔB alone (TRF2 ΔB ) or with TRF2 ΔB plus Flag–TIN2 (TRF2 ΔB + Flag–TIN2) were incubated with the [ 32 P]-labeled probes described in A, after which Flag-tagged protein/DNA complexes were captured with the M2 antibody and the amount of radioactivity recovered was counted. Results in triplicates show the percent of each probe recovered by the antibody (mean ± standard deviation; n = 3). The percent of probe C2 recovered from the TRF2 ΔB alone-transfected cell extract defines the value of the background (<0.2%).

Article Snippet: Plasmids pcDNA3.1-Flag-TRF2 ΔB and pcDNA3.1-Flag-TRF2 were made from their pCMV1 equivalents, by transfer of their TRF2 cassettes to vector pcDNA3.1(–) (Invitrogen). pCMV1-Flag-TRF2 ΔB was made by the transfer of a SacII–BamHI fragment from plasmid pTetFLAGhTRF2 45-501 (a gift from Titia de Lange, Rockefeller University, NY, USA) ( ) to pCMV1.

Techniques: Binding Assay, Transfection, Incubation, Labeling, Radioactivity, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Transfected Flag-TRF2 ΔB forms a complex that binds selectively to telomeric DNA fragments that carry a 3′-overhang. ( A ) Graphical representation of probes 4M-3′, 4M-bl and 4M-5′. Shaded areas represent telomeric DNA. The probes carried four binding sites for the Myb domain of TRF2 (ds-TTAGGGTTA motifs numbered 1–4) followed by different end structures: 3′-telomeric overhang (4M-3′), blunt (4M-bl) or 5′-telomeric overhang (4M-5′). [ 32 P]-phosphate was at the 5′-end of the top strand. ( B ) TRF2 constructs used in transient transfection. Full-length TRF2 contains a basic domain that binds ss-DNA independently of sequence (Basic), a TRF homology region (TRFH/Dimerization) that serves as dimerization interface, a domain of interaction with TIN2 (aa 350–367), and a Myb domain that binds to ds-TTAGGGTTA (Myb). Interaction with TIN2 allows the recruitment of TRF2 in Shelterin complexes. In these complexes, TPP1 and TIN2 act scaffolds linking TRF2 to the ss-DNA binding protein POT1, which binds to 5′-TTAGGGTTAG-3′. TRF2 ΔB lacks amino acids 2-44 containing the sequence-independent ss-DNA binding domain of TRF2. TRF2 ΔB (R361P) contains an additional mutation that blocks the association of TRF2 ΔB with TIN2. All constructs were tagged at the N-terminus with the Flag epitope. ( C ) Western blot analysis of HeLa cells transiently transfected with the different TRF2 ΔB constructs. Whole cell extracts prepared for EMSA (50 µg) were probed with the anti-Flag M2 antibody. ( D ) Transfected TRF2 ΔB is in a complex that binds selectively to telomeric DNA fragments carrying a 3′-overhang. Top panel: WCEs from mock-transfected (lane 1) and TRF2 ΔB -transfected HeLa cells (lanes 2–4) were incubated with the indicated [ 32 P]-probes (4M-3′, 4M-blunt, 4M-5′) and the protein/DNA complexes that formed were resolved by electrophoresis in TAM buffer. T2, TRF2-containing complex T2. NS, non-specific band. Bottom panel: after incubation of the extracts with the indicated probes, protein/DNA complexes containing Flag-TRF2 ΔB were captured using magnetic beads coated with an anti-Flag antibody, were eluted with an excess of 3XFLAG peptide, and were subsequently resolved by electrophoresis in TAM buffer. ( E ) Effects of R361P mutation on formation of complex T2. HeLa cells were mock-transfected or transfected with Flag-TRF2 ΔB or Flag-TRF2 ΔB (R361P). WCEs were prepared and incubated with probe 4M-3’, after which point protein/DNA complexes were resolved by electrophoresis in a native polyacrylamide gel containing TAM buffer. Complex T2 failed to be detected in extracts of HeLa cells transfected with the R361P mutant.

Article Snippet: Plasmids pcDNA3.1-Flag-TRF2 ΔB and pcDNA3.1-Flag-TRF2 were made from their pCMV1 equivalents, by transfer of their TRF2 cassettes to vector pcDNA3.1(–) (Invitrogen). pCMV1-Flag-TRF2 ΔB was made by the transfer of a SacII–BamHI fragment from plasmid pTetFLAGhTRF2 45-501 (a gift from Titia de Lange, Rockefeller University, NY, USA) ( ) to pCMV1.

Techniques: Transfection, Binding Assay, Construct, Sequencing, Mutagenesis, FLAG-tag, Western Blot, Incubation, Electrophoresis, Magnetic Beads

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Complex T2 formed by Flag-tagged TRF2 is a member of the Shelterin family. ( A ) Western blot analysis of HT1080-TRF2 and HT1080-Vector cells. WCEs prepared for EMSA (50 µg each) were analyzed by western blotting to detected total TRF2 (anti-TRF2 antibody) and the stably transfected Flag-TRF2 (anti-Flag M2 antibody). Membranes were re-probed with an antibody against β-actin. ( B ) Complex T2 formed by Flag-TRF2 contains the Shelterin scaffolding component TIN2. Extracts of HT1080-TRF2 cells were incubated with the indicated probes, after which point protein/DNA complexes were resolved by native electrophoresis in a composite gel containing TAM buffer. Five minutes prior to loading, the indicated antibodies were added to samples in lanes 4–8 (1 µg each of anti-Flag M2 antibody, normal mouse IgG, or antibodies against TIN2, c-Fos, or Vimentin). Short arrow indicates positions of the supershifted complex T2. NS, non-specific band. ( C ) Loss of Shelterin components prevents formation of complex T2. HT1080-TRF2 cells were transiently transfected with siRNA smartpools against TPP1, TIN2, POT1 or TRF1 or with non-targeting (NT) siRNA. Nuclear extracts prepared from HT1080-Vector cells (Vector) or from HT1080-TRF2 cells (TRF2) transfected with the different siRNA were incubated with probe 4M-3′, after which protein/DNA complexes were separated by native electrophoresis in composite gel containing TAM buffer. As expected, complex T2 was more abundant in cells expressing Flag-TRF2 (lane 1 versus 2), was selective for telomeric DNA fragments that carried a 3′-overhang (lane 7 versus 8), and was supershifted by the anti-Flag antibody (lane 9 versus 10). Complex T2 was reduced in HT1080-TRF2 cells transfected with siRNA smartpools directed against TIN2, TPP1 or POT1 (lanes 3–5 versus lane 2). Short arrow indicates positions of the supershifted complex T2. NS, non-specific band. ( D ) Relative abundance of the TPP1, TIN2, POT1 and TRF1 mRNA in the siRNA-transfected cells. For each transfection, abundance of the targeted mRNA was measured by real-time PCR, comparing cells transfected with the targeting and non-targeting (NT) siRNA. For each mRNA, abundance in cells treated with the non-targeting siRNA was set to 1. To show that targeting was specific, TRF2 and GAPDH mRNAs were also measured, both of which found to be unaffected by the different siRNA treatments.

Article Snippet: Plasmids pcDNA3.1-Flag-TRF2 ΔB and pcDNA3.1-Flag-TRF2 were made from their pCMV1 equivalents, by transfer of their TRF2 cassettes to vector pcDNA3.1(–) (Invitrogen). pCMV1-Flag-TRF2 ΔB was made by the transfer of a SacII–BamHI fragment from plasmid pTetFLAGhTRF2 45-501 (a gift from Titia de Lange, Rockefeller University, NY, USA) ( ) to pCMV1.

Techniques: Western Blot, Plasmid Preparation, Stable Transfection, Transfection, Scaffolding, Incubation, Electrophoresis, Expressing, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Modeling the interactions of Shelterin complexes with ds- and ss-telomeric DNA. ( A ) Simultaneous interactions of Shelterin complexes with both ds- and ss-telomeric DNA. Binding of these multi-subunit complexes to DNA requires a binding site for POT1 (5′-TTAGGGTTAG-3′) and at least one binding site for the Myb domains of either TRF1 or TRF2 (ds-TTAGGGTTA motif). Once bound to telomeric DNA, other Myb domains present in the complexes could further stabilize the protein/DNA complex by establishing additional contacts with ds-telomeric DNA. B) Implications of this model on the role(s) played at telomeres by the Shelterin complexes. The DNA binding specificity of Shelterin complexes would be expected to recruit these complexes to regions of telomeres where ss- and ds-telomeric DNA are present in close proximity, including the 3′-telomeric overhang (i), telomeric DNA bubbles (ii), and the D-loop at the base of T-loops (iii). At these locations, Shelterin complexes would be ideally positioned to control the accessibility of the 3′-telomeric overhang (i) to telomerase and T-loop forming machinery. Unoccupied Myb domains in complexes bound to the 3′-telomeric overhang could potentially be available to mediate long-range interactions with upstream ds-telomeric DNA (dotted arrow). These long-range interactions could help initiate the formation of T-loops. Finally, through their simultaneous interactions with both ds- and ss-telomeric DNA, Shelterin complexes could help stabilize pre-existing D-loops and T-loops (iii) as well as telomeric DNA bubbles (ii). Stabilization of these bubbles could promote T-loop formation if the stabilized bubbles are persisting long enough to have a chance to collide with the 3′-telomeric overhang.

Article Snippet: Plasmids pcDNA3.1-Flag-TRF2 ΔB and pcDNA3.1-Flag-TRF2 were made from their pCMV1 equivalents, by transfer of their TRF2 cassettes to vector pcDNA3.1(–) (Invitrogen). pCMV1-Flag-TRF2 ΔB was made by the transfer of a SacII–BamHI fragment from plasmid pTetFLAGhTRF2 45-501 (a gift from Titia de Lange, Rockefeller University, NY, USA) ( ) to pCMV1.

Techniques: Binding Assay