Journal: The Journal of Biological Chemistry

Article Title: Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere *

doi: 10.1074/jbc.M112.415984

Figure Lengend Snippet: hRap1 binds to model DNA templates. Schematic representations of the minichromosome (A), replication fork with a 25-nt gap (B), and Holliday junction templates (C). Black regions correspond to the telomeric DNA. Tungsten shadowcast images of hRap1 illustrate protein bound to the minichromosome (D), replication fork (E), and telomeric Holliday junction template (F). Data are shown in reverse contrast. Scale bar in D and F are 100 nm and is 50 nm in E. Shown is EMSA with 4% PAGE demonstrates hRap1 bound to the nontelomeric template with a 3′ overhang in the presence and absence of anti-His6 antibody (G).

Article Snippet: A nontelomeric template with a 3′ overhang was generated by treating EcoRI-digested pGLGAP, which does not contain telomeric repeats ( 29 ), with T7 exonuclease at room temperature for 40 s followed by incubation on ice for 5 min (New England Biolabs).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere *

doi: 10.1074/jbc.M112.415984

Figure Lengend Snippet: hRap1 recognizes the 3′ ds-ss junction structures independent of sequence. A illustrates that both hRap1 and TRF2 have a strong and similar preference for binding to the ss-ds junction at replication fork with a 25-nt gap and the crossover at the HJ. B illustrates the strong preference for hRap1 binding to the end of the minichromosome containing a 3′ ss extension as contrasted to binding internally along the ds telomeric segment. C showed the strong preference for hRAP1 binding to the minichromosome containing a 3′ ss extension as contrasted to the same but blunt-ended DNA. D shows that both hRap1 and TRF2 prefer to bind at the ss-ds junction of DNAs with a 3′ overhang as contrasted to a 5′ overhang. E compares the binding to DNAs containing 3′ overhangs joined to either telomeric ds segments or non-telomeric. F compares the t-loop formation percentages of hRap1 and hTRF2 on the minichromosome. Each binding experiment was done in triplicate, and at least 100 molecules were counted. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: A nontelomeric template with a 3′ overhang was generated by treating EcoRI-digested pGLGAP, which does not contain telomeric repeats ( 29 ), with T7 exonuclease at room temperature for 40 s followed by incubation on ice for 5 min (New England Biolabs).

Techniques: Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere *

doi: 10.1074/jbc.M112.415984

Figure Lengend Snippet: The hTRF2-Rap1 complex recognizes replication forks and Holliday junctions. The TRF2-Rap1 complex localizes to the junction site of the replication fork (A) and the telomeric HJ DNA (B). Junction preference of the protein bound molecules on the DNA templates is in C, whereas D represents the sequence preference of the hTRF2-Rap1 complex on the HJ DNAs. Each EM binding reaction was done in triplicate, and 100 molecules each were counted. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: A nontelomeric template with a 3′ overhang was generated by treating EcoRI-digested pGLGAP, which does not contain telomeric repeats ( 29 ), with T7 exonuclease at room temperature for 40 s followed by incubation on ice for 5 min (New England Biolabs).

Techniques: Sequencing, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere *

doi: 10.1074/jbc.M112.415984

Figure Lengend Snippet: The binding properties of the TRF2-Rap1 complex along the minichromosome. Tungsten shadowcast images of the TRF2-Rap1 complex on the minichromosome illustrate the protein bound to the ds-ss junction and/or to the ds telomeric DNA (A–C). An example of the TRF2-Rap1 complex bringing the ends of the minichromosome together to form a circle is depicted in D. Bars in A–D are equivalent to 100 nm. In high magnification images, the ds telomeric DNA appears to pass through the TRF2-Rap1 complex (E). Analysis of the binding preference of the TRF2-Rap1 complex along the minichromosome DNA is in F, showing significant binding to the internal duplex telomeric sequences. G further compares the binding percentages of hRap1, hTRF2, and the complex to the ds telomeric DNA segments of the minichromosomes. The effect of DNA sequence on the binding of the TRF2-Rap1 is shown in H. I demonstrates the comparison of the t-loops formed by the TRF2-Rap1 complex and hTRF2 at 10 nm protein concentration. Corresponding p values are shown by the different number of asterisks on each graph as described in the legend to Fig. 2.

Article Snippet: A nontelomeric template with a 3′ overhang was generated by treating EcoRI-digested pGLGAP, which does not contain telomeric repeats ( 29 ), with T7 exonuclease at room temperature for 40 s followed by incubation on ice for 5 min (New England Biolabs).

Techniques: Binding Assay, Sequencing, Protein Concentration

Journal: The Journal of Biological Chemistry

Article Title: Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere *

doi: 10.1074/jbc.M112.415984

Figure Lengend Snippet: Mass and oligomeric state of hRap1, hTRF2, and the TRF2-Rap1 complex on telomeric Holliday junctions Mass, mean area, and the oligomeric state of each protein were obtained from the two-dimensional projection analysis of the tungsten shadowcast images as in Figs. 1 and . Masses were calculated based on using ferritin as a size standard. Area distributions with Gaussian fits are shown in supplemental Fig. 5 .

Article Snippet: A nontelomeric template with a 3′ overhang was generated by treating EcoRI-digested pGLGAP, which does not contain telomeric repeats ( 29 ), with T7 exonuclease at room temperature for 40 s followed by incubation on ice for 5 min (New England Biolabs).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere *

doi: 10.1074/jbc.M112.415984

Figure Lengend Snippet: Affinity of hRap1, hTRF2, and the TRF2-Rap1 complex on telomeric and nontelomeric DNA templates The dissociation constants of hRap1, hTRF2, and the TRF2-Rap1 complex on the telomeric and nontelomeric DNA templates with a 3′ overhang or on the duplex telomere DNA are presented derived from electrophoretic experiments shown in the supplemental Figs. 6 and 7 . The Hill coefficient values of each protein on the model telomeric DNA are shown with the error values.

Article Snippet: A nontelomeric template with a 3′ overhang was generated by treating EcoRI-digested pGLGAP, which does not contain telomeric repeats ( 29 ), with T7 exonuclease at room temperature for 40 s followed by incubation on ice for 5 min (New England Biolabs).

Techniques: Derivative Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Rap1 Interacts Directly with Telomeric DNA and Regulates TRF2 Localization at the Telomere *

doi: 10.1074/jbc.M112.415984

Figure Lengend Snippet: Model for the TRF2-Rap1 complex loading onto and protecting the telomere. (1), the nontelomeric and the telomeric DNA are presented in solid lines and in dashed lines (2), respectively. The new telomeric DNA is synthesized in S phase (3). hTRF2 and hRap1 bind to the newly synthesized ds telomeric DNA as a complex and then end resection occurs forming the 3′ overhang (4). The TRF2-Rap1 complexes slide along the ds telomeric DNA toward the newly formed 3′ overhang (5). End protection by the TRF2-Rap1 complex occurs through t-loop formation or simply by blocking the exposed end (6). In the absence of hTRF2, hRap1 binds to the junction site to prevent NHEJ (or HDR in mice) and in the absence of hRap1, hTRF2 prevents DDR by forming t-loops.

Article Snippet: A nontelomeric template with a 3′ overhang was generated by treating EcoRI-digested pGLGAP, which does not contain telomeric repeats ( 29 ), with T7 exonuclease at room temperature for 40 s followed by incubation on ice for 5 min (New England Biolabs).

Techniques: Synthesized, Blocking Assay