Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Biphasic TRF2 recruitment to non-telomeric damage sites in nuclei of HeLa cells. (A) PAR stimulation by PARG inhibition (PARGi) promotes GFP–TRF2 accumulation at low input-power damage sites (indicated by arrowheads). Box plot shows quantification of the relative increase of GFP signals at damage sites. (B) Time-course analysis of GFP–TRF2 recruitment to laser-induced DNA damage sites (between arrowheads). (C) Quantification of GFP signals at damage sites in B. N=16. (D) Detection of endogenous TRF2 at damage sites. PARP inhibition (PARPi) suppresses phase I, but has no effect on phase II, TRF2 recruitment. (E) Quantification of the effects of PARP inhibitors (NU1025 and olaparib) on immediate (1 min, phase I) and late (30 min, phase II) GFP–TRF2 recruitment. (F) Time course analysis of the effect of IDP depletion on dispersion of TRF2 at damage sites in HeLa cells transfected with control siRNA (siControl) or FET siRNAs (siFET). Left: quantification of signal intensity changes of GFP–TRF2 (blue) and dark line (red). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Inhibition, Dispersion, Transfection, Control

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Distinct TRF2 domain requirement for phase I and II recruitment. (A) Schematic diagrams of TRF2 deletion mutants. (B) Time course analysis of damage site localization (between arrowheads) of wild-type and TRF2 deletion mutants in the nuclei of HeLa cells. (C) Box plot shows quantification of TRF2 mutant GFP signals at damage sites at 1 min (phase I) and 30 min (phase II) post-damage induction. (D) Box plot shows quantification on the effects of the N-terminal amino acid substitutions on phase I recruitment of TRF2. Arginine-to-alanine mutations (RA), arginine-to-lysine substitution (RK) and the HJ binding mutation (H31A) were tested. WT, wild type. Amino acid sequences of N-terminal domain mutations are shown on the right. In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Mutagenesis, Binding Assay

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Phase II recruitment is affected by TERT and is dependent on the iDDR region in the hinge domain of TRF2. (A) Box plot shows that TERT depletion using siRNA inhibits phase II recruitment of TRF2 to DNA damage sites in nuclei of HeLa cells. (B) Schematic diagrams of chimeric TRF1/2 mutants (as previously described in Okamoto et al., 2013) used in the experiments represented in panels C–F. (C) Left: representative cell images of the recruitment of chimeric mutants to damage sites (between arrowheads) at ∼1 min (phase I) and ∼30 min (phase II) after damage induction. Right: box plots show quantification of the GFP–TRF2 signal increase at phase I and phase II at damage sites. (D) Comparison of the GFP signal at damage sites in HeLa cells expressing iDDR and TIN2 deletion mutants at 30 min after damage induction. (E) The effect of MRE11 and NBS1 siRNA (siMRE11 and siNBS1) depletion on phase I and II recruitment of GFP–TRF2 was examined comparing to control siRNA (siControl). HeLa cells were fixed and stained with anti-MRE11 antibody (red) to confirm the depletion. Box plot shows quantification of the GFP–TRF2 signal increase at damage sites in control or MRE11 and NBS1 siRNA-treated cells. (F) ChIP-qPCR analysis of GFP–TRF2 binding at I-PpoI cut sites. TRF2 binding was examined in the absence or presence of I-PpoI, and with and without MMS as indicated. Cells were further treated with DMSO or PARP inhibitor (PARPi) (left panel). Cells expressing GFP only were used as a negative control. Alternatively, cells were transfected with control (siCon) or MRE11 and NBS1 siRNA in the presence of I-PpoI with or without MMS (right panel). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Bar graphs show mean±s.d., *P<0.01, **P<0.001, ***P<0.0001. Scale bars: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Comparison, Expressing, Control, Staining, ChIP-qPCR, Binding Assay, Negative Control, Transfection

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: TRF2 specifically promotes non-sister chromatid HR repair. (A) The effect of TRF2 depletion on DSB repair using the I-SceI HR system. Complementation analysis of TRF2-depleted cells was performed using the wild type and chimeric TRF1/2 mutants. BRCA1 depletion was used as a positive control. Comparable expression levels of the recombinant TRF2 proteins were confirmed using western blot analysis (right). Histone H3 serves as a loading control. (B) The effect of TRF2 depletion on different DSB repair pathways was examined using SCE, classic NHEJ (C-NHEJ) and alternative NHEJ (Alt-NHEJ) assays. (C) Schematic showing similarity between strand invasion in D-loop formation at telomeres and at DSB sites by TRF2. (D) Schematic showing biphasic mechanism of TRF2 recruitment to damage sites. Phase I involves PARP-dependent recruitment through the basic domain. Phase II is mediated by the MYB/SANT domain, which is also dependent on the iDDR region and the Mre11 complex. Bar graphs show mean±s.d.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Positive Control, Expressing, Recombinant, Western Blot, Control