Journal: Nucleic Acids Research

Article Title: DNA-PK controls Apollo’s access to leading-end telomeres

doi: 10.1093/nar/gkae105

Figure Lengend Snippet: DNA-PK kinase activity is not required for Apollo recruitment to telomeres. ( A ) Immunoblots for HA-Apollo (anti-HA), TRF2 and phosphorylated Chk2 in SV40LT-immortalized TRF2 F/F Lig4 -/− MEFs after retroviral transduction transduced with an empty vector (EV) or HA-Apollo, 108 h after Hit & Run Cre-mediated deletion of TRF2 and/or 24 h DNA-PKi treatment. (B and C) Representative immunofluorescence-fluorescence in-situ hybridization (IF-FISH) images and quantification of HA-Apollo localization at telomeres in the same MEFs as described in (A) 120 h after Hit & Run Cre transduction or without any treatment. Apollo and telomeres were detected using anti-HA (red) or Alexa488-OO-(TTAGGG) 3 probe (green), respectively. Approximately 300 nuclei were analyzed for each condition over n = 3 independent experiments (100 nuclei per experiment). Median bar is indicated. ( D ) Immunoblots for HA-Apollo (anti-DCLRE1B, Atlas) and actin in SV40-LT-immortalized DNA-PKcs +/+ , DNA-PKcs −/− or DNA-PKcs KD/KD MEFs transduced with an empty vector (EV) or HA-Apollo. * aspecific band. ( E and F ) IF-FISH analysis and quantification of HA-Apollo localization at telomeres in the SV40-LT-immortalized DNA-PKcs +/+ , DNA-PKcs −/− or DNA-PKcsK KD/KD MEFs transduced with an empty vector (EV) or HA-Apollo, analyzed as described in (B, C). For HA-Apollo, 300 nuclei over n = 3 independent experiments are shown (100 nuclei per experiment), with median bar. As negative control, 296 nuclei (at least 90 nuclei per experiment) were analyzed in parallel for EV. ( G ) Co-immunoprecipitation (Co-IP) of Myc-mTRF2 (WT or F120A) with HA-mApollo in 293T cells without or after 24 h treatment with DNA-PKi. Pull down was performed with anti-HA antibody. HA IP (first and second panels) and Input (third and fourth panels) were analyzed with anti Myc (first and third panels) or HA (second and fourth panels) antibodies. MYC-mTRF2-F120A (F120A) was used as negative control. Statistical analysis by non-parametric Kruskal–Wallis ANOVA test for multiple comparisons (C, F). Statistical significance was indicated by **** P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; n.s., not significant.

Article Snippet: Western blot was performed with 5% milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T) using the following antibodies: β-actin (#3700; Cell Signaling), Chk2 (BD 611570, BD Biosciences), DCLRE1B/Apollo (HPA064934, Atlas Antibodies), DNA-PKcs (SC-1552, SC-5282, SC-390495 and SC-390849; Santa Cruz Biotechnology), HA (HA.11, #901502 Biolegend), Ku70 (sc-17789 or sc-1487, Santa Cruz Biotechnology), Myc (9B11, mAb 2276; Cell Signaling), TRF2 (#13136, Cell Signaling) and secondary anti-Mouse/anti-Rabbit IgG HRP (Cytiva).

Techniques: Activity Assay, Western Blot, Retroviral, Transduction, Plasmid Preparation, Immunofluorescence, Fluorescence, In Situ Hybridization, Negative Control, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nucleic Acids Research

Article Title: DNA-PK controls Apollo’s access to leading-end telomeres

doi: 10.1093/nar/gkae105

Figure Lengend Snippet: Apollo interaction with DNA-PK mediates telomere protection. ( A ) Schematics of mArtemis and mApollo. The MBL, the β-CASP, the known Lig4, PTIP (Pax transactivation-domain interacting protein) and TRF2-interacting domains, and the DNA-PKcs interacting regions are indicated. ( B ) MUSCLE alignment of human and mouse Artemis and Apollo C-terminal tails. Amino acids are colored according to physico-chemical properties (Zappo). Highlighted, the contact points (CP) of Artemis with DNA-PKcs and the predicted DNA-PK-interacting region of Apollo, with a patch of positively charged amino acids followed by a patch of negatively charged ones. ( C ) Co-IP of endogenous DNA-PKcs and TRF2 with HA-mApollo-WT (WT) or HA-mApollo-ΔPK (ΔPK) in 293T cells. Pull-down was performed with anti-HA antibody. HA IP and input were analyzed by immunoblotting with anti-DNA-PKcs (top panels), TRF2 (middle panels) or HA (lower panel) antibodies. ( D ) Immunoblot of SV40LT-immortalized Apollo F/F MEFs transduced with either EV, HA-Apollo-WT or HA-Apollo-ΔPK 108 h after Hit & Run Cre-mediated deletion of endogenous Apollo. (E and F) IF-FISH analysis and quantification of Apollo localization at telomeres in the same MEFs as in ( D ). Graph represents 100 nuclei for each condition from n = 3 independent experiments (300 nuclei in total), with medians. (G and H) CO-FISH metaphase analysis and quantification of leading-end telomere fusions on MEFs treated as in (D). Graph represents 30 metaphases over n = 3 independent experiments with medians. Statistic by non-parametric Kruskal–Wallis ANOVA test for multiple comparisons (F, H). Statistical significance was indicated by **** P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; n.s., not significant.

Article Snippet: Western blot was performed with 5% milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T) using the following antibodies: β-actin (#3700; Cell Signaling), Chk2 (BD 611570, BD Biosciences), DCLRE1B/Apollo (HPA064934, Atlas Antibodies), DNA-PKcs (SC-1552, SC-5282, SC-390495 and SC-390849; Santa Cruz Biotechnology), HA (HA.11, #901502 Biolegend), Ku70 (sc-17789 or sc-1487, Santa Cruz Biotechnology), Myc (9B11, mAb 2276; Cell Signaling), TRF2 (#13136, Cell Signaling) and secondary anti-Mouse/anti-Rabbit IgG HRP (Cytiva).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transduction

Journal: Nucleic Acids Research

Article Title: DNA-PK controls Apollo’s access to leading-end telomeres

doi: 10.1093/nar/gkae105

Figure Lengend Snippet: Proposed model for Apollo recruitment and access to leading-end telomeres after replication. ( A ) Apollo is recruited at telomeres by TRF2 binding. The physical presence of inactive DNA-PK at the blunt DNA end prevents any nucleolytic attack of the ends, including Apollo’s. After autophosphorylation of DNA-PKcs on the ABCDE/Thr2609 cluster and Apollo interaction with DNA-PKcs, Apollo gains access to the telomere ends and perform the initial resection required for the generation of the 3′overhang and for telomere protection. ( B ) In the absence of Apollo, when Apollo cannot be recruited by TRF2 or cannot interact with DNA-PKcs, the leading-end telomeres are not resected and are fused via alt-EJ. ( C ) In the absence of DNA-PK autophosphorylation, due to inhibition of the kinase activity of DNA-PK and/or to mutations affecting the kinase activity and/or the ABCDE/Thr2609 cluster of DNA-PK, Apollo cannot get access to the telomere ends, the leading-end telomeres are not resected and are fused via the alt-EJ.

Article Snippet: Western blot was performed with 5% milk in PBS containing 0.1% (v/v) Tween-20 (PBS-T) using the following antibodies: β-actin (#3700; Cell Signaling), Chk2 (BD 611570, BD Biosciences), DCLRE1B/Apollo (HPA064934, Atlas Antibodies), DNA-PKcs (SC-1552, SC-5282, SC-390495 and SC-390849; Santa Cruz Biotechnology), HA (HA.11, #901502 Biolegend), Ku70 (sc-17789 or sc-1487, Santa Cruz Biotechnology), Myc (9B11, mAb 2276; Cell Signaling), TRF2 (#13136, Cell Signaling) and secondary anti-Mouse/anti-Rabbit IgG HRP (Cytiva).

Techniques: Binding Assay, Inhibition, Activity Assay