Cancer Cell Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague CZ-14220, Czech Republic.
LifeB, Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno CZ-62500, Czech Republic.
Nucleic Acids Res. 2023 Feb 22;51(3):1154-1172. doi: 10.1093/nar/gkac1269.
Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.
蛋白磷酸酶镁依赖性 1 型 δ（PPM1D）通过去磷酸化肿瘤抑制蛋白 p53 来终止细胞周期检查点。通过靶向染色质上的其他底物，PPM1D 有助于控制 DNA 损伤反应和 DNA 修复。我们使用邻近生物素化 followed by proteomic analysis，鉴定了 PPM1D 与保护端粒 DNA 的 shelterin 复合物之间的新相互作用。此外，共聚焦显微镜显示内源性 PPM1D 定位于端粒。进一步，我们发现 ATR 在端粒双链断裂诱导后将 TRF2 磷酸化 S410，并且这种修饰在 PPM1D 抑制或缺失后增加。TRF2 磷酸化刺激其与 TIN2 的相互作用 both in vitro and at telomeres。相反，诱导表达 PPM1D 会损害 TIN2 和 TPP1 在端粒处的定位。最后，抑制 PPM1D 后，DNA 修复因子 53BP1 向端粒断裂的募集强烈减少，并且可以通过表达 TRF2-S410A 突变体来挽救。我们的结果表明，TRF2 磷酸化促进了 TIN2 在 shelterin 复合物内的结合，并调节了端粒处的 DNA 修复。
