Legartová Soňa, Fagherazzi Paolo, Goswami Pratik, Brazda Vaclav, Lochmanová Gabriela, Koutná Irena, Bártová Eva
Department of Cell Biology and Epigenetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
Department of Cell Biology and Epigenetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic; Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
Biochimie. 2023 Jan;204:154-168. doi: 10.1016/j.biochi.2022.09.013. Epub 2022 Sep 24.
An essential factor of the DNA damage response is 53BP1, a multimeric protein that inhibits the resection-dependent double-strand break (DBS) repair. The p53 protein is a tumor suppressor known as a guardian of the genome. Although the interaction between 53BP1 and its p53 partner is well-known in regulating gene expression, a question remains whether genome injury can affect the interaction between 53BP1 and p53 proteins or p53 binding to DNA. Here, using mass spectrometry, we determine post-translational modifications and interaction properties of 53BP1 and p53 proteins in non-irradiated and γ-irradiated cells. In addition, we used Atomic Force Microscopy (AFM) and Fluorescent Lifetime Imaging Microscopy combined with Fluorescence Resonance Energy Transfer (FLIM-FRET) for studies of p53 binding to DNA. Also, we used local laser microirradiation as a tool of advanced confocal microscopy, showing selected protein accumulation at locally induced DNA lesions. We observed that 53BP1 and p53 proteins accumulate at microirradiated chromatin but with distinct kinetics. The density of 53BP1 (53BP1pS1778) phosphorylated form was lower in DNA lesions than in the non-specified form. By mass spectrometry, we found 22 phosphorylations, 4 acetylation sites, and methylation of arginine 1355 within the DNA-binding domain of the 53BP1 protein (aa1219-1711). The p53 protein was phosphorylated on 8 amino acids and acetylated on the N-terminal domain. Post-translational modifications (PTMs) of 53BP1 were not changed in cells exposed to γ-radiation, while γ-rays increased the level of S6ph and S15ph in p53. Interaction analysis showed that 53BP1 and p53 proteins have 54 identical interaction protein partners, and AFM revealed that p53 binds to both non-specific and TP53-specific sequences (AGACATGCCTA GGCATGTCT). Irradiation by γ-rays enhanced the density of the p53 protein at the AGACATGCCTAGGCATGTCT region, and the binding of p53 S15ph to the TP53 promoter was potentiated in irradiated cells. These findings show that γ-irradiation, in general, strengthens the binding of phosphorylated p53 protein to the encoding gene.
DNA损伤反应的一个关键因素是53BP1，它是一种多聚体蛋白，可抑制依赖切除的双链断裂（DBS）修复。p53蛋白是一种肿瘤抑制因子，被称为基因组守护者。尽管53BP1与其p53伙伴之间的相互作用在调节基因表达方面广为人知，但一个问题仍然存在，即基因组损伤是否会影响53BP1与p53蛋白之间的相互作用或p53与DNA的结合。在这里，我们使用质谱法确定了未照射和γ照射细胞中53BP1和p53蛋白的翻译后修饰和相互作用特性。此外，我们使用原子力显微镜（AFM）以及结合了荧光共振能量转移（FLIM-FRET）的荧光寿命成像显微镜来研究p53与DNA的结合。我们还使用局部激光微照射作为先进共聚焦显微镜的工具，显示特定蛋白质在局部诱导的DNA损伤处积累。我们观察到53BP1和p53蛋白在微照射的染色质处积累，但动力学不同。在DNA损伤处，磷酸化形式的53BP1（53BP1pS1778）密度低于未特定化形式。通过质谱分析，我们在53BP1蛋白（aa1219 - 1711）的DNA结合域内发现了22个磷酸化位点、4个乙酰化位点以及精氨酸1355的甲基化。p53蛋白在8个氨基酸上发生磷酸化，在N端结构域发生乙酰化。在暴露于γ辐射的细胞中，53BP1的翻译后修饰（PTM）没有变化，而γ射线增加了p53中S6ph和S15ph的水平。相互作用分析表明，53BP1和p53蛋白有54个相同的相互作用蛋白伙伴，AFM显示p53与非特异性和TP53特异性序列（AGACATGCCTAGGCATGTCT）都结合。γ射线照射增强了AGACATGCCTAGGCATGTCT区域的p53蛋白密度，并且在照射细胞中p53 S15ph与TP53启动子的结合增强。这些发现表明，一般来说，γ射线照射会增强磷酸化p53蛋白与编码基因的结合。
