Post-translational Modifications in DNA Damage Response: a Structural Perspective

DNA 损伤反应的翻译后修饰：结构视角

• 批准号: 8627747
• 负责人: Georges Mer
• 金额: $ 35.78万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-26 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8627747
• 关键词: Acute; Address; Affinity; Apoptosis; Avidity; BRCA1 gene; Binding; Biological Assay; Bypass; Cell Cycle; Cell Cycle Progression; Cell Death; Cell physiology; Cells; Cellular Assay; Chromatin; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Data; Detection; Disease; Double Strand Break Repair; Drug resistance; Evaluation; Event; Excision; Genome; Genomics; Goals; Health; Histone H2A; Histone H4; Histones; Human; In Vitro; Knowledge; Laboratories; Lysine; Maintenance; Malignant Neoplasms; Measures; Mediator of activation protein; Methods; Methylation; Molecular; Mono-S; Mutation; Nonhomologous DNA End Joining; Nucleosome Core Particle; Nucleosomes; Pathway interactions; Peptides; Phenotype; Phosphotransferases; Polymerase; Post-Translational Protein Processing; Predisposition; Process; Proteins; Public Health; Recruitment Activity; Regulation; Research; Resistance; Role; Signal Transduction; Site; Specificity; Structure; Surface; Therapeutic; Time; Ubiquitin; Work; base; biophysical techniques; cancer cell; cancer therapy; design; human DNA damage; in vivo; interest; link protein; mutant; neoplastic cell; prevent; protein complex; protein structure function; repaired; response; stoichiometry; tumor; ubiquitin ligase

Our long-term goal is to help decipher how cells process DNA lesions in the maintenance of genomic integrity. The DNA damage response (DDR) involves protein complexes that sense DNA lesions and coordinate cell cycle progression along with DNA repair, apoptosis and DNA damage tolerance pathways. Reversible post-translational modifications (PTMs) of histone and non-histone proteins are essential for the DDR machinery to assemble at DNA damage sites but the mechanisms are poorly understood. Our proposed research focuses on probing, from a structural perspective, how PTMs participate in the control of time- and space-dependent protein associations in the DDR. Specifically, we will investigate the roles of lysine methylation and ubiquitylation in the repair of DNA double-strand breaks (DSBs) and in DNA damage tolerance by translesion DNA synthesis (TLS). 53BP1 is an important mediator of DNA repair by non-homologous end joining. In response to DSBs, 53BP1 associates with histone H4 dimethylated at lysine 20 (H4K20me2), which, in the absence of damage, is thought to be complexed to the lysine demethylase JMJD2A. Upon DSB induction, several ubiquitin ligases such as RNF8 are recruited to damage sites where they ubiquitylate histones and JMJD2A. The ubiquitylation of JMJD2A triggers its degradation, facilitating 53BP1 interaction with H4K20me2 and relocalization to DSBs. Under Aim 1, we will probe the recruitment mechanisms of 53BP1, JMJD2A and structurally related protein PHF20. We will characterize interactions of these proteins with the nucleosome core particle. Under Aim 2, we will investigate how mono-ubiquitylation of the DNA clamp PCNA in response to DNA damage activates damage tolerance by TLS. We will probe how the TLS DNA polymerase Rev1 and TLS are activated by PCNA ubiquitylation. Our hypothesis is that ubiquitylation alters PCNA–Rev1 interaction and functions as a double-switch to turn on TLS. Our studies will be the basis for designing in vivo assays that can elevate our knowledge of the DDR. Alteration of H4K20 methylation is a hallmark of human tumor cells and mutations in several DDR proteins are linked to cancer predispositions in humans. Because loss of 53BP1 reverses cancer phenotypes of BRCA1 mutant cells, inhibition of 53BP1 recruitment to DSB sites could have potential for cancer therapy. Rev1 and TLS contribute to the acquired therapeutic resistance of cancer cells.

我们的长期目标是帮助破译细胞如何处理DNA损伤以维持基因组的完整性。DNA损伤反应（DDR）涉及感知DNA损伤并与DNA修复、凋亡和DNA损伤耐受途径一起沿着协调细胞周期进程的蛋白复合物。组蛋白和非组蛋白的可逆翻译后修饰（PTM）对于DDR机制在DNA损伤位点组装是必不可少的，但其机制知之甚少。我们的研究重点是从结构的角度探讨PTM如何参与DDR中时间和空间依赖性蛋白质关联的控制。具体来说，我们将研究赖氨酸甲基化和泛素化在DNA双链断裂（DSB）修复和跨损伤DNA合成（TLS）的DNA损伤耐受中的作用。53 BP 1是DNA非同源末端连接修复的重要介质。在对DSB的反应中，53 BP 1与组蛋白H4在赖氨酸20处二甲基化（H4 K20 me 2）相关联，在没有损伤的情况下，H4 K20 me 2被认为与赖氨酸脱甲基酶JMJD 2A复合。在DSB诱导后，几种泛素连接酶如RNF 8被募集到损伤位点，在那里它们使组蛋白和JMJD 2A泛素化。JMJD 2A的泛素化引发其降解，促进53 BP 1与H4 K20 me 2的相互作用并重新定位到DSB。在目标1下，我们将探索53 BP 1，JMJD 2A和结构相关蛋白PHF 20的募集机制。我们将描述这些蛋白质与核小体核心颗粒的相互作用。在目标2下，我们将研究DNA钳PCNA的单泛素化如何响应DNA损伤激活TLS的损伤耐受。我们将探讨TLS DNA聚合酶Rev 1和TLS如何被PCNA泛素化激活。我们的假设是，泛素化改变了PCNA-Rev 1的相互作用，并作为一个双开关打开TLS的功能。我们的研究将是设计体内试验的基础，可以提高我们对DDR的认识。H4 K20甲基化的改变是人类肿瘤细胞的标志，并且几种DDR蛋白的突变与人类的癌症易感性有关。由于53 BP 1的缺失逆转了BRCA 1突变细胞的癌症表型，因此抑制53 BP 1募集到DSB位点可能具有癌症治疗的潜力。Rev 1和TLS有助于癌细胞的获得性治疗抗性。