Structural Biology of Lysine Methylation in DNA Damage and Checkpoint Signaling

DNA 损伤和检查点信号转导中赖氨酸甲基化的结构生物学

• 批准号: 8016109
• 负责人: Georges Mer
• 金额: $ 30.41万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8016109
• 关键词: Acetylation; Affinity; Antigens; Apoptosis; BRCT Domain; Binding; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell physiology; Cells; Collaborations; Complex; Coupling; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Repair; DNA damage checkpoint; DNA repair protein; Data; Disulfides; Double Strand Break Repair; Epigenetic Process; Eukaryota; Fission Yeast; Genetic; Genome Stability; Glioma; Health; Histone H3; Histone H4; Histones; Human; Hybrids; In Vitro; Knowledge; Light; Link; Lysine; Malignant Neoplasms; Malignant neoplasm of brain; Methylation; Modification; Molecular Probes; Mutation; NMR Spectroscopy; Organism; Orthologous Gene; Peptides; Phosphorylation; Post-Translational Protein Processing; Prevention; Process; Protein p53; Proteins; Public Health; Recruitment Activity; Regulation; Research; Role; Series; Serine; Signal Transduction; Site; Specificity; Structure; Testing; Time; Transducers; Tumor Suppressor Proteins; Work; base; cancer therapy; crosslink; design; dimer; histone acetyltransferase; human 53BP1 protein; human H2AX protein; in vivo; neoplastic cell; prevent; repaired; research study; response; structural biology; three dimensional structure

DESCRIPTION (provided by applicant): Our long-term objective is to help decipher the mechanism of DNA double-strand break (DSB) repair, a process indispensable in maintaining genomic stability in all organisms, and an important barrier to cancer in higher eukaryotes. Reversible site-specific post-translational modifications of histone and non-histone proteins are essential for the DNA repair machinery to assemble at sites of DNA DSBs and to the concomitant checkpoint activation leading to cell cycle arrest or apoptosis, but how these modifications contribute to the DNA damage response is poorly understood. Our proposed research focuses on understanding the role of lysine methylation in DNA DSB and cell cycle checkpoint signaling. We will probe several molecular interactions driven by the methylation of histone H4 at lysine 20 (H4-K20) and p53 at lysine 370 (p53-K370), and their possible synergistic coupling to other post-translational modifications (e.g. phosphorylated histone H2AX). In particular, we will study the interactions of methylated histone H4-K20 and methylated p53-K370 with human proteins 53BP1, JMJD2A and PHF20. 53BP1 is a key transducer of the cell response to DNA DSBs that participates in the assembly of DNA repair proteins and in checkpoint activation regulated by p53. JMJD2A is a histone demethylase whose function is linked to p53-dependent DNA damage-induced apoptosis. PHF20 is a component of the MLL1/MOF histone acetyltransferase complex that acetylates histone H4 in a p53 dependent manner in connection to DNA damage. For each study, we will apply a combination of biophysical experiments centered on nuclear magnetic resonance (NMR) spectroscopy and crystallographic three-dimensional (3D) structure determination of selected domains in complex with their associated targets. In Aim1, we will test the hypothesis that 53BP1 simultaneously recognizes methylated H4-K20 and phosphorylated histone H2AX using tandem tudor and tandem BRCT domains, respectively. This dual binding mode would explain how 53BP1 is recruited to DNA damage sites. In Aims 2 and 3, we will test the hypotheses that methylation of p53 at lysine 370 triggers a tight interaction of p53 with 53BP1 and JMJD2A, preventing p53 from binding DNA. This would provide a mechanism for the known inhibition of p53 transcriptional activity by methylation of lysine 370. In Aim 4, we have identified a new motif in PHF20 that we call disulfide cross-linked tudor dimer. This tudor domain is found in tandem with an MBT domain. We will characterize the tudor, MBT and tandem MBT-tudor domains of PHF20 in complex with methylated H4-K20 and p53-K370 peptides. Our structural and interaction studies at the atomic level will be the basis for informed design of in vivo experiments that will elevate our knowledge of DNA DSB signaling and repair. Alteration of histone H4 methylation at Lys20 is a hallmark of human tumor cells and mutation of p53 occurs in about 50% of human cancers. In the long term, our studies involving methylated histone H4 and p53 may help control the DNA damage response therapeutically for the prevention and treatment of cancer. PUBLIC HEALTH RELEVANCE The proposed studies will contribute to the elucidation of fundamental cellular processes involved in detecting and repairing DNA double-strand breaks, one of the most harmful types of DNA damage. The work has relevance to public health because by understanding the mechanisms of these important processes, we may be able to find ways to prevent and treat human malignancies, particularly cancer.

描述（由申请人提供）：我们的长期目标是帮助破译DNA双链断裂（DSB）修复的机制，这是维持所有生物体基因组稳定性不可或缺的过程，也是高等真核生物癌症的重要屏障。组蛋白和非组蛋白的可逆位点特异性翻译后修饰对于DNA修复机制在DNA dsb位点组装以及伴随的检查点激活导致细胞周期阻滞或凋亡至关重要，但这些修饰如何促进DNA损伤反应尚不清楚。我们提出的研究重点是了解赖氨酸甲基化在DNA DSB和细胞周期检查点信号传导中的作用。我们将探索由组蛋白H4在赖氨酸20位点（H4- k20）和p53在赖氨酸370位点（p53- k370）甲基化驱动的几种分子相互作用，以及它们与其他翻译后修饰（例如磷酸化组蛋白H2AX）可能的协同偶联。特别是，我们将研究甲基化组蛋白H4-K20和甲基化p53-K370与人蛋白53BP1、JMJD2A和PHF20的相互作用。53BP1是细胞对DNA dsb反应的关键换能器，参与DNA修复蛋白的组装和p53调控的检查点激活。JMJD2A是一种组蛋白去甲基化酶，其功能与p53依赖性DNA损伤诱导的细胞凋亡有关。PHF20是MLL1/MOF组蛋白乙酰转移酶复合物的一个组成部分，该复合物以p53依赖的方式使组蛋白H4乙酰化，与DNA损伤有关。对于每一项研究，我们将结合以核磁共振（NMR）光谱为中心的生物物理实验和晶体三维（3D）结构确定为中心的复杂结构域及其相关目标。在Aim1中，我们将验证53BP1分别通过串联tudor和串联BRCT结构域同时识别甲基化的H4-K20和磷酸化的组蛋白H2AX的假设。这种双重结合模式可以解释53BP1是如何被招募到DNA损伤位点的。在目标2和目标3中，我们将验证赖氨酸370处p53甲基化触发p53与53BP1和JMJD2A紧密相互作用的假设，从而阻止p53结合DNA。这将为赖氨酸370甲基化抑制p53转录活性提供一种已知的机制。在Aim 4中，我们在PHF20中发现了一个新的基序，我们称之为二硫交联都铎二聚体。这个都铎域是与MBT域串联发现的。我们将表征PHF20与甲基化的H4-K20和p53-K370肽复合物的tudor、MBT和串联MBT-tudor结构域。我们在原子水平上的结构和相互作用研究将为体内实验的知情设计奠定基础，这将提高我们对DNA DSB信号传导和修复的认识。Lys20位点组蛋白H4甲基化的改变是人类肿瘤细胞的一个标志，大约50%的人类癌症发生p53突变。从长远来看，我们对甲基化组蛋白H4和p53的研究可能有助于从治疗上控制DNA损伤反应，从而预防和治疗癌症。拟议的研究将有助于阐明涉及检测和修复DNA双链断裂（最有害的DNA损伤类型之一）的基本细胞过程。这项工作与公共卫生有关，因为通过了解这些重要过程的机制，我们可能能够找到预防和治疗人类恶性肿瘤，特别是癌症的方法。