Deciphering the chromatin-based DNA damage response pathway

项目总结 DNA损伤是对我们基因组的持续威胁。我们的身体已经进化出一种监督机制，即 DNA损伤反应(DDR)途径，以保护我们的细胞免受遗传毒性侮辱。DDR通路导联缺陷 对DNA断裂进行不忠实的修复，导致突变、染色体重排和 基因组不稳定。尽管关于染色质如何感知和响应DNA损伤已知很多，但 关于染色质受损机制的描述不佳，存在着重大的知识差距 转导信号以招募效应蛋白到DNA断裂。这样的知识是必须理解的 细胞如何通过在时间和时间内招募正确的修复蛋白复合体来准确地执行正确的途径 空间方式对于维持我们基因组的完整性是必不可少的。这项研究计划旨在解决 这是以染色质为基础的DDR途径中的这个基本问题。我们的专长在于基因组编辑、功能 蛋白质组学、分子生物学、细胞生物学和生物化学。我们结合了我们的蛋白质纯化技术， 共聚焦显微镜，激光诱导的单细胞微照射，基于细胞的报告分析，蛋白质标记，以及 用于整体多学科方法的定量质谱学。米拉项目的目标是 通过1)测定以染色质为基础的DDR途径的 DDR途径的表观遗传学特征，2)新的DNA修复蛋白的功能特征和3) 阐明染色质损伤如何协调不同的DNA修复途径的机制 在染色质受损的背景下的细胞周期。利用分离纯化偶联蛋白质组学方法 基于细胞的功能分析，我们已经鉴定了20多个新的DNA修复蛋白。我们的长期合作 目标是在基于染色质的DDR途径内建立物理和遗传网络，并了解其 对人类健康的影响。总的来说，这些研究将为DNA修复领域开辟一个新的舞台，提供洞察力 深入研究癌症的病因学和基因组不稳定相关的遗传病，将为 翻译研究和治疗策略的发展。

PROJECT SUMMARY DNA damage is a constant threat to our genome. Our body has evolved a surveillance mechanism, namely the DNA damage response (DDR) pathway, to protect our cells from genotoxic insults. Defective DDR pathway leads to unfaithful repair of DNA breaks which results in accumulation of mutations, chromosome rearrangement, and genome instability. Although much is known about how chromatin senses and responds to DNA damage, a significant gap in knowledge exists with the poorly characterized mechanism by which damaged chromatin transduces the signal to recruit effector proteins to DNA breaks. Such knowledge is imperative to understand how cells precisely execute the correct pathway by recruiting the right repair protein complex in a temporal and spatial manner which is essential for maintaining our genome integrity. This research program aims to address this fundamental question in the chromatin-based DDR pathway. Our expertise lies in genome editing, functional proteomics, molecular biology, cell biology, and biochemistry. We combine our techniques of protein purification, confocal microscopy, laser-induced single cell micro-irradiation, cell-based reporter assays, protein tagging, and quantitative mass spectrometry for an overall multidisciplinary approach. The goals of this MIRA project are to obtain a comprehensive molecular understanding of the chromatin-based DDR pathway by 1) determining the epigenetic profile in the DDR pathway, 2) characterizing the functions of the novel DNA repair proteins and 3) elucidating the mechanism of how damaged chromatin orchestrates different DNA repair pathways during the cell cycle in the context of damaged chromatin. Using fractionation purification-coupled proteomic approach and cell-based functional assays, we have identified more than twenty novel DNA repair proteins. Our long-term goals are to build a physical and genetic network within the chromatin-based DDR pathway and understand its impact in human health. Overall, these studies will open up a new arena for the DNA repair field, provide insight into the etiology of cancer and genome instability-related genetic diseases that will lay the foundation for translational research and therapeutic strategy development.