Delineating molecular mechanisms of DNA repair through protein engineering

DNA修复是一个基本的生物学过程，是所有生物基因组稳定性和完整性的重要保障。在蛋白质水平上，DNA修复组分主要受两种翻译后修饰的调控：泛素化和磷酸化。它们共同构成了检测DNA损伤的信号事件，向受损的染色质募集蛋白质，并激活修复机制以恢复受损的DNA。然而，对于泛素化和磷酸化过程中个体蛋白-蛋白相互作用（PPI）如何在综合信号网络中引发生物学功能的理解仍然很少。因此，迫切需要一种可以很容易地适应不同系统进行功能分析的合成PPI改性剂。为了解决这个问题，我设计了一种蛋白质工程策略来开发新的基于蛋白质的PPI抑制剂和激活剂，这使得泛素化系统中不同蛋白质家族的新的生化机制和新的生物学功能得以发现。展望未来，我的研究计划的长期目标是建立一个生物分子工程平台，用于开发有效和特异性的PPI调节剂，以前所未有的精度探测和重新连接DNA修复信号，以了解潜在的分子机制。在接下来的五年里，我的短期目标是了解中心DNA修复E3泛素连接酶BRCA1- bard1如何触发细胞效应以保持基因组完整性，以及多个BRCA1蛋白复合物如何反折叠磷酸化信号以协调DNA双链断裂（DSBs）的修复，DSBs是最具细胞毒性的DNA损伤类型。为了实现这些目标，我们将设计各种蛋白质支架，用于合成BRCA1-BARD1酶活性和复合物组装的抑制剂和激活剂。这些合成分子将使我们对BRCA1-BARD1在DNA损伤信号转导和DSB修复途径选择中的作用的理解取得重大进展。此外，新的泛素化底物和新的磷酸依赖的BRCA1-BARD1相互作用将被发现，使我们和DNA修复研究界能够解剖人类细胞中DSB损伤信号传导的基本分子结构。因此，这项工作将对我们在DNA修复和其他信号通路中调节其他Ub和磷酸化依赖性PPIs的长期目标具有重要意义。至关重要的是，高素质的人员（HQP）将在蛋白质工程和合成生物学方面接受培训，发展技能和专业知识，这些技能和专业知识是利用最近的基因组学革命和解决关键生物过程中的基本问题所迫切需要的。

DNA repair is a fundamental biological process and an essential safeguard of genome stability and integrity for all living organisms. At the protein level, DNA repair components are mainly regulated by two types of post-translational modifications: ubiquitination and phosphorylation. Together, they constitute signaling events that detect DNA damage, recruit proteins to the damaged chromatin, and activate the repair machinery to restore damaged DNA. However, it is still poorly understood how individual protein-protein interaction (PPI) in ubiquitination and phosphorylation elicits biological functions in the context of integrated signaling networks. Thus, there is an urgent need of synthetic PPI modifiers that can be easily adapted into different systems for functional analysis. To solve this problem, I have devised a protein engineering strategy to develop novel protein-based PPI inhibitors and activators, which enabled discovery of new biochemical mechanisms and new biological functions for diverse protein families in the ubiquitination system. Going forward, the long-term goal of my research program is to establish a biomolecular engineering platform for the development of potent and specific PPI modulators to probe and rewire DNA repair signaling with unprecedented precision for underlying molecular mechanisms. Over the next five years, my short-term goal is to understand how the central DNA repair E3 ubiquitin ligase BRCA1-BARD1 triggers cellular effects to preserve genome integrity and how multiple BRCA1 protein complexes deconvolute phosphorylation signals to orchestrate repair of DNA double-strand breaks (DSBs), the most cytotoxic type of DNA damage. To achieve these objectives, we will engineer a variety of protein scaffolds for synthetic inhibitors and activators of BRCA1-BARD1 enzymatic activity and complex assembly. Theses synthetic molecules will empower significant advances of our understanding for BRCA1-BARD1's role in DNA damage signal transduction and DSB repair pathway choice. Moreover, new ubiquitination substrates and new phospho-dependent interactions of BRCA1-BARD1 will be identified, allowing us and the DNA repair research community to dissect the fundamental molecular architecture of DSB damage signaling in human cells. This work will therefore have critical implications for our long-term goal of modulating other Ub- and phospho-dependent PPIs in DNA repair and other signaling pathways. Crucially, highly qualified personnel (HQP) will be trained in protein engineering and synthetic biology, developing skills and expertise that are urgently needed to capitalize on the recent genomics revolution and address fundamental questions in critical biological processes.