Delineating molecular mechanisms of DNA repair through protein engineering

DNA修复是一个基本的生物学过程，也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修复成分主要受两种翻译后修饰的调节：泛素化和磷酸化。它们共同构成信号事件，检测DNA损伤，向受损的染色质招募蛋白质，并激活修复机制以恢复受损的DNA。然而，在整合信号网络的背景下，泛素化和磷酸化过程中的单个蛋白质-蛋白质相互作用(PPI)如何引发生物功能的研究仍然很少。因此，迫切需要合成的PPI修饰剂，这种修饰剂可以很容易地适应不同的体系进行功能分析。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型的基于蛋白质的PPI抑制剂和激活剂，这使得在泛素化系统中发现不同蛋白质家族的新的生化机制和新的生物学功能。展望未来，我研究计划的长期目标是建立一个生物分子工程平台，用于开发有效和特定的PPI调节器，以前所未有的精度探测和重连DNA修复信号，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央DNA修复E3泛素连接酶BRCA1-BARD1如何触发细胞效应，以保持基因组的完整性，以及多个BRCA1蛋白复合体如何解卷磷酸化信号，以协调修复DNA双链断裂(DSB)，这是最具细胞毒性的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BRCA1-BARD1酶活性和复杂组装的合成抑制剂和激活剂。这些合成的分子将使我们对BRCA1-BARD1‘S在DNA损伤信号转导和双链断裂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依赖于磷酸的BRCA1-BARD1相互作用将被确定，使我们和DNA修复研究社区能够剖析人类细胞中DSB损伤信号的基本分子结构。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依赖Ub和磷酸的PPI的长期目标具有关键意义。至关重要的是，高素质的人员(HQP)将接受蛋白质工程和合成生物学方面的培训，开发利用最近的基因组学革命和解决关键生物学过程中的根本问题所迫切需要的技能和专业知识。

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.