Delineating molecular mechanisms of DNA repair through protein engineering

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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.