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.