DNA–Protein Cross-Linking Sequencing for Genome-Wide Mapping of Abasic Sites at Single-Nucleotide Resolution

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Project Summary Endogenous metabolism and environmental exposure can result in a battery of DNA lesions. Failure to repair these DNA lesions gives rise to genome instability and mutation accrual, which lead to accelerated aging, cancer, and neurodegenerative diseases. Abasic site (AP site) is one of the most prevalent forms of DNA damage. Although AP sites are known to be repaired by AP endonuclease in human cells, the dynamics of AP site induction and repair across the genome and the cellular factors modulating these processes remain elusive. In this application, we aim to explore how site-specific induction and repair of AP sites are affected by toxicant exposure and epigenetic alterations. We propose three specific aims to achieve our objective: 1) we will develop a DNA-protein cross-linking followed by next-generation sequencing (DPC-Seq) method for mapping, at single- base resolution, AP sites in the human genome after toxicant exposure; 2) we will explore how the formation and repair of AP sites are influenced by epigenetic state of chromatin; and 3) we will employ DPC-seq and whole- genome mutation analysis to examine how AP sites contribute to mutational signatures observed in cancer genomes. To accomplish this proposal and enable my transition to an independent academic career, I assembled a mentoring committee with expertise in animal models, bioinformatics, mutagenesis, and toxicology. The proposed research is built upon my research experience/skill sets, strong preliminary data, and the proposed mentoring plan. The outcome of the proposed research will establish a new method for mapping AP sites at single-base resolution and provide important insights into the occurrence and repair of AP sites and how they are influenced by toxicant exposure and genomic contexts. The proposed research will also reveal the roles of AP sites in cancer etiology, which will ultimately lead to better strategies for the prevention and therapeutic interventions of human cancer. Moreover, this proposal will fill gaps in my training and collect the data for my independent publications and research grant applications, thereby enabling me to transition to become an independent scientist in the field of environmental toxicology.