PROJECT SUMMARY Somatic mutations are the driving force behind carcinogenesis and other age-related diseases, yet the mechanisms underlying their genesis in humans are ambiguous. This proposal seeks to resolve fundamental processes of nuclear mutagenesis and ascertain the utility of monitoring somatic mutation as a biomarker of individual environmental exposure within a well-characterized population. These objectives are now feasible thanks to the development of a novel mutation detection technology termed CypherSeq, which utilizes circular barcoded templates, rolling circle amplification, and massively parallel sequencing to accurately quantify somatic mutation throughout the genome with unprecedented sensitivity. First, the CypherSeq method will be used to establish a genome-wide profile of somatic mutation in disease-free humans, and test how the frequency, distribution, and spectrum of mutation change with age throughout the genome. Second, proliferating and quiescent human cells will be treated with known mutagens, and induced mutagenesis will be tracked by CypherSeq. These experiments will explore the relationships between cell proliferation, transcription, DNA repair, and somatic mutation, while also uncovering highly mutable regions of the genome, which might serve as sites to monitor acute and chronic mutagen exposure in human populations. Further, this Aim will also test the hypothesis that proliferation is required for repair and mutation fixation within non- transcribed regions of the genome. Lastly, in the third Aim, robust mutational target sites will be characterized and used to monitor mutation frequency in blood draws from individuals who have endured a long-term, carcinogenic exposure to smoky coal. This cross-sectional study will test the hypothesis that underlying the carcinogenicity of smoky coal is its potential to increase the frequency of somatic mutation in exposed individuals. As such, it is expected that increased lifetime exposure to smoky coal will positively correlate with mutation induction. Successful completion of the proposed Aims would provide new insights into human mutagenesis, and highlight the potential utility of monitoring in vivo mutation induction as a biomarker of mutagenic environmental exposure; and thus identify individuals with an elevated risk of developing cancer. Ultimately, mutation-based biomarkers have the potential to stratify cancer risk, providing a basis to direct medical intervention, lifestyle changes (i.e. limiting mutagen exposure), early diagnosis, and/or the application of chemopreventive measures, and thus save lives.