Mutations are the underlying cause of cancer and contribute to metastasis and resistance to cancer therapeutics. In human cells, both perturbing transcription and inducing DNA-topoisomerase adducts are known to cause DNA damage. However, the mechanisms generating transcription- and DNA-topoisomerase adduct-associated mutations in human cells, the types of mutations caused by these processes, and their contribution to mutations in cancer are poorly understood. Using a novel mutation reporter in human cells, we observed a mutation spectrum dominated by 2- to 5-base pair (bp) deletions and distinct larger deletions. This mutation spectrum mirrors that of DNA topoisomerase 1-dependent, transcription-associated mutagenesis in S. cerevisiae, which indicates transcription-induced DNA-topoisomerase adducts produce these mutations in human cells. We additionally found that 2- to 5-bp deletions are enriched within highly expressed genes in primary breast cancers, suggesting DNA-topoisomerase adducts are important contributors to cancer etiology as deletions of ≥ 2 bp constitute between 3% and 12% of all inactivating mutations in tumor suppressor genes. The goal of this proposal is to characterize the basic determinants of transcription-associated, DNAtopoisomerase adduct-induced mutagenesis in human cells and assess its contribution to mutagenesis in cancer. Aim1 will determine the mechanistic basis of transcription-associated, DNA-topoisomerase adductinduced mutagenesis in human cells and define the spectrum of mutations generated by this process. This will be accomplished by measuring mutation rates and spectra utilizing mutation reporters for which we will modulate transcription via CRISPRi and CRISPRa and/or increase DNA-topoisomerase adducts by utilizing topoisomerase variants and inhibitors. Also, we will determine the contribution of various end joining DNA double strand break repair pathways and R-loop resolution to promoting or limiting specific types of DNAtopoisomerase adduct-induced mutations. Aim 2 will determine the genome-wide distribution of both DNATOP1 adducts at single nucleotide resolution using a novel genomics approach and DNA-TOP1 adductdependent mutations via whole genome sequencing of human cells. These unbiased, complementary distributions of adducts and mutations will be compared to the location of genomic features such as transcripts and R-loops to assess their influence on the occurrence and spectrum of DNA-TOP1 adduct-induced mutations. The distributions of these adducts and mutations will be further compared to the distribution DNATOP1 adduct signature mutations in sequenced human tumors to estimate the contribution of DNA-TOP1 adducts to mutagenesis in cancer. Completion of these aims will provide insight into the roles DNAtopoisomerase adducts in cancer etiology, determine the primary mechanism(s) that generate transcriptionassociated mutations, and create experimental systems that will facilitate future studies on the effects that DNA structures, genetic determinates, and environmental exposures have on this understudied mutagenic pathway.