Project Summary/Abstract Here, we propose to study cancer etiology in the context of DNA damage caused by a methylating agent, N- nitrosodimethylamine (NDMA), a probable human carcinogen that has been found in water, food, and drugs. NDMA is both mutagenic and toxic, and our overriding hypothesis is that its biological consequences are shaped interactions among three repair pathways: direct reversal, mismatch repair (MMR), and homologous recombination (HR). One of the key DNA lesions created by NDMA is O6MeG, which mispairs readily with thymine. The direct reversal protein O6-methylguanine DNA methyltransferase (MGMT) removes the offending methyl group, restoring the structure of guanine. Interestingly, O6MeG can become toxic when acted upon by Mismatch Repair (MMR), although the underlying mechanism remains to be fully elucidated in vivo. Normally, MMR recognizes mismatches behind the replication fork and removes the newly synthesized strand to give the cell another chance at accurate replication. In the case of O6MeG, it has been posited that MMR removes the strand opposite the lesion forming a single strand gap that upon the subsequent replication cycle becomes a broken fork (i.e., a double strand break [DSB]) that requires HR for repair. Despite the prevalence of this model in the literature, these predicted processes have been largely untested in vivo, a key gap in the literature that will be addressed in part by using an in-house genetically engineered mouse model, namely RaDR, for which HR yields a fluorescent signal. An advantage of RaDR is that it can also be used for lineage tracing, which makes it possible to monitor clonal expansion. Using the RaDR mice, we made the remarkable discovery that MGMT strongly suppresses clonal expansion. Our overriding hypothesis is that MMR promotes toxicity and inflammation, providing selective pressure for clonal expansion. Specific Aim 1 is to test the MMR model by measuring the replication dependence of DSBs and HR to elucidate possible dependence on two cycles of replication, which would be consistent with gap-driven fork breakdown. Specific Aim 2 is to quantify clonal expansion, to leverage 2-photon microscopy for whole-organ 3D imaging of clonal outgrowths, and to peer into clonal outgrowths via spatial transcriptomics to reveal underlying mechanisms of clonal expansion. Specific Aim 3 is to quantify the impact of MGMT, MMR, and their interaction on NDMA-induced inflammation and cancer in the liver. The proposed work will not only elucidate the ways by which interactions among DNA repair pathways modulate susceptibility to DNA damage, but it will also be one of the first studies of how DNA repair shapes the risk of clonal expansion, a fundamentally important step in carcinogenesis. Together, these studies will have a significant and lasting impact by advancing our understanding of the molecular forces that shape disease with important implications to public health and the clinic.