Abstract In Saccharomyces cerevisiae, Sam1, and its paralog Sam2, play roles in the methyl cycle catalyzing the biosynthesis of AdoMet. Despite Sam1 and Sam2 having high levels of homology and findings that both proteins localize to the cytoplasm, differences in abundance and regulation of expression speak to the differential function and use of these proteins by the cell. In humans, three genes, MAT1A, MAT2A, and MAT2B, encode subunits of the homologous AdoMet synthetases. These genes, and their product AdoMet, have been implicated in multiple cancer types, but the mechanism of action is not well understood because both increases and decreases in expression are associated with cancers. Our group has conducted studies of SAM gene dosage and determined the impacts of changes in AdoMet synthetase genes on genome stability. SAM1 and SAM2 clearly operate by two distinct mechanisms to impart different impacts on genome stability. This proposal contains the next logical steps to improve our understanding of the SAM genes and impacts of changes to the methyl cycle on cellular processes including genome stability. Through the first iteration of this grant, we have found that the increased instability in sam2Δ/sam2Δ cells is linked to increased dNTP levels, and studies proposed here aim to define the mechanism of increased dNTPs and how they increase recombination and breakage/NDJ events. We also found that the increased stability in sam1Δ/sam1Δ cells is not due to increased GSH or polyamines, or decreased ROS, and therefore hypothesize stability may come from the increased AdoMet levels in methylations or via suppressing more reactive methyl donors. We seek to define the protective effects of increased AdoMet, as well as changes in the proteomes of our mutants due to changes in AdoMet-dependent methyltransferase function. We also propose studies to better understand the regulation and functionality of the Sam1 and Sam2 proteins, to provide context to the opposite effects observed due to loss of one or the other. Together, these studies will aid our understanding of the differences in the roles of the unlinked SAM1 and SAM2 genes and how altered expression of the homologous genes and/or changes in AdoMet levels might be implicated in disease development and treatments. Finally, these studies will train numerous undergraduate students in hypothesis- based research utilizing the genetically malleable yeast system ideal for teaching genetics and molecular biology and focusing on their development as scientists and scientifically literate citizens. This proposal meets the stated objectives of the R15 AREA program by involving undergraduate students in hypothesis driven research and strengthening the research environment at NKU.