ABSTRACT - PROJECT 3 DNA damage and its sequelae are now recognized as major drivers of aging. However, many questions remain regarding the nature of age-promoting DNA damage, the role of mutations in non-cancer age-related pathology, and how genomic damage and its sequelae drive age-related phenotypes and pathologies. Project 3 will determine -- in depth, breadth and primarily at the level of cellular responses -- how genes that participate in genome maintenance assure tissue health and ultimately longevity. The primary (but not the only) consequence-generating cell fate responses are cellular senescence and apoptosis. The extent to which cells in aged organisms acquire mutations and/or experience these cellular consequences due to DNA damage are poorly understood. We will explore the relationship between DNA damage-induced cell fates and mutations (with Project 2), molecular consequences of DNA damage (with Project 1), and human gene variants implicated in longevity (with Project 4). Specifically, we will determine cell fate responses of cells carrying genome maintenance genotypes associated with premature and delayed aging identified by Projects 1 and 4, focusing on multifunctional repair genes that participate in repair processes important for relieving replication or transcriptional stress. We will interrogate cellular senescence and apoptosis (and other forms of cell death), and markers of cell function. With Project 2, we will determine whether some genomic changes (e.g., aneuploidy, INDELS) preferentially elicit one cell fate over another. We will use simple and complex culture systems, focusing primarily on the skin, brain and liver and use single cell analyses to determine how different types of DNA damage affect cellular heterogeneity and variability. We will also determine the cellular responses to genotoxic stress in the backgrounds of genome maintenance genotypes and in response to genomic damage in wild-type and mutant mouse models, and naturally aged mice. We will exploit our mouse model in which senescent cells can be eliminated, or use pharmacological means to eliminate senescent cells, to determine how modifying this cell fate affects hallmarks of aging. Finally, once we have a candidate list of human gene variants associated with aging and longevity from Project 4, we will use human embryonic stem cells harboring these variants to assess the cells and their differentiated progeny for their cell fate responses to genomics stressors. Together, this Project will allow us to integrate the genomic and cellular responses to DNA damage during natural, accelerated and delayed aging in mouse and human cells, and provide mechanistic bases for predicting the efficacy of interventions into aging phenotypes and pathologies.