Summary Numerous studies in human populations, human tissue, animal models and cell culture demonstrate that environmental genotoxic and oxidative stress are associated with accelerated telomere shortening and dysfunction. Telomeres at chromosome ends are essential for genome stability and sustained cell proliferation, and dysfunctional telomeres contribute to degenerative diseases and carcinogenesis in humans. The goals of this project are to advance exciting discoveries and highly innovative work from two NIEHS funded R01 awards investigating the consequences of nucleobase damage and excision repair at telomeres. The overarching hypothesis for this R35 proposal is that telomere shortening and dysfunction caused by environmental genotoxic and oxidative stress, occurs via formation of specific base lesions and toxic repair intermediates that directly interfere with telomere replication and maintenance. Working with collaborators we pioneered a highly innovative chemoptogenetic tool that selectively induces DNA lesions at telomeres. This technology is transformative because targeting well-defined base damage to telomeres allows us to unequivocally attribute phenotypic changes and health outcomes to the induced telomere lesions, eliminating confounding effects of damage elsewhere. We fully validated this system for the targeted formation of a common oxidative guanine lesion at telomeres, and remarkably, we discovered that the chronic generation this lesion induces profound hallmarks of telomere dysfunction that mimic genetic loss of telomere shelterin proteins. This project will probe and uncover the mechanisms of DNA lesion induced telomere loss and dysfunction. A major strategy is to extend and modify this flexible technology in a phased approach for introducing base damage, toxic repair intermediates, bulky monoadducts, and other lesion types. We will measure various cellular and telomeric endpoints after lesion induction and will use candidate and unbiased approaches to identify proteins required to protect telomeres against the various forms of environmentally relevant DNA damage. This chemoptogenetic tool has been adapted for use in model organisms, and as the R35 evolves we will translate what we learn in cell culture to experiments in transgenic zebrafish and mice. Using this system, we will generate telomeric damage in key organs and cell types and will measure the impact on organ function and health. This program will lead to significant advances in mechanistic understanding of how environmentally relevant forms of telomeric DNA lesions impact telomere function, cellular function, and organism health. Ultimately, knowledge gained from this program will be highly valuable for developing new strategies that 1) preserve telomeres to ameliorate the effects of genotoxic and oxidative stress in healthy cells or conversely, that 2) inhibit telomere maintenance in malignant cells to arrest proliferation.