Summary The goal of this project is to test the hypothesis that oxidatively damaged deoxyribonucleotide triphosphates (dNTPs) cause telomere instability by inhibiting telomere elongation and repair, thereby compromising cell survival and proliferation. Oxidative stress caused by intrinsic and environmental factors, leads to an excess of reactive oxygen species (ROS), which damage cellular components and contribute to the pathogenesis of many diseases. Previous studies show oxidative stress accelerates telomere loss, but the mechanisms are unknown. In biochemical studies, we discovered that the use of oxidized dNTPs by telomerase during telomere extension is mutagenic and terminates further telomere elongation. Consistent with this, our recent data provide novel evidence that elevating oxidized dNTPs by depleting the sanitase MTH1 induces telomere dysfunction and cell death in human cells. Strikingly, this occurs preferentially in telomerase positive cells harboring critically short telomeres, suggesting that telomerase activity modulates sensitivity to elevated oxidized dNTPs. Aim 1 will biochemically define the efficiency of telomerase utilizaiton of oxidized dNTPs, and will measure the degree of selectivity for correct dNTP use over incorrect and damaged dNTPs; also known as telomerase fidelty. These kinetic parameters will be important for understanding the frequency of oxidized dNTP use during telomere elongation in cells. Aim 2 will examine how oxidized dNTPs alter telomeric sequences, length and integrity, as a funciton of telomerase activity in human cell lines. We will use next generation single molecule real time DNA sequencing to detect DNA damage and mutations in telomeres. The biological outcome will be determined by measuring endpoints of cell survival and senescence. Aim 3 will examine how oxidized dNTPs alter the efficiency of base excision repair at telomeric ends. Previous studies suggest that incomplete processing of damaged nucleobases at telomeres, rather than the initial damage itself, causes telomere shortening, and that oxidized dNTPs abort base excision repair. To test this we developed a novel technology to selectively induce DNA base damage at telomeres with an innovative fluorogen activated peptide targeting system. We will measure endpoints of cell survival and telomere integrity upon the induction of telomere damage in the presence and absence of the MTH1 sanitase, which hydrolyzes oxidized dNTPs. Given that MTH1 and telomerase are both being pursued as targets for killing cancer cells, the results of this project will have important translational implications for cancer therapy. In addition, these studies will fill a significant void in our understanding of how oxidation of DNA precursors alters telomerase activity and telomere maintenance, and of how telomere biology modulates cellular sensitivity to inhibition of nucleotide pool sanitation. Ultimately, knowledge gained from this study will be highly valuable for developing new strategies that 1) preserve telomeres to ameliorate the effects of oxidative stress in healthy cells or conversely, that 2) inhibit telomere maintenance in malignant cells to arrest proliferation.