Reactive oxygen species (ROS) are natural byproducts of oxygen metabolism. Environmental assaults can dramatically elevate ROS, overwhelming the defenses of cellular antioxidants, and triggering damage to essential macromolecules particularly DNA. Oxidative damage is linked to numerous disease states, but despite much research, fundamental questions remain on how cells avert the detrimental impacts of ROS. Mounting evidence implicates telomere proteins in functions beyond the chromosome terminus. Both Protection of Telomeres 1 (POT1) and the telomerase catalytic subunit TERT traffic in and out of the nucleus and are implicated in various aspects of the response to oxidative damage in mammalian cells. However, unmasking potential non-telomeric functions is problematic because of their critical roles in telomere maintenance and stability. In this renewal application, the model eukaryote Arabidopsis thaliana is employed to study how telomere-associated proteins (TAPs) respond to and mitigate oxidative stress. Arabidopsis encodes two highly divergent POT1 paralogs, AtPOT1a and AtPOT1b, which exhibit separation-of-function with respect to canonical telomere biology, and hence present a unique opportunity to elucidate the full complement of POT1 functions. The proposal builds on additional preliminary showing that AtPOT1b accumulates in the cytoplasm, and loss of AtPOT1b significantly elevates ROS and activates numerous cellular defenses against oxidative stress. AtTERT carries a mitochondrial localization signal, suggesting that it too may regulate the response to ROS. The central hypothesis of this proposal is that the TAPs, AtPOT1a, AtPOT1b and AtTERT, serve important roles in the cellular defense against oxidative damage. This hypothesis will be examined through two Specific Aims.
For Aim 1, the breadth of POT1 functions in promoting genome integrity will be assessed by testing how POT1a and POT1b regulate oxidative damage in telomeric and non-telomeric DNA, and how POT1b cooperates with POT1a and TERT to avert genome destabilization. The role of ATR signaling in reproductive progenitor cells will be examined in pot1b mutants. Finally, the evolutionary origin of stress response functions in POT1 proteins will be explored.
In Aim 2, three complementary strategies will probe the molecular mechanism and interaction partners of TAPs in non-telomeric pathways. These include monitoring the subcellular trafficking of TAPs in response to stress, quantitative mass spec to identify protein binding partners and stress-induced post-translational modifications, and a novel suppressor screen to explore the genetic pathway that enables POT1b and TERT to promote plant development. These studies will increase understanding of the cross- talk between TAPs and the stress response, open new horizons for exploring the non-canonical functions of POT1, and provide a roadmap to explore rare splice variants of human POT1 that cannot associate with telomeres, and yet are associated with increased predisposition to cancer.
Oxidative damage can severely compromise the genome, but recent studies indicate that telomere proteins may help to mitigate the consequences of this assault. Studies in model organisms established the paradigms for human telomere biology, and continue to uncover novel telomere components and regulatory mechanisms. In this tradition, we will exploit the genetic tractability of Arabidopsis and the rich multi-omics resources of the plant stress response to explore the non-canonical pathways telomere-associated proteins employ to promote genome integrity and organismal development in the face of oxidative stress.
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