PROJECT SUMMARY/ABSTRACT All genetic information is stored in DNA that is intricately wrapped by proteins to form chromosomes. Telomeres are the nucleoprotein complexes that cap and protect the ends of chromosomes to prevent them from fraying, fusing together, and degrading. In addition to capping and protecting the ends of chromosomes, telomeres regulate the recruitment of telomerase, a specialized enzyme that synthesizes telomere DNA to collaborate with replicative polymerases and ensure complete chromosome replication. Over the past four years, multiple point-mutations have been identified in telomere end-binding proteins in patients diagnosed with a range of disorders, including many different types of cancer. These observations indicate that subtle changes in the structure and/or function of telomere proteins contributes to genome instability. POT1 (Protection of Telomeres 1) is the most mutated telomere protein associated with human disorders. POT1 forms a heterodimeric complex with another telomere end-binding protein, TPP1, to perform diverse but equally critical functions. Specifically, POT1-TPP1 binds to the extreme 3’ end of telomeres and helps recruit telomerase to the telomere and regulates telomerase-mediated telomere synthesis. In addition, the POT1-TPP1 proteins shield telomere DNA from being recognized and repaired by DNA damage machinery. Finally, the POT1-TPP1 heterodimer exhibits extraordinary sequence specificity that provides discrimination against binding to RNA or to DNA with non-telomere sequence. The objective of the present proposal is to investigate the multiple and diverse roles of POT1-TPP1 in telomere maintenance. We will combine structure-function studies to determine the molecular interactions that dictate POT1-TPP1 functions and we will corroborate the mechanistic studies with cellular outcome. We will introduce several disease-associated point mutations to both POT1 and TPP1 and characterize alterations in protein structure, as well as protein-protein and protein-nucleic acid interactions. The results from this investigation will reveal both structural and functional alterations introduced by pathogenic mutations and will be used to better understand the diverse functions of the native heterodimer. The work performed in this study will shed light on the fundamental assembly, organization, and functional motions that regulate chromosome end protection.