Molecular mechanisms of intersecting human telomeric functions Project Summary/Abstract Telomeres perform three essential functions in human cells. First, telomeres protect chromosomes against catastrophic end-to-end fusions by shielding them from the DNA damage sensing pathways. A six-membered protein complex called shelterin binds specifically to telomeric DNA to afford end protection. Second, telomeres facilitate end replication, allowing proliferating cells like stem/progenitor cells and cancer cells to replenish chromosome ends. A complex ribonucleoprotein enzyme called telomerase helps solve end replication. Telomerase facilitates end replication by adding telomeric DNA to chromosome ends using its RNA template. While telomerase activation in somatic cells is a hallmark of cancer, telomerase dysfunction in stem cells results in diseases called telomeropathies. Shelterin must protect chromosome ends from illicit DNA fusions but allow telomerase to access the same ends. A shelterin protein called TPP1 is instrumental in recruiting telomerase to telomeres, allowing shelterin to facilitate end protection and end replication, but the molecular mechanism of how TPP1 switches from end-protection mode to end-replication mode during S-phase is not known. Third, telomeres help homologous chromosomes undergo pairing and meiotic crossover to facilitate gamete production. To perform this function, telomeres attach to the nuclear envelope with the help of a meiosis-specific protein complex called TERB1-TERB2-MAJIN and connect with the cytoskeletal force- generating machinery. This allows chromosomes to move, enabling homologous chromosomes to pair up and undergo meiotic crossover. Paired meiotic chromosomes must be protected from telomeric recombination at the nuclear envelope, but how this occurs is not known at the molecular level. Illuminating the molecular interplay between the three telomeric functions will enrich our understanding of how genome integrity is upheld and suggest novel therapeutic avenues for diseases like cancers, telomeropathies, and infertility. This proposal aims to apply the knowledge of telomeres, telomerase, and meiotic assemblies, and expertise in biochemistry, crystallography, and cell biology to understand how human telomeres perform their three critical functions, especially in the contexts where these clash with one another. It also aims to discover new factors responsible for upholding these functions and reveal their underlying biochemical activities. Finally, this proposal aims to dissect the molecular mechanism of disease caused by the disruption of these functions and test new strategies for therapeutic development. The proposed approach to dissect the molecular mechanisms of intersecting telomeric processes will enhance our knowledge of how telomeres enable genome integrity and suggest therapeutic opportunities to tackle telomere dysfunction in disease.