Summary/Abstract Human chromosomes end in telomeres, repetitive DNA sequences that are bound by the Shelterin protein complex (1). During semi-conservative DNA replication the extreme ends of a chromosome are unable to be duplicated, leading to successive chromosome shortening. Once telomeres reach a critical length, cells enter senescence or undergo apoptosis (2). To counteract chromosome shortening, continuously dividing cells, such as germ cells, stem cells, and most cancer cells, express telomerase, an RNA-containing reverse transcriptase (3). Telomerase is a unique enzyme that processively adds telomeric repeats, copied from its RNA component, to the single-stranded DNA overhang of chromosome ends (4). The molecular mechanisms that govern telomerase processivity are poorly defined, but are critical to understand telomere maintenance. The Shelterin complex carries out two key functions at telomeres; it prevents telomeres from being recognized as sites of DNA damage, and it recruits telomerase to telomeres (5,6). Telomerase recruitment to telomeres is a tightly regulated process. Telomerase resides in Cajal bodies, specialized RNA-processing compartments in the nucleus, throughout most of the cell cycle. During S-phase, telomerase is recruited to telomeres to maintain telomere length (7). Although the protein-protein interactions required for telomerase to associate with telomeres are well understood, the spatio- temporal control of telomerase recruitment is poorly defined (7). Potential mechanisms for regulating telomerase recruitment include alterations in composition of telomerase and the shelterin complex or post-translational modification of its components. Telomere maintenance plays an important role in multiple human diseases. Deficiencies in telomerase assembly, activity, or recruitment to telomeres cause dyskeratosis congenita, pulmonary fibrosis, and aplastic anemia, severe human conditions characterized by stem cell failure (8). In addition, 90% of cancers rely on telomerase activity to allow them to divide indefinitely (9). Therefore, understanding the basic biology of telomerase recruitment to telomeres and telomerase catalysis could lead to novel approaches to modulate this process as a therapeutic approach for several human diseases. I propose to analyze the molecular mechanisms underlying telomerase recruitment to telomeres and telomerase catalysis using genome editing and a combination of cell biological, proteomic, biochemical, and single-molecule approaches. In particular I will: 1. Determine the molecular mechanisms that drive telomerase recruitment to telomeres in S- Phase. Using genome-edited cell lines expressing tagged telomerase and shelterin components, I will conduct live cell imaging of telomerase trafficking to telomeres, analyze the assembly state of telomerase and the shelterin complex throughout the cell cycle using cell biological and proteomic approaches, and identify kinases that modulate telomerase traffic...