Telomeres, the ends of eukaryotic chromosomes, consist of long tandem arrays of double-stranded G-rich repeats that run out as single-stranded overhangs to the 3'-ends of chromosomes. Telomeres have two essential functions; serving as templates for replication by the enzyme telomerase and protecting chromosome ends from DNA repair systems. Telomere function is essential for chromosome stability and cell growth, and telomere integrity impacts cellular aging. Human telomeres are coated by the proteins TRF1 and TRF2, which bind double-stranded DNA. Until recently, TRF1 and TRF2 (along with their unique associated factors) were believed to function independently to regulate telomere length and protect chromosome ends, respectively. However, new studies show that TRF1 and TRF2 exist as partially redundant complexes, each bound to a common set of telomeric proteins: TIN2, TPP1 (TINT1/PIP1/PTOP), and POT1. These new findings of redundant complexes at telomeres challenge the established paradigms on how telomere function is regulated at the molecular level. The object of this project is to determine how these partially redundant complexes function (either discretely or together) to regulate telomere length and protect chromosome ends. The starting point will be a molecular dissection of the interacting domains between the components. TIN2 is the focal point of the partially redundant complexes. TIN2 binds TRF1, TRF2, and TPP1. TPP1 links the complex to POT1. To determine the contribution of each interaction to telomere function, alleles of TIN2 and TPP1 will be generated in which one or more interactions are ablated by point mutations, but other interactions are maintained. Stable cell lines expressing the mutant alleles will be generated and functional analyses performed to determine the effect of each mutation on telomere length regulation and/or chromosome end protection in vivo. Although these complexes appear redundant they may be distinguished by association with novel proteins. Thus, to identify new proteins that might distinguish the complexes, two-hybrid screens and coimmunoprecipitation analysis will be performed. Ultimately, the molecular dissection of the individual components, their unique binding partners, and their function will be essential for understanding telomere function.
Telomeres, the specialized structures at the ends of eukaryotic chromosomes, are essential for the stability of those chromosomes. Telomere function is controlled by multisubunit protein complexes that bind to the telomeric DNA. In this project the investigator will elucidate the molecular mechanisms by which these protein complexes regulate and protect these essential structures.
Dr. Smith considers mentoring students an important and productive component of university research. Individuals she has mentored include women and minorities and range from high school students to graduate students. She has found that the microscopic techniques she uses to visualize human cells and chromosomes especially capture the interest of younger researchers. For this research she has incorporated projects with features specifically designed to appeal to, and be appropriate for, this category of researchers.
