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Show UNDERGRADUATE RESEARCH ABSTRACTS SPRING 2007 Angela Hanson M.P. Horvath 29 Structural Analysis Reveals Conformational Flexibility in Telomere Capping Protein-DNA Complexes Angela Hanson (M.P. Horvath) Department of Biology University of Utah Telomeres consist of simple, tandemly repeated sequences that are species-specific. In Oxytricha nova, our model organism, telomeres consist of 5'-d(TTTTGGGG/CCCCAAAA) double strand repeats where the GT rich strand protrudes as a single strand 3'-terminal extension. Previous images show a telomere binding protein (On TEBP ??) binds one GT-rich repeat sequence at the 3'-terminus. Because telomere single strand extensions often exist as multiple repeats, we wondered if multiple proteins could bind at neighboring DNA sites and if so, what would such structures look like? We have tested repeat lengths of 3, 6, and 9 [5'-d(TTTTGGGG)-3'-OH], allowing for multiple binding sites for (On TEBP ?). Each single strand telomere length was mixed with a corresponding amount of protein, i.e. 3 repeats received 3-fold protein, and allowed to complex over night. The DNA-protein structures were then purified through a size exclusion chromatography column to separate any unbound protein from DNA-protein complexes. We explored these DNA-protein structures using solution-based methods such as small angle x-ray scattering (SAXS) and analytical ultracentrifugation (AUC). SAXS and AUC techniques have been very informative. Each method revealed that our structure assumes multiple conformations and is surprisingly flexible. Unoccupied sites along the single strand DNA could offer a molecular interpretation for such flexibility within the complex. Ultimately, we anticipate solving the structure using x-ray crystallography techniques; however our initial attempts have yielded poor results. Based on our findings from solution analysis, we now know our solution is not reaching a desirable saturation point for crystal growth. This has guided us to revise our strategy and seek out new methods to saturate all available DNA binding sites. Resolution of these complexes through x-ray crystallography will yield a molecular image of telomere structure making it possible to relate telomere evolution and telomerase activities. |
