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Show DNA-Protein Complexes Hint at the Ancestral Role for Telomere-capping Proteins Angela Hansen and Martin Horvath Department of Biology Telomeres consist of simple, tandemly repeated sequences that are species-specific. In Oxytricha nova the sequence is 5'-d(TTTTGGGG)-3'-OH and extends out of the 3'-hydroxal ends of the double helix as a single strand extension. The alpha and beta subunits of telomere end binding protein of Oxytricha nova (On TEBP) bind to the single strand telomere extension forming a DNA-protein complex. Based on previous thermodynamic testing by the Horvath Lab, telom-ere DNA can favorably bind multiple telomere end binding proteins of Oxytricha nova (On TEBP-alpha). Our hypothesized structure should include the alpha (On TEBP-alpha) subunit binding tightly to the terminal repeat sequence, 5'-d(TTTTGGGG)-3'-OH, of the telomere single strand DNA. Each subsequent (On TEBP-alpha) unit will bind to the interminal telomere repeat sequence. Repeated bindings should creating favorable protein-DNA interactions, as well as, favorable pro-teinprotein interactions through non-binding protein subunits. To elucidate the structure we will express the telomere end binding protein (On TEBPalpha) using a bacterial host. The protein samples will then be purified using fast protein liquid chromatography (FPLC) by means of ion exchange and size exclusion chromatography. Telomere DNA oligonucleotides will be synthesized of varying lengths of the repeat sequence n[5'-d(TTTTGGGG)-3'-OH]. We intend to test repeat lengths of at least 3, 6 or 9[5'd(TTTTGGGG)-3'-OH], allowing for multiple binding sites for (On TEBP-alpha). The telomere DNA will be purified using high performance liquid chromatography (HPLC) by way of reversephase chromatography. The purified protein and DNA lengths will be mixed and analyzed form structural information. The telomere DNA and (On TEBP-alpha) compounds will be (1) crystallized to determine the DNA-protein complex structure though x-ray crystallography. Additionally, DNA-protein complexes will be examined using (2) small angle x-ray scattering (SAXS). Upon confirmation of the possibility of our hypothesis we anticipate developing new hypotheses, perhaps relating our findings toward telomere evolution and telomerase activities. It is possible that multiple structural possibilities of the DNA-protein complex could occur, which would inhibit crystal growth. However SAXS techniques should be informative, by providing comparative measurements of largely variant structural motifs. This research is supported by funding from The National Institute of Health grant to M.P. H (R01 GM067994). The University of Utah DNA/Peptide Research Core facility receives support from the National Cancer Institute (5P30 CA42014). Angela Hansen is supported by funding from The University of Utah, Department of Biology, Bioscience Undergraduate Research Program. |