Proteomic Approach to G-Quartets and Cell Aging

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The goal of this R03 project is to develop tools for identification of celt proteins that bind to G-quartet structures formed by telomeric repeat sequences of human chromosomes. The proposed research takes two "directed" proteomic approaches to capture of G-quartet binding proteins from cell lysates. In the first approach, oligonucleotides with telomeric repeat sequences will be immobilized at the surfaces of capillaries for affinity capillary electrophoresis. Captured proteins will then be analyzed by matrix assisted laser desorption/ionization (MALDI) mass spectrometry. In the second approach, the oligonucleotides will be attached to substrate surfaces for surface enhanced laser desorption/ionization (SELDI) mass spectrometric analysis. The long-term goal of the proposed research is to study the role of G-quartet DNA structures in cellular processes related to aging. Telomeres are nucleoprotein complexes that cap the ends of eukaryotic chromosomes, affording protection from degradation by nucleases or fusion with the ends of other chromosomes. The G-rich strands of telomeric DNA form highly stable, intramolecular G-quartet structures in vitro. Although in vivo formation of G-quartet structures by telomeric DNA has yet to be confirmed, there is compelling evidence that G-quartets may play a role in telomere elongation, genetic recombination, and gene regulation. Proteins that bind to G-quartet structures formed by telomeric DNA sequences in vitro, including human DNA topoisomerase and several helicases, have been associated with genetic instability. The proposed research is an essential first step towards elucidation of the relationship between G-quartet binding proteins and telomeric processes in cells.