Proteomic Approach to G-Quartets and Cell Aging

这个R 03项目的目标是开发工具，用于识别与人类染色体端粒重复序列形成的G-四联体结构结合的细胞蛋白。该研究采用两种“定向”蛋白质组学方法从细胞裂解物中捕获G-四联体结合蛋白。在第一种方法中，将具有端粒重复序列的寡核苷酸固定在毛细管表面用于亲和毛细管电泳。然后通过基质辅助激光解吸/电离（MALDI）质谱法分析捕获的蛋白质。 在第二种方法中，寡核苷酸将附着到基底表面，以进行表面修饰。 增强激光解吸/电离（SELDI）质谱分析。拟议研究的长期目标是研究G-四重DNA结构在与衰老相关的细胞过程中的作用。端粒是核蛋白复合物，其覆盖真核生物染色体的末端，提供保护以免被核酸酶降解或与核蛋白融合。 其他染色体的末端。端粒DNA的富含G的链形成高度稳定， 体外分子内G-四重体结构。虽然在体内形成G-四联体结构 虽然G-四联体与端粒DNA的关系尚未得到证实，但有令人信服的证据表明，G-四联体可能在端粒延伸、遗传重组和基因调控中发挥作用。与端粒DNA序列在体外形成的G-四联体结构结合的蛋白质，包括人DNA 拓扑异构酶和几种解旋酶与遗传不稳定性有关。这项研究是阐明G-四联体结合蛋白与细胞端粒过程之间关系的重要第一步。

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.