Single Stranded DNA Recognition in Telomeres

端粒中的单链 DNA 识别

• 批准号: 7728589
• 负责人: DEBORAH S. WUTTKE
• 金额: $ 29.47万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7728589
• 关键词: Address; Affect; Age; Aging; Amino Acid Sequence; Animal Model; Apoptosis; Area; Binding; Biochemical; Biological Assay; Biological Models; Bypass; Cell Aging; Cell Proliferation; Cell Survival; Cell division; Cells; Chromosome abnormality; Chromosomes; Complex; Cyclin B; DNA; DNA Binding; DNA Binding Domain; DNA Damage; DNA biosynthesis; Elements; Enzymes; Fission Yeast; Funding; Genome Stability; Health; Holoenzymes; Human; In Vitro; Knowledge; Length; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Nucleoproteins; Organism; Peptide Sequence Determination; Play; Proliferating; Protein Family; Proteins; Recruitment Activity; Regulation; Research; Resolution; Role; SS DNA BP; Saccharomyces cerevisiae; Single-Stranded DNA; Specificity; Structure; Telomerase; Telomere Capping; Telomere Maintenance; Yeasts; cancer cell; enzyme activity; genetic regulatory protein; insight; novel; prevent; programs; reconstitution; response; senescence; telomere; tool; yeast genetics

Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual GT-rich 32 single-strand overhang that requires a special cap to prevent inappropriate recognition by the DNA damage machinery. Furthermore, telomeres are not fully replicated by the canonical DNA replication machinery. Highly proliferating cells overcome this limitation through the action of the replicative enzyme telomerase. The implementation of telomere capping and the regulation of telomerase are critical to cellular survival. This research program combines biochemical and structural strategies to understand how telomere factors perform these activities. Our program is built around a central player in telomere maintenance, the family of proteins that target the 32 single-stranded overhang region of the telomere via specialized sequence-specific single-stranded DNA-binding domains. These telomere end-protection (or TEP) proteins are required for normal cellular proliferation, playing a vital role in telomere maintenance by providing a capping function as well as regulating the action of telomerase at the telomere. The regulation of telomerase action at telomeres by TEP proteins is performed in concert with regulatory components of the telomerase holoenzyme, but the mechanism by which this occurs is largely unknown. In Aim 1, we present a focused research plan to understand how telomerase activity is regulated by these factors in the model system S. cerevisiae. We will obtain mechanistic insights into telomerase regulation using an in vitro reconstituted telomerase assay and direct binding assays. Aim 2 expands on our discovery in the previous funding period of a new DNA-binding domain (DBD) in the TEP protein from a second model organism, S. pombe. Our knowledge of the mechanism of ssDNA recognition by TEP proteins is incomplete and the high-resolution structure of the SpPot1-DBD will help identify the common elements of ssDNA-recognition, as well as the molecular mechanisms that mediate alternate specificity. This integrated research program will provide novel insights into the activity of a biologically critical family of proteins by addressing key questions with high-resolution structural and biochemical tools.

线性染色体终止在称为端粒的专用核蛋白结构中， 这是基因组稳定性和细胞增殖所必需的。端粒以不寻常的方式结束 GT充足的32个单链悬垂，需要一个特殊的帽子以防止不适当的识别 通过DNA损伤机械。此外，端粒未完全由规范复制 DNA复制机械。高度增殖的细胞通过 复制酶端粒酶的作用。端粒上限的实施和 端粒酶的调节对于细胞存活至关重要。 该研究计划结合了生化和结构性策略，以了解 端粒因素执行这些活动。我们的程序是围绕端粒中的中心参与者构建的 维护，针对32个单链悬垂区域的蛋白质家族 通过专门序列特异性的单链DNA结合域进行端粒。这些 正常细胞增殖需要端粒终端保护（或TEP）蛋白 通过提供上限功能并调节端粒维护中的重要作用 端粒酶在端粒中的作用。 TEP对端粒的端粒酶作用调节 蛋白质与端粒酶全酶的调节成分一起进行，但 发生这种情况的机制在很大程度上是未知的。 在AIM 1中，我们提出了一项重点研究计划，以了解端粒酶活动的方式 在模型系统S.酿酒酵母中受这些因素的调节。我们将获得机械见解 使用体外重构的端粒酶测定和直接结合进入端粒酶调节 测定。 AIM 2扩展了我们在新的DNA结合的前资金期间的发现 第二个模型生物体的TEP蛋白中的域（DBD），S。Pombe。我们的知识 TEP蛋白识别ssDNA的机制是不完整的，高分辨率 SPPOT1-DBD的结构将有助于确定ssDNA识别的共同元素， 以及介导替代特异性的分子机制。 该集成的研究计划将为活动的活动提供新颖的见解 通过使用高分辨率结构的关键问题来解决生物学上关键的蛋白质家族 和生化工具。