Telomere G- and C-strand synthesis: mechanisms and regulation

端粒 G 链和 C 链合成：机制和调控

• 批准号: 9278233
• 负责人: NEAL F LUE
• 金额: $ 32.47万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-11 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9278233
• 关键词: Affinity; Binding; Biochemical; Biochemical Genetics; Candida; Cells; Chromosomes; Clinical; Complex; DNA; DNA Binding; DNA biosynthesis; DNA-Directed DNA Polymerase; Development; Disease; Enzymes; Excision; Genome Stability; Goals; Holoenzymes; Human; In Vitro; Investigation; Length; Liver Fibrosis; Longevity; Lung; Maintenance; Malignant Neoplasms; Mediating; Modeling; Molecular; Nucleic Acid Binding; Nucleoproteins; Outcome; Pancytopenia; Play; Property; Proteins; RNA; RNA primers; Reaction; Recombinants; Regulation; Research; Reverse Transcription; Ribonucleases; Role; Saccharomycetales; Series; Site; Structure; Surface; System; Tail; Techniques; Telomerase; Testing; Time; Yeast Model System; Yeasts; age related; base; clinical application; comparative; experimental study; fungus; genetic regulatory protein; human disease; in vivo; insight; miniaturize; novel diagnostics; novel therapeutics; public health relevance; reconstitution; single-molecule FRET; stoichiometry; telomere

DESCRIPTION (provided by applicant): Project Summary/Abstract Telomeres are specialized nucleoprotein structures located at the termini of linear eukaryotic chromosomes that are critical for genome stability. The lengths of telomere reserve often play a major role in dictating the replicative life span of cells. Many human diseases are now known to be caused by aberrations in proteins that regulate telomere synthesis. The two strands of telomeres, known as the G- and C-strand, are synthesized sequentially by telomerase and DNA polymerase �, respectively. Telomerase mediates the extension of the G-strand through reverse transcription of an integral RNA template component. The newly synthesized G-strand in turn serves as the template for the synthesis of the C-strand by the Pol � complex. A key regulator of telomere DNA synthesis is the Cdc13-Stn1-Ten1 (CST) complex, a conserved RPA-like complex that binds the telomere G-strand with high affinity and sequence-specificty, and that regulates both telomerase and Pol �. The goal of this research is to understand the mechanisms of the telomerase, Pol �, and CST complex with respect to the regulation of telomere G and C-strand synthesis. We will utilize factors derived from several Candida species as models. These versions of the three complexes are particularly amenable to biochemical analyses, allowing us to reconstitute several critical interactions that were difficlt to analyze in other systems. Studies of these biochemically tractable factors have led to a series of new and well-defined hypotheses concerning their mechanisms of action. These hypotheses will be tested through an integrated approach that incorporates biochemical, genetic, and single-molecule FRET techniques. The first aim is to dissect the nucleic acid-binding mechanisms of the Est3-TEN complex (comprised of two critical and conserved domains in the telomerase holoenzyme) and assess their contribution to telomerase activity and processivity in vitro and in vivo. The second aim is to dissect the mechanisms of C-strand synthesis by Pol �, especially with respect to initiation site selection and primer length regulation. The third aim is to characterize the physical interactions between the CST complex and Pol �, and define the mechanisms by which CST stimulates telomere C-strand synthesis in vitro and in vivo. The targets of these investigations are conserved between budding yeast and humans. The anticipated outcome is a deeper understanding of mechanisms that regulate telomere DNA synthesis, which should inform the development of telomere-based clinical applications.

描述（由申请人提供）： 项目摘要/摘要端粒是位于线性真核染色体术语上的专业核蛋白质结构，这对于基因组稳定性至关重要。端粒储备的长度通常在决定细胞的复制寿命方面起主要作用。现在已知许多人类疾病是由调节端粒合成的蛋白质的畸变引起的。端粒（称为G-和C链）的两条链分别由端粒和DNA聚合酶顺序合成。端粒酶通过积分RNA模板分量的逆转录介导G链的扩展。新合成的G链反过来又用作Pol-Complex的C链合成的模板。端粒DNA合成的关键调节剂是CDC13-STN1-TEN1（CST）复合物，这是一种保守的RPA样复合物，其具有高亲和力和序列特异性的端粒G链，并调节端粒酶和pol-''。这项研究的目的是了解端粒酶，pol和CST复合物的机理，以调节端粒G和C链合成。我们将利用源自几种念珠菌物种的因素作为模型。这三个复合物的这些版本特别适合生化分析，从而使我们能够重建几种在其他系统中难以分析的关键相互作用。对这些生物化学因素的研究导致了一系列有关其作用机制的新的新定义的假设。这些假设将通过综合生化，遗传和单分子货物技术的综合方法进行检验。第一个目的是剖析EST3-TEN复合物的核酸结合机制（由端粒酶全酶中的两个关键和组成域组成），并评估其对端粒酶活性以及体外和体内加工性的贡献。第二个目的是通过pol解剖C链合成的机理，尤其是在主动选择位点选择和底漆长度调节方面。第三个目的是表征CST复合物与Pol之间的物理相互作用，并定义CST在体外和体内刺激端粒C链合成的机制。这些研究的靶标在萌芽的酵母和人类之间是保守的。预期的结果是对调节端粒DNA合成的机制的深入了解，这应该为基于端粒的临床应用的发展提供信息。