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

端粒 G 链和 C 链合成：机制和调控

基本信息

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
项目类别：
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

项目摘要

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末端的特殊核蛋白结构，对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发挥着重要作用。由调节端粒合成的蛋白质畸变引起端粒的两条链（称为 G 链和 C 链）依次合成。端粒酶和 DNA 聚合酶 � 分别通过整合 RNA 模板成分的逆转录介导 G 链的延伸，新合成的 G 链又充当 Pol 合成 C 链的模板。 � 端粒 DNA 合成的关键调节因子是 Cdc13-Stn1-Ten1 (CST) 复合体，这是一种保守的 RPA 样复合体，以高亲和力结合端粒 G 链。和序列特异性，并且调节端粒酶和 Pol � 本研究的目标是了解端粒酶、Pol � 和 CST 复合物在端粒 G 和 C 链合成调节方面的机制。使用来自几种念珠菌物种的因子作为模型。这三种复合物的这些版本特别适合生化分析，使我们能够重建在其他系统研究中难以分析的几种关键相互作用。生化上易处理的因素导致了一系列关于其作用机制的新的、明确的假设，这些假设将通过结合生化、遗传和单分子 FRET 技术的综合方法进行测试。 Est3-TEN 复合物（由端粒酶全酶中的两个关键和保守结构域组成）的核酸结合机制，并评估它们对体外和体内端粒酶活性和持续合成能力的贡献。第二个目标是剖析 Pol � 合成 C 链的机制，特别是在起始位点选择和引物长度调节方面。第三个目标是表征 CST 复合物和 Pol � 之间的物理相互作用，并定义其机制。这些研究的目标在芽殖酵母和人类之间是保守的，预期结果是对调节端粒 DNA 合成的机制有更深入的了解，这应该为端粒 DNA 合成的发展提供信息。基于端粒的临床应用。