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: 8697590
• 负责人: NEAL F LUE
• 金额: $ 34.26万
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8697590
• 关键词: Accounting; 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; Relative (related person); Research; Reverse Transcription; Ribonucleases; Role; Saccharomycetales; Series; Site; Structure; Surface; System; Tail; Techniques; Telomerase; Testing; Time; Yeast Model System; Yeasts; abstracting; age related; base; clinical application; comparative; fungus; genetic regulatory protein; human disease; in vivo; insight; miniaturize; novel diagnostics; public health relevance; reconstitution; research study; 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）： 端粒是位于真核生物线性染色体末端的特殊核蛋白结构，对基因组的稳定性至关重要。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。端粒的两条链，称为G链和C链，分别由端粒酶和DNA聚合酶依次合成。端粒酶通过逆转录整合RNA模板组分介导G链的延伸。新合成的G链反过来又作为模板，由Pol？复合物合成C链。端粒DNA合成的关键调节因子是Cdc 13-Stn 1-Ten 1（CST）复合物，这是一种保守的RPA样复合物，以高亲和力和序列特异性结合端粒G链，并调节端粒酶和Pol？。本研究的目的是了解端粒酶、Pol？和CST复合物在调节端粒G和C链合成方面的机制。我们将利用来自几个念珠菌属物种的因素作为模型。这三种复合物的这些版本特别适合于生化分析，使我们能够重建在其他系统中难以分析的几种关键相互作用。对这些生物化学上易处理的因子的研究已经导致了一系列关于其作用机制的新的和明确的假设。这些假设将通过一个综合的方法，结合生物化学，遗传和单分子FRET技术进行测试。第一个目的是剖析Est 3-TEN复合物（由端粒酶全酶中的两个关键和保守的结构域组成）的核酸结合机制，并评估它们对端粒酶活性和体外和体内持续合成能力的贡献。第二个目的是剖析C链合成的机制，特别是在起始位点的选择和引物长度的调节。第三个目的是表征CST复合物和Pol？之间的物理相互作用，并确定CST在体外和体内刺激端粒C链合成的机制。这些研究的目标在芽殖酵母和人类之间是保守的。预期的结果是更深入地了解调节端粒DNA合成的机制，这将为基于端粒的临床应用的发展提供信息。