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

描述(由申请人提供)： 端粒是一种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复制寿命方面起着重要作用。现在已知，许多人类疾病是由调节端粒合成的蛋白质的异常引起的。端粒的两条链，被称为G链和C链，分别由端粒酶和DNA聚合酶�顺序合成。端粒酶通过逆转录一个完整的RNA模板组分来调节G链的延伸。新合成的G-链反过来作为模板，用于通过POL�复合体合成C-链。端粒DNA合成的一个关键调控因子是cdc13-stn1-ten1(Cst)复合体，它是一个保守的类似RPA的复合体，以高亲和力和序列特异性结合端粒G链，调节端粒酶和Pol�。本研究的目的是了解端粒酶、POL�和cst复合体在调节端粒G和C链合成方面的作用机制。我们将使用来自几个念珠菌物种的因子作为模型。这三个复合体的这些版本特别适合进行生化分析，使我们能够重建在其他系统中难以分析的几个关键相互作用。对这些生化易处理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说。这些假说将通过结合生化、遗传和单分子FRET技术的综合方法进行检验。第一个目的是剖析Est3-Ten复合体(由端粒酶全酶中两个关键和保守的结构域组成)的核酸结合机制，并评估它们在体外和体内对端粒酶活性和加工能力的贡献。第二个目的是剖析POL�合成C链的机制，特别是关于起始点的选择和引物长度的调节。第三个目的是研究Cst复合体与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。这些研究的目标是在萌芽酵母和人类之间保守的。预期的结果是更深入地了解调节端粒DNA合成的机制，这应该会为基于端粒的临床应用的发展提供信息。

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.