RPA and RPA-like Complexes at Telomeres

端粒上的 RPA 和类 RPA 复合物

• 批准号: 10626908
• 负责人: DEBORAH S. WUTTKE
• 金额: $ 30.27万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-07 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626908
• 关键词: Address; Adopted; Affinity; Aging; Apoptosis; Automobile Driving; Binding; Biochemical; Biology; Bone marrow failure; Cell Proliferation; Cells; Characteristics; Chromatin; Chromosomal Stability; Chromosomes; Complement; Complex; Crowns; Cryoelectron Microscopy; DNA; DNA Binding; DNA Damage; DNA Maintenance; DNA Primase; DNA biosynthesis; DNA replication fork; Data; Defect; Disease; Event; Foundations; Functional disorder; Genetic Recombination; Genome; Genomics; Geometry; Goals; Health; Hereditary Disease; Human; Inherited; Left; Link; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Molecular Conformation; Mus; Mutate; Mutation; Nucleoproteins; Oligonucleotides; Phenotype; Play; Proliferating; Proteins; Research; Resolution; Role; Series; Single-Stranded DNA; Site; Specificity; Stretching; Structure; Surface; System; Tandem Repeat Sequences; Telomerase; Telomere Maintenance; Telomere Shortening; Terminator Regions; Tertiary Protein Structure; Testing; Tumor Suppression; Yeasts; chromosome replication; design; genome integrity; human disease; in vivo; insight; interest; monomer; mutant; particle; people of color; programs; protein complex; recruit; repaired; replication factor A; response; senescence; telomere

Project Summary/Abstract Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes that are required for chromosome stability and cellular proliferation. These structures are essential for human health because dysregulation of either telomere protection or telomerase activity causes many inherited and acquired human diseases, with telomere dysfunction also closely tied to cancer and aging. Chromosomal ends consist of tandem repeats of TG-rich sequences that terminate in a highly conserved 3¢ single-stranded DNA (ssDNA) overhang. Management of this single-stranded overhang is one of the most critical aspects of telomere maintenance. When left unprotected, this overhang initiates DNA damage responses, leading to catastrophic events that permanently damage the genome and result in apoptosis or senescence. Recent data point to the intricate integration of the general DNA-maintenance and telomere machineries. Understanding the interplay of the genomic and telomere-specific ssDNA-binding factors is key to understanding the basic biology of chromosome maintenance and the catastrophic consequences of its misregulation. This program is focused on two protein complexes that manage ssDNA in many chromosomal contexts, including at telomeres and replication forks. Human CTC1/STN1/TEN1(hCST) is a heterotrimeric protein complex that protects and maintains sites of G-rich ssDNA throughout the genome. It acts prominently at telomeres, binding the conserved G-rich overhang to coordinate the termination of telomerase activity and recruitment of C-strand fill in by DNA pola-primase. Mutants of hCST are associated with a range of human diseases characteristic of proliferation defects. As a first step in understanding the mechanism of action of hCST, we have solved the high-resolution structure of hCST bound to ssDNA using cryoEM. This structure provides surprising insights into hCST function and is an excellent starting point to address key mechanistic questions regarding its function, such as its interaction with ssDNA, addressed in Aim 1, and the importance of the decamer structure in cells, investigated in Aim 2. Our structure reveals that hCST most strikingly resembles replication protein A (RPA), also a heterotrimeric complex involved in the non-specific binding of ssDNA during replication, repair and recombination. The driving hypothesis for Aim 3 is based on the observations that, in the context of certain mutants, RPA and CST activities can substitute for one another. This leads to the testable hypothesis that these RPA mutants reveal a cryptic G-specific binding activity contained within the protein. The structural, functional and biochemical parallels between the CST and RPA complexes suggest a highly tuned interplay of their activities that allow for their crosstalk in the management of difficult G-rich regions of chromatin, and this will be addressed in this aim.

项目摘要/摘要 端粒是所需的真核染色体末端的专门核蛋白结构 用于染色体稳定性和细胞增殖。这些结构对于人类健康至关重要，因为 端粒保护或端粒酶活性的失调导致许多人遗传并获得了人类 疾病，端粒功能障碍也与癌症和衰老密切相关。染色体末端包括 富含TG的序列的串联重复序列，该序列终止于高度组成3¢单链DNA（ssDNA） 悬垂。对这个单链的悬垂的管理是端粒最关键的方面之一 维护。当不受保护时，此悬垂会引发DNA损伤反应，导致灾难性 永久损害基因组并导致凋亡或感应的事件。最近的数据指向 一般DNA维护和端粒机械的复杂整合。了解相互作用 基因组和端粒特异性ssDNA结合因素是理解基本生物学的关键 染色体维持及其危害后果的灾难性后果。 该程序的重​​点是在许多染色体环境中管理ssDNA的两个蛋白质复合物， 包括端粒和复制叉。人CTC1/STN1/TEN1（HCST）是异三个蛋白 在整个基因组中保护和维护富含G的ssDNA位点的复合物。它显着地在 端粒，结合配置的g-富含g的悬垂，以协调端粒酶活性的终止和 通过DNA pola-primase填充C链的募集。 HCST的突变体与一系列人有关 疾病的增殖缺陷特征。作为理解行动机制的第一步 HCST，我们已经使用冷冻剂解决了与ssDNA结合的HCST的高分辨率结构。这个结构 提供对HCST功能的惊喜见解，是解决钥匙机制的绝佳起点 有关其功能的问题，例如其与ssDNA的互动，在AIM 1中解决，以及的重要性 在AIM 2中研究的细胞中的decamer结构。我们的结构表明，HCST最引人注目 类似于复制蛋白A（RPA），也是一种参与非特异性结合的异三聚体复合物 复制，修复和重组期间的ssDNA。 AIM 3的驱动假设是基于 观察到，在某些突变体的背景下，RPA和CST活动可以互相代替。 这导致了可检验的假设，即这些RPA突变体揭示了加密G特异性结合活性 包含在蛋白质中。 CST和RPA之间的结构，功能和生化相似之处 建筑群暗示了他们的活动的高度调谐相互作用，使他们的串扰在管理中 染色质的艰难G富含G的区域，这将在此目标中解决。