RPA and RPA-like Complexes at Telomeres

端粒上的 RPA 和类 RPA 复合物

• 批准号: 10404051
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10404051
• 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; Cryoelectron Microscopy; DNA; DNA Binding; DNA Damage; DNA Maintenance; DNA Primase; DNA biosynthesis; DNA replication fork; Data; Defect; Dental crowns; Disease; Event; Foundations; Functional disorder; Genetic Recombination; Genome; Genomics; Geometry; Goals; Hand; Health; Hereditary Disease; Human; Inherited; Left; Link; Maintenance; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Molecular Conformation; Mus; Mutate; Mutation; Nucleoproteins; Oligonucleotides; Phenotype; Play; Proteins; Research; Resolution; Role; Series; Single-Stranded DNA; Site; Specificity; Stretching; Structure; Surface; System; Tandem Repeat Sequences; Telomerase; Telomere Maintenance; Telomere Shortening; Tertiary Protein Structure; Testing; Tumor Suppression; Yeasts; base; design; genome integrity; human disease; in vivo; insight; interest; 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.

项目概要/摘要 端粒是真核染色体末端所需的特殊核蛋白结构 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会导致许多遗传性和后天性人类遗传性疾病 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 序列的串联重复，终止于高度保守的 3 美分单链 DNA (ssDNA) 悬垂。这种单链突出的管理是端粒最关键的方面之一 维护。如果不加保护，这种突出端就会引发 DNA 损伤反应，从而导致灾难性的后果。 永久性损害基因组并导致细胞凋亡或衰老的事件。最新数据指向 一般 DNA 维护和端粒机制的复杂整合。了解之间的相互作用 基因组和端粒特异性 ssDNA 结合因子是理解基础生物学的关键 染色体的维护及其错误调节的灾难性后果。 该项目专注于在许多染色体环境中管理 ssDNA 的两种蛋白质复合物， 包括端粒和复制叉。人 CTC1/STN1/TEN1(hCST) 是一种异源三聚体蛋白 保护和维持整个基因组中富含 G 的 ssDNA 位点的复合物。它的作用突出于 端粒，结合保守的富含 G 的突出端以协调端粒酶活性的终止和 DNA pola-primase 募集 C 链填充。 hCST 突变体与一系列人类 以增殖缺陷为特征的疾病。作为了解作用机制的第一步 hCST，我们使用冷冻电镜解析了与 ssDNA 结合的 hCST 的高分辨率结构。这个结构 提供了对 hCST 功能的令人惊讶的见解，并且是解决关键机制的绝佳起点 有关其功能的问题，例如目标 1 中讨论的其与 ssDNA 的相互作用，以及 细胞中的十聚体结构，在目标 2 中进行了研究。我们的结构揭示了 hCST 最引人注目 类似于复制蛋白 A (RPA)，也是一种异源三聚体复合物，参与非特异性结合 复制、修复和重组过程中的单链DNA。目标 3 的驱动假设基于 观察结果表明，在某些突变体的背景下，RPA 和 CST 活性可以相互替代。 这导致了可检验的假设，即这些 RPA 突变体揭示了神秘的 G 特异性结合活性 包含在蛋白质内。 CST 和 RPA 之间的结构、功能和生化相似之处 复合体表明它们的活动之间存在高度协调的相互作用，从而允许它们在管理中进行串扰 染色质富含 G 的区域是困难的，这一点将在这个目标中得到解决。