RPA and RPA-like Complexes at Telomeres

项目摘要/摘要 端粒是真核细胞染色体末端所必需的特殊核蛋白结构。 为了染色体的稳定和细胞的增殖。这些结构对人类健康是必不可少的，因为 端粒保护或端粒酶活性的失调导致许多遗传性和获得性人类 疾病，端粒功能障碍也与癌症和衰老密切相关。染色体末端由以下部分组成 富含TG的序列的串联重复序列，终止于高度保守的3？单链DNA(SsDNA) 悬垂。对这种单链悬垂的管理是端粒最关键的方面之一 维修。如果不加以保护，这种悬垂会引发DNA损伤反应，导致灾难性的 永久性破坏基因组并导致细胞凋亡或衰老的事件。最近的数据表明 普通DNA维护和端粒机制的复杂集成。了解以下方面的相互影响 基因组和端粒特异的单链DNA结合因子是理解红斑狼疮基础生物学的关键。 染色体维持及其错误调控的灾难性后果。 这个项目的重点是两个蛋白质复合体，它们在许多染色体环境中管理单链DNA， 包括在端粒和复制叉处。人CTC1/STN1/TEN1(HCST)是一种异源三聚体蛋白 保护和维护整个基因组中富含G的单链DNA位点的复合体。它在很大程度上 端粒，结合保守的富含G的悬垂以协调端粒酶活性的终止和 DNA Pola-Primase对C链的募集作用。Hcst的突变与一系列人类 以增殖缺陷为特征的疾病。作为了解其作用机制的第一步 通过对HCST的研究，我们用低温电子显微镜解决了HCST与单链DNA结合的高分辨结构。这个结构 提供对HCST功能的惊人洞察力，是解决关键机制问题的极佳起点 关于它的功能的问题，如它与单链DNA的相互作用，在目标1中讨论，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最显著的 类似于复制蛋白A(RPA)，也是参与非特异性结合的异三聚体复合体 复制、修复和重组过程中的单链DNA。目标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活性可以相互替代。 这导致了一种可测试的假设，即这些RPA突变体显示出隐蔽的G特异性结合活性 包含在蛋白质中。CST和RPA在结构、功能和生化上的相似性 复合体暗示他们的活动高度协调的相互作用，允许他们在管理中的串扰 困难的富含G的染色质区域，这将在这一目标中得到解决。

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.