RPA and RPA-like Complexes at Telomeres

项目总结/摘要 端粒是真核生物染色体末端的特殊核蛋白结构， 用于染色体稳定和细胞增殖。这些结构对人类健康至关重要， 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 富含TG序列的串联重复序列，终止于高度保守的3 ′单链DNA（ssDNA） 悬垂。这种单链突出端的管理是端粒最关键的方面之一 上维护当不受保护时，这种突出端会引发DNA损伤反应，导致灾难性的 永久性损伤基因组并导致细胞凋亡或衰老的事件。最近的数据表明， 一般DNA维护和端粒机制的复杂整合。理解以下因素的相互作用 基因组和端粒特异性的ssDNA结合因子是理解 染色体的维持及其失调的灾难性后果。 该计划的重点是在许多染色体背景下管理ssDNA的两种蛋白质复合物， 包括端粒和复制叉。人CTC 1/STN 1/TEN 1（hCST）是异源三聚体蛋白质 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， 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， 通过DNA pola-primase募集C-链填充。hCST的突变体与一系列人类 以增殖缺陷为特征的疾病。作为了解其作用机制的第一步， hCST，我们已经解决了高分辨率结构的hCST结合到ssDNA使用cryoEM。这种结构 提供了令人惊讶的见解hCST功能，是一个很好的起点，以解决关键机制 关于其功能的问题，如目标1中提到的与ssDNA的相互作用，以及 目标2中研究的细胞中的十聚体结构。我们的结构显示，hCST最引人注目的是 类似于复制蛋白A（RPA），也是一种参与非特异性结合的异源三聚体复合物， ssDNA在复制、修复和重组过程中的作用。目标3的驱动假设基于 观察到，在某些突变体的情况下，RPA和CST活动可以相互替代。 这导致了一个可检验的假设，即这些RPA突变体揭示了一种神秘的G特异性结合活性 包含在蛋白质中。CST和RPA之间的结构、功能和生化相似性 复合体表明它们的活动具有高度协调的相互作用，这使得它们在管理 困难的G丰富的染色质区域，这将在本目标中得到解决。

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.