Investigating sliding clamps and their contribution to genome stability

研究滑动夹及其对基因组稳定性的贡献

基本信息

批准号：
10373968
负责人：
Joseph Magrino
金额：
$ 3.17万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-14 至 2024-03-13
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10373968
关键词：
Address Affect Affinity Binding Biochemical Biological Assay Calorimetry Cell Cycle Cell Cycle Progression Cells Chemoresistance Client Closure by clamp Complex Cryoelectron Microscopy Cysteine DNA DNA Damage DNA Repair DNA Repair Pathway DNA Sequence Alteration DNA Structure DNA biosynthesis Development Disease Dissociation Drug Design Ensure Equilibrium Genetic Code Genome Genome Stability Genomic Instability I-antigen Isoleucine Link Maintenance Malignant Neoplasms Modeling Molecular Molecular Conformation Mutation Oncogenic Pathway interactions Play Proliferating Cell Nuclear Antigen Proteins RAD9A gene Regulation Research Resected Residencies Resolution Role SOS Response Series Serine Site Slide Specificity Structure Time Titrations Tumor Markers Variant Work X-Ray Crystallography chromatin remodeling ds-DNA experience experimental study genetic information genome integrity improved insight interdisciplinary approach mutant premature repaired residence tumor diagnostic

项目摘要

Project Summary All cells must replicate their genome once per cell cycle. To ensure proper duplication, cells integrate hundreds of factors that copy, surveil, and repair our genetic information. Proliferating Cell Nuclear Antigen [PCNA] and Rad9-Rad1-Hus1 [9-1-1] are ring-shaped clamps that act as master “conductors” that regulate many of the factors that replicate and maintain our DNA. PCNA is a homotrimeric ring that coordinates the replisome during DNA synthesis to work in tandem with DNA repair, chromatin remodeling, and cell cycle progression. When cells experience dsDNA breaks, they use the heterotrimeric clamp 9-1-1 to coordinate specific “SOS” repair factors. The collaborative efforts of both clamps are critical for genome stability. Many cancers are linked to inappropriate clamp coordination and changes in their expression. Because sliding clamps are central to many oncogenic pathways, we must address how they regulate themselves and their client partners. This proposal aims to address the following questions about sliding clamps: 1) How do sliding clamps coordinate their various partners? 2) Does the time sliding clamps spend on DNA influence genome stability? and 3) What determines site-specific loading of sliding clamps? I propose a multidisciplinary approach to address these questions about sliding clamps by investigating two-disease causing PCNA variants [PCNA-S228I [serine to isoleucine] and PCNA-C148S [cysteine to serine]] and the loading mechanism of 9-1-1. I hypothesize that sliding clamps control genome integrity via site-specific loading, proper partner interactions, and residence-time on DNA. I further hypothesize that PCNA-S228I and PCNA-C148S disrupt genome integrity by either promoting premature DNA dissociation or disrupting partner interactions. Finally, I hypothesize that the Rad17 subunit alters the clamp loader structure to specifically load the 9-1-1 clamp at sites of DNA damage. In aims 1 and 2, I will use PCNA-S228I and PCNA-C148S to address how clamps “choose” their partners and regulate their time on DNA. I will use x-ray crystallography, unfolding experiments, and a series of functional assays to determine how each variant compromises genome stability. In aim 3, I will determine the loading mechanism of clamp 9-1-1 to address how clamps are loaded to specific sites in the genome. I will use cryo-electron microscopy to determine how Rad17-RFC binds to clamp 9-1-1. Collectively, my work will broaden our insight into the factors that cause genome instability which may augment the development of personalized chemotherapeutics.

项目概要所有细胞必须在每个细胞周期复制一次基因组，以确保正确的复制，细胞会整合。复制、监视和修复我们的遗传信息的数百个因子。 [PCNA] 和 Rad9-Rad1-Hus1 [9-1-1] 是环形夹具，充当主“导体”，调节许多复制和维持 DNA 的因子中的一个是协调复制体的同源三聚体环。 DNA 合成过程中与 DNA 修复、染色质重塑和细胞周期进程协同工作。当细胞经历 dsDNA 断裂时，它们使用异源三聚体夹 9-1-1 来协调特定的“SOS” 两个钳子的协同作用对于基因组稳定性至关重要。不适当的夹具协调及其表达的变化，因为滑动夹具是许多的核心。致癌途径，我们必须解决他们如何监管自己及其客户合作伙伴的问题。旨在解决有关滑动夹具的以下问题： 1）滑动夹具如何协调其各种功能 2) 滑动夹在 DNA 上花费的时间是否会影响基因组稳定性？ 3) 是什么决定了这一点？我提出了一种多学科方法来解决这些问题通过研究导致两种疾病的 PCNA 变异 [PCNA-S228I [丝氨酸到异亮氨酸] 和 PCNA-C148S【半胱氨酸转丝氨酸】]以及9-1-1的加载机制。通过位点特异性加载、适当的伙伴相互作用和停留时间来控制基因组完整性我进一步发现 PCNA-S228I 和 PCNA-C148S 通过任一方式破坏基因组完整性。促进 DNA 过早解离或破坏伴侣相互作用最后，我发现 Rad17 亚基改变了钳装载机结构，以在 DNA 位点上特异性装载 9-1-1 钳在目标 1 和 2 中，我将使用 PCNA-S228I 和 PCNA-C148S 来解决夹具如何“选择”它们的问题。我将使用 X 射线晶体学、展开实验和一系列的方法来控制他们的 DNA 时间。确定每个变异如何损害基因组稳定性的功能分析在目标 3 中，我将确定我将使用夹具 9-1-1 的加载机制来解决如何将夹具加载到基因组中的特定位点。冷冻电子显微镜来确定 Rad17-RFC 如何结合钳 9-1-1 总的来说，我的工作将拓宽。我们对导致基因组不稳定的因素的洞察可能会促进个性化的发展化疗。