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

• 批准号: 10551232
• 负责人: Joseph Magrino
• 金额: $ 3.27万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 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/10551232
• 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; Isoleucine; Link; Maintenance; Malignant Neoplasms; Modeling; Molecular; Molecular Conformation; Mutation; Oncogenic; Pathway interactions; Proliferating Cell Nuclear Antigen; Proteins; RAD9A gene; Regulation; Research; Resected; Residencies; Resolution; Role; SOS Response; Series; Serine; Shapes; Site; Slide; Specificity; Structure; Therapeutic; Time; Titrations; Treatment Efficacy; 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; therapeutic target; tumor diagnostic

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.

项目摘要 所有细胞在每个细胞周期都必须复制一次基因组。为了确保正确的复制，细胞整合 数以百计的复制、监视和修复我们遗传信息的因子。增殖细胞核抗原 [PCNA]和Rad 9-Rad 1-Hus 1 [9-1-1]是环形夹，它们作为主“导体”，调节许多 复制和维持我们DNA的因素。PCNA是一个同源三聚体环， 在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，它们使用异三聚体夹9-1-1来协调特定的“SOS”。 修复因子这两个夹子的协同努力对基因组稳定性至关重要。许多癌症都与 不适当的钳位协调和它们表达的变化。因为滑动夹对于许多人来说都是核心 致癌通路，我们必须解决他们如何调节自己和他们的客户合作伙伴。这项建议 旨在解决以下有关滑动夹具的问题：1）滑动夹具如何协调其各种 合伙人2)滑动钳花在DNA上的时间会影响基因组的稳定性吗？（3）什么决定了 滑动夹的现场特定加载？我提出了一个多学科的方法来解决这些问题， 通过研究两种引起疾病的PCNA变体[PCNA-S228 I [丝氨酸到异亮氨酸]和 PCNA-C148 S [半胱氨酸至丝氨酸]]和9-1-1的加载机制。我假设滑动夹 通过位点特异性加载、适当的伴侣相互作用和停留时间来控制基因组完整性 DNA.我进一步假设PCNA-S228 I和PCNA-C148 S通过以下方式破坏基因组完整性： 促进过早的DNA解离或破坏伴侣相互作用。最后，我假设 Rad 17亚基改变钳装载器结构以在DNA位点特异性装载9-1-1钳 损害在目标1和目标2中，我将使用PCNA-S228 I和PCNA-C148 S来解决夹钳如何“选择”它们的 并在DNA上控制他们的时间。我将使用X射线晶体学，展开实验，和一系列 功能测定以确定每个变体如何损害基因组稳定性。在目标3中，我将确定 夹具9-1-1的加载机制，以解决夹具如何加载到基因组中的特定位点。我会用 冷冻电子显微术以确定Rad 17-RFC如何结合到钳9-1-1。总的来说，我的工作将扩大 我们对导致基因组不稳定性的因素的洞察，这可能会增加个性化的发展， 化疗药物

项目成果

A thermosensitive PCNA allele underlies an ataxia-telangiectasia-like disorder.

• DOI: 10.1016/j.jbc.2023.104656
• 发表时间: 2023-05
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Magrino, Joseph;Munford, Veridiana;Martins, Davi Jardim;Homma, Thais K.;Page, Brendan;Gaubitz, Christl;Freire, Bruna L.;Lerario, Antonio M.;Vilar, Juliana Brandstetter;Amorin, Antonio;Leao, Emilia K. E.;Kok, Fernand;Menck, Carlos F. M.;Jorge, Alexander A. L.;Kelch, Brian A.
• 通讯作者: Kelch, Brian A.