The role of PRMT5 in preventing intra-chromosomal deletions in cancer cells

PRMT5 在预防癌细胞染色体内缺失中的作用

• 批准号: 10728560
• 负责人: Renee Bouley
• 金额: $ 7.88万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10728560
• 关键词: Acetyltransferase; Active Sites; Affect; Automobile Driving; BRCA1 gene; BRCA2 gene; Biochemical; Biological Assay; Biological Models; Catalogs; Cell Cycle; Cell Proliferation; Cells; Chromosomal Rearrangement; Chromosome Deletion; Chromosome abnormality; Complex; DNA Damage; DNA Double Strand Break; DNA Repair; DNA annealing; Data; Development; Direct Repeats; Double Strand Break Repair; Elements; Enzymes; Eukaryota; Event; Evolution; Excision; Fission Yeast; Frequencies; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Recombination; Goals; HTATIP gene; Histones; Homology Modeling; Human; In Vitro; Link; Malignant Neoplasms; Maps; Mediating; Methylation; Methyltransferase; Mitosis; Modeling; Mutate; Mutation; Mutation Analysis; Mutation Detection; Nonhomologous DNA End Joining; Pathogenicity; Pathway interactions; Peptides; Pharmaceutical Preparations; Post-Translational Protein Processing; Process; RAD52 gene; Repetitive Sequence; Reporting; Role; S phase; Side; Somatic Mutation; Techniques; Testing; Validation; Work; Yeasts; analog; arginine methyltransferase; artificial intelligence algorithm; brca gene; cancer cell; carcinogenesis; chromatin remodeling; design; driver mutation; enzyme activity; experimental study; gene repair; genetic information; homologous recombination; in vivo; innovation; metaplastic cell transformation; mutant; neoplastic cell; prevent; recombinational repair; repaired; response; small molecule inhibitor; yeast genetics

Project Summary/Abstract PRMT5 is an arginine methyltransferase with key roles in cancer. The gene has pleiotropic functions ranging from gene regulation and development to modulation of DNA double strand break (DSBs) repair. In S-phase and mitosis, DSBs are repaired primarily by homologous recombination (HR) a process in which missing genetic information is copied from another undamaged chromosomal region. In humans, error-free HR is mediated by BRCA1, BRCA2 and RAD51. RAD52, an accessory gene, facilitates an error-prone HR sub-pathway that can produce intrachromosomal deletions (ICDs). PRMT5 appears to modulate chromatin remodeling at the DSB through the error-free BRCA1/2-RAD51 pathway. In fission yeast (S. pombe), we identified physical and genetic interactions between PRMT5 and RAD52. Deletion of PRMT5 increases the frequency of ICDs while deletion of RAD52 decreases ICDs suggesting that they have opposite functions. In cancer cells PRMT5 mutations increases the size of ICDs. Using artificial intelligence algorithms, we discovered multiple likely pathogenic mutations including three driver mutations in the active site of PRMT5 that are likely to destabilize the function of the enzyme. We hypothesize that PRMT5 inhibits ICDs by biasing repair of DNA double strand breaks toward conservative pathways. We developed innovative in vivo assays to probe HR repair mechanisms that produce ICDs. The repair mechanisms and factors involved are conserved from yeast to humans making the S. pombe model system highly tractable. A homology analysis reveals that all identified human mutated residues are present in yeast. In Aim1 we will place PRMT5 in the DNA damage repair epistatic pathway using mutational analysis and sensitivity to various DNA damage drugs. Additionally, we will employ in vivo repair assays to understand the HR pathways by which ICDs are produced in the absence of the PRMT5 function. In Aim 2 we will analyze PRMT5 mutations identified in cancer cells using the Catalogue of Somatic Mutations in Cancer (COSMIC). We will employ modeling techniques and enzymatic assays to test how these mutations affect the enzymatic function of PRMT5 and DSB repair. Most of these mutations are conserved in yeast making this analysis tractable. The findings will be further validated in human cells.

项目概要/摘要 PRMT5 是一种精氨酸甲基转移酶，在癌症中发挥着关键作用。该基因具有多效性功能 从基因调控和发育到 DNA 双链断裂 (DSB) 修复的调节。在S期和 有丝分裂期间，DSB 主要通过同源重组 (HR) 进行修复，在该过程中，缺失的基因 信息是从另一个未受损的染色体区域复制的。在人类中，无差错 HR 是由以下因素调节的： BRCA1、BRCA2 和 RAD51。 RAD52 是一种辅助基因，它促进了一个容易出错的 HR 子通路，可以 产生染色体内缺失（ICD）。 PRMT5 似乎可以调节 DSB 处的染色质重塑 通过无错误的 BRCA1/2-RAD51 途径。 在裂殖酵母（S. pombe）中，我们鉴定了 PRMT5 和 RAD52 之间的物理和遗传相互作用。删除 PRMT5 的缺失增加了 ICD 的频率，而 RAD52 的缺失则降低了 ICD 的频率，这表明它们具有 相反的功能。在癌细胞中，PRMT5 突变会增加 ICD 的大小。使用人工智能 算法中，我们发现了多种可能的致病突变，包括活性基因中的三个驱动突变 PRMT5 的位点可能会破坏酶功能的稳定性。 我们假设 PRMT5 通过偏向修复 DNA 双链断裂来抑制 ICD 走向保守的道路。 我们开发了创新的体内检测方法来探测产生 ICD 的 HR 修复机制。修复 所涉及的机制和因素从酵母到人类都是保守的，使得粟酒裂殖酵母模型系统高度 易于处理。同源性分析表明，所有已鉴定的人类突变残基都存在于酵母中。 在 Aim1 中，我们将使用突变分析将 PRMT5 置于 DNA 损伤修复上位途径中， 对各种 DNA 损伤药物的敏感性。此外，我们将采用体内修复测定来了解 在缺乏 PRMT5 功能的情况下产生 ICD 的 HR 途径。在目标 2 中我们将分析 使用癌症体细胞突变目录 (COSMIC) 在癌细胞中鉴定出 PRMT5 突变。我们 将采用建模技术和酶测定来测试这些突变如何影响酶功能 PRMT5 和 DSB 修复。大多数这些突变在酵母中是保守的，使得这种分析易于处理。这 研究结果将在人体细胞中得到进一步验证。