UDG Superfamily Glycosylases and the Chemotherapeutic Response

UDG 超家族糖基化酶和化疗反应

• 批准号: 10462782
• 负责人: Mary Elizabeth Tarantino
• 金额: $ 5.18万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462782
• 关键词: 1 year old; Acetylation; Address; Affect; Award; Base Excision Repairs; Base Pairing; Binding; Biological; Catalysis; Cell Line; Cell Survival; Cells; ChIP-seq; Chemicals; Chromatin; Clinical; Coupled; Cytosine; DNA; DNA Packaging; DNA Repair; DNA Repair Enzymes; DNA strand break; Deacetylase; Deamination; Dinucleoside Phosphates; Educational workshop; Embryo; Embryonic Development; Enzymatic Biochemistry; Enzymes; Epigenetic Process; Excision; Fluorouracil; Genes; Genome; Genomics; Goals; Histones; Human; Individual; Knock-out; Knowledge; Lesion; Lysine; Maintenance; Malignant Neoplasms; Measures; Mentors; Metabolic Diseases; Mitochondrial DNA; Molecular; Molecular and Cellular Biology; Mus; Mutation; Nucleosome Core Particle; Pathway interactions; Pharmaceutical Preparations; Physicians; Post-Translational Protein Processing; Process; Proteins; RNA; Role; SIRT1 gene; Scientist; Series; Site; Specificity; Substrate Specificity; Testing; Thymine DNA Glycosylase; Training; Universities; Uracil; Western Blotting; Work; base; biochemical tools; career; career development; chemotherapeutic agent; chromatin immunoprecipitation; colon cancer cell line; cytotoxicity; experimental study; knock-down; member; novel; nucleobase; potential biomarker; predicting response; predictive marker; preference; repaired; response; symposium; tool; tumor; uracil-DNA glycosylase

Project Summary/Abstract Although uracil (U) is a canonical nucleobase in RNA, it has mutagenic potential in DNA. U lesions are corrected by the DNA base excision repair (BER) pathway. BER is initiated by a glycosylase specific to the lesion that removes the nucleobase, such as a uracil DNA glycosylase. The resulting abasic site then is processed by downstream enzymes to restore the DNA. Human cells express three uracil glycosylases belonging to the uracil DNA glycosylase (UDG) superfamily: (1) uracil DNA glycosylase (UNG); (2) single-strand selective monofunctional uracil DNA glycosylase (SMUG1); and (3) thymine DNA glycosylase (TDG). All three enzymes are capable of excising U as well as the chemotherapeutic 5-fluorouracil (5FU). Hence all three superfamily members have been implicated in the response of cells to 5FU treatment. UNG, SMUG1, and TDG also have known or candidate sites of lysine acetylation. Preliminary studies have demonstrated that UDG superfamily glycosylases display drastically different excision of U from DNA when packaged into a nucleosome core particle (NCP), consisting of 145 base pairs wrapped around a core of eight histone proteins. These results have led to three fundamental questions: (1) does post-translational acetylation modulate the activity of UNG, SMUG1, and TDG? (2) If the glycosylase activities are drastically different for excision of U from NCP, does this difference in activity extend to other lesions, such as 5FU? And (3) does post-translational acetylation determine the cellular response to 5FU? This proposal seeks to test the hypothesis that modulation of UDG superfamily glycosylase activity by post-translational acetylation affects the cellular response to 5FU. In Aim 1, the effect of lysine acetylation on UDG superfamily glycosylase activity will be determined. Using biochemical tools, quantitative measures of binding and catalysis on DNA substrates containing both U and 5FU will be obtained. In Aim 2, the contribution of the UDG superfamily glycosylases to 5FU response will be examined. Using a series of cell lines, glycosylase expression will be knocked down and the effect of acetylation will be tested by regulating expression of the histone/protein deacetylase SIRT1. These cell lines will be treated with 5FU and cell viability will be measured. Furthermore, the DNA occupancy for each of these enzymes will be probed by sequencing following chromatin immunoprecipitation (ChIP-Seq). These experiments will elucidate how these glycosylases can be used in assessing the tumor sensitivity to a chemotherapeutic in addition to identifying novel mechanisms for enhanced tumor killing. The long-term goal for this award is to transition into a career as a physician-scientist, exploring the intersection of metabolic disease and mitochondrial DNA repair. A sponsor team has been assembled with expertise in enzymology, DNA repair, cellular and molecular biology, and metabolic disease. This team also will mentor in the transition to clinical work. Further training will be acquired from workshops, courses, and clinical Grand Rounds offered both at and outside of Brown University coupled with opportunities to attend and present at conferences. These tools are essential for technical training and career development.

项目摘要/摘要 尽管尿嘧啶（U）是RNA中的典型核酶，但它在DNA中具有诱变潜力。纠正了u病变 通过DNA碱基切除修复（BER）途径。 BER是由特异性的糖基酶发起的 去除核碱基，例如尿嘧啶DNA糖基化酶。然后由此产生的abasic网站通过 下游酶以恢复DNA。人类细胞表达属于尿嘧啶的三种尿嘧啶糖基酶 DNA糖基酶（UDG）超家族：（1）尿嘧啶DNA糖基酶（UNG）； （2）单链选择性 单功能尿嘧啶DNA糖基化酶（SMUG1）； （3）胸腺胺DNA糖基化酶（TDG）。所有三种酶 能够切除U以及化学治疗性5-氟尿嘧啶（5FU）。因此，这三个超家族 成员与细胞对5FU治疗的反应有关。 UNG，SMUG1和TDG也有 赖氨酸乙酰化的已知或候选部位。初步研究表明UDG超家族 糖基酶在包装到核小体核心粒子中显示出U与DNA的截然不同 （NCP），由包裹在八个组蛋白蛋白的核心周围的145个碱基对组成。这些结果导致了 三个基本问题：（1）翻译后乙酰化会调节UNG，SMUG1和 TDG？ （2）如果从NCP切除U的糖基化酶活性截然不同，则此差异在 活动扩展到其他病变，例如5FU？ （3）翻译后乙酰化决定了细胞 对5FU的响应？该建议旨在检验UDG超家族糖基化酶调节的假设 翻译后乙酰化的活性会影响细胞对5FU的反应。在AIM 1中，赖氨酸的作用 将确定对UDG超家族糖基化酶活性的乙酰化。使用生化工具，定量 将获得对包含U和5FU的DNA底物的结合和催化的度量。在AIM 2中 将检查UDG超家族糖基酶对5FU反应的贡献。使用一系列细胞系， 糖基化酶表达将被击倒，乙酰化的作用将通过调节表达来测试 组蛋白/蛋白质脱乙酰基酶SIRT1。这些细胞系将用5FU处理，细胞活力将是 测量。此外，这些酶的DNA占用率将通过测序以下探测 染色质免疫沉淀（CHIP-SEQ）。这些实验将阐明这些糖基化酶如何 除了确定新的机制外，用于评估肿瘤对化学治疗的敏感性 增强的肿瘤杀死。该奖项的长期目标是转变为医生科学家的职业， 探索代谢疾病和线粒体DNA修复的交集。赞助商团队已经 在酶学，DNA修复，细胞和分子生物学以及代谢疾病方面具有专业知识。 该团队还将指导过渡到临床工作。将从研讨会获得进一步的培训， 课程和临床大回合提供了布朗大学和外部的临床大赛以及机会 参加会议并参加会议。这些工具对于技术培训和职业发展至关重要。