UDG Superfamily Glycosylases and the Chemotherapeutic Response

UDG 超家族糖基化酶和化疗反应

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要尽管尿嘧啶 (U) 是 RNA 中的典型核碱基，但它在 DNA 中具有诱变潜力。 U病变得到纠正通过 DNA 碱基切除修复 (BER) 途径。 BER 由特定于病变的糖基酶启动，去除核碱基，例如尿嘧啶DNA糖基化酶。然后将生成的 abasic 站点进行处理下游酶来恢复 DNA。人类细胞表达三种属于尿嘧啶的尿嘧啶糖基化酶 DNA糖基化酶(UDG)超家族：(1)尿嘧啶DNA糖基化酶(UNG)； (2)单链选择性单功能尿嘧啶 DNA 糖基化酶 (SMUG1)； (3)胸腺嘧啶DNA糖基化酶(TDG)。所有三种酶能够切除 U 以及化疗药物 5-氟尿嘧啶 (5FU)。因此所有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位点。初步研究表明UDG超家族当糖基化酶包装到核小体核心颗粒中时，其从 DNA 中切除 U 的能力截然不同 (NCP)，由包裹在八个组蛋白核心周围的 145 个碱基对组成。这些结果导致三个基本问题：(1) 翻译后乙酰化是否调节 UNG、SMUG1 和 TDG？ (2) 如果从 NCP 中切除 U 的糖基化酶活性显着不同，这种差异是否会导致活性扩展到其他病变，例如 5FU？ (3) 翻译后乙酰化是否决定细胞对5FU的反应？该提案旨在检验以下假设：UDG 超家族糖基化酶的调节翻译后乙酰化活性影响细胞对 5FU 的反应。在目标 1 中，赖氨酸的作用将测定乙酰化对UDG超家族糖基化酶活性的影响。使用生化工具，定量将获得对含有 U 和 5FU 的 DNA 底物的结合和催化测量。在目标 2 中，将检查 UDG 超家族糖基化酶对 5FU 反应的贡献。使用一系列细胞系，糖基化酶表达被敲低，通过调节表达来测试乙酰化的效果组蛋白/蛋白脱乙酰酶 SIRT1。这些细胞系将用 5FU 处理，细胞活力将受到影响。测量。此外，将通过以下测序来探测每种酶的 DNA 占用率染色质免疫沉淀 (ChIP-Seq)。这些实验将阐明这些糖基酶如何除了确定新的机制外，还用于评估肿瘤对化疗的敏感性增强肿瘤杀伤作用。该奖项的长期目标是转变为医师科学家的职业，探索代谢疾病与线粒体 DNA 修复的交叉点。赞助团队已汇集了酶学、DNA 修复、细胞和分子生物学以及代谢疾病方面的专业知识。该团队还将指导向临床工作的过渡。将从讲习班获得进一步的培训，布朗大学内外提供的课程和临床大查房以及机会出席并出席会议。这些工具对于技术培训和职业发展至关重要。