UDG Superfamily Glycosylases and the Chemotherapeutic Response

项目概要/摘要 虽然尿嘧啶（U）是RNA中的典型核碱基，但它在DNA中具有致突变潜力。U型病变得到纠正 DNA碱基切除修复（BER）途径。BER由病变特异性糖基化酶启动， 去除核碱基，如尿嘧啶DNA糖基化酶。然后通过以下步骤处理所得的脱碱基位点： 下游的酶来修复DNA人细胞表达三种尿嘧啶糖基化酶，属于尿嘧啶糖基化酶。 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（SMUG 1）;和（3）胸腺嘧啶DNA糖基化酶（TDG）。所有这三种酶 能够切除U以及化疗剂5-氟尿嘧啶（5 FU）。因此这三个超家族 这些成员与细胞对5 FU处理的反应有关。UNG、SMUG 1和TDG也有 已知或候选的赖氨酸乙酰化位点。初步研究表明，UDG超家族 糖基化酶在包装入核小体核心颗粒时显示出显著不同的从DNA中切除U的能力 (NCP)由145个碱基对组成，包裹在8个组蛋白的核心上。这些结果导致 三个基本问题：（1）翻译后乙酰化是否调节UNG、SMUG 1和 TDG？(2)如果从NCP中切除U的糖基化酶活性显著不同， 活动扩展到其他病变，如5 FU？（3）翻译后乙酰化是否决定了细胞的 对5 FU的反应？该提议试图检验UDG超家族糖基化酶的调节 翻译后乙酰化的活性影响细胞对5 FU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。使用生物化学工具，定量 将获得对含有U和5 FU的DNA底物的结合和催化的测量。在目标2中， 将检查UDG超家族糖基化酶对5 FU反应的贡献。使用一系列细胞系， 糖基化酶表达将被敲低，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SIRT 1。这些细胞系将用5 FU处理，并且细胞活力将被测定。 测定了此外，将通过以下测序来探测这些酶中的每一种的DNA占有率： 染色质免疫沉淀（ChIP-Seq）。这些实验将阐明这些糖基化酶是如何 用于评估肿瘤对化疗药物的敏感性，以及鉴定肿瘤对化疗药物敏感性的新机制。 增强肿瘤杀伤。这个奖项的长期目标是过渡到一个职业生涯作为一个物理学家，科学家， 探索代谢疾病和线粒体DNA修复的交叉点。一个赞助商团队已经 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。将通过讲习班获得进一步培训， 课程，以及布朗大学内外提供的临床大查房， 参加并出席会议。这些工具对于技术培训和职业发展至关重要。

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.