UDG Superfamily Glycosylases and the Chemotherapeutic Response

项目摘要/摘要 虽然尿嘧啶(U)在RNA中是一个典型的碱基，但它在DNA中具有诱变潜力。U个病变已得到纠正 通过DNA碱基切除修复(BER)途径。BER是由病变特有的糖基酶启动的 去除碱基，如尿嘧啶DNA糖基酶。所产生的基本站点然后由 下游的酶来恢复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糖基酶(SMUG1)；(3)胸腺嘧啶DNA糖基酶(TDG)。所有三种酶 能切除U和化疗药物5-氟尿嘧啶(5FU)。因此，这三个超级家族 一些成员被认为与细胞对5FU处理的反应有关。Ung、SMUG1和TDG也有 已知的或候选的赖氨酸乙酰化位点。初步研究表明，UDG超家族 当糖基酶被包装成核小体核心颗粒时，显示出从DNA中截取U的截留率截然不同 (NCP)，由145个碱基对组成，包裹在8个组蛋白的核心上。这些结果导致了 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的糖基酶活性有很大的不同，这种差异在 活性延伸至其他病变，如5FU？以及(3)翻译后乙酰化是否决定了细胞 对5FU的反应？这一建议旨在检验UDG超家族糖基酶的调节 翻译后乙酰化的活性影响细胞对5FU的反应。在目标1中，赖氨酸的作用 将测定乙酰化对UDG超家族糖基酶活性的影响。使用生化工具，定量 将获得在同时含有U和5FU的DNA底物上的结合和催化措施。在目标2中， UDG超家族糖基酶对5FU反应的贡献将被检测。使用一系列的细胞系， 糖基酶的表达将被打倒，乙酰化的效果将通过调控表达来检验 组蛋白/蛋白脱乙酰基酶SIRT1。这些细胞系将被5FU处理，细胞活力将被 量过了。此外，这些酶中每一种的DNA占有率将通过以下测序进行探测 染色质免疫沉淀(CHIP-SEQ)。这些实验将阐明这些糖基酶是如何 用于评估肿瘤对化疗药物的敏感性，以及确定新的治疗机制 增强肿瘤杀伤力。该奖项的长期目标是过渡到内科科学家的职业生涯， 探索代谢性疾病和线粒体DNA修复的交叉。一支赞助商团队已经 拥有酶学、DNA修复、细胞和分子生物学以及代谢性疾病方面的专业知识。 该团队还将在过渡到临床工作的过程中提供指导。将从讲习班获得进一步的培训， 布朗大学内外都提供课程和临床培训，并提供机会 出席出席会议并在会上发言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.