Chemistry and Mechanism of Direct DNA Repair Proteins

描述(申请人提供)：甲基化是DNA和RNA最丰富的修饰。它也代表了生物学中最重要的化学信号之一。AlkB家族蛋白是新发现的一种酶，它使用一种新的氧化去甲基化过程来从DNA/RNA碱基上去除甲基。原型是E.coliAlkB，是一种DNA/RNA碱基损伤修复酶，催化N1-甲基腺嘌呤(1-mea)、N3-甲基胞嘧啶(3-MEC)和外环DNA碱基损伤的直接逆转。在人类基因组中已经确定了九个序列同源物。其中一些蛋白质在DNA/RNA修复、肥胖/糖尿病和各种癌症中发挥关键作用。氧化去甲基化机制也被其他参与关键表观遗传过程的人类蛋白质所使用，例如DNA中的组蛋白去甲基化和5-MEC羟化。在这一更新应用中，我们建议结合化学、结构和生物学方法来揭示与AlkB家族蛋白相关的详细去甲基化机制和生物学功能。我们开发并应用了一种化学交联策略来稳定AlkB家族蛋白质的不稳定和瞬时蛋白质-DNA相互作用。使用这一策略，我们将捕获和表征氧化修复中间体，这应该会给出这些以及相关加氧酶的清晰机制图。我们还建议在没有DNA损伤的情况下表征蛋白质与DNA的相互作用，从而进一步深入了解这些蛋白质使用的损伤搜索机制。为补充结构研究，本文将采用解和理论相结合的方法。这种已被证明成功表征AlkB-DNA和ABH2-DNA复合体的交联策略将被应用于研究其他AlkB人类同源物，特别是前列腺癌标记物ABH3和FTO，FTO是肥胖和能量平衡的主要因素。此外，我们还开发了一种光活性非天然氨基酸，它可以高效、准确地结合到活哺乳动物细胞中的蛋白质上。与这种新的探针进行光交联将使我们能够下拉并识别ABH3和FTO的特定配对蛋白或底物。最后，将开发FTO的小分子抑制剂，用于进一步剖析FTO的细胞功能，并作为潜在的治疗导向来调节人体能量平衡。 与公共健康相关：AlkB家族蛋白使用一种新的氧化去甲基化机制来从甲基化的DNA/RNA碱基中去除甲基。人类AlkB同源物在DNA修复、肥胖和各种癌症中发挥着关键作用。这项拟议的研究将有助于更好地理解与这些蛋白质相关的去甲基化机制和生物学功能。

DESCRIPTION (provided by applicant): Methylation is the most abundant modification of DNA and RNA. It also represents one of the most important chemical signals in biology. The AlkB family proteins are newly discovered enzymes that use a novel oxidative demethylation process to remove a methyl group from DNA/RNA bases. The prototype, the E. coli AlkB, is a DNA/RNA base damage repair enzyme that catalyzes direct reversal of N1-methyladenine (1-meA), N3-methylcytosine (3-meC), and exocyclic DNA base lesions. Nine sequence homologues have been identified in the human genome. Some of these proteins play critical roles in DNA/RNA repair, obesity/diabetes, and various cancers. The oxidative demethylation mechanism is also used by other human proteins that are involved in key epigenetic processes such as histone demethylation and 5-meC hydroxylation in DNA. In this renewal application, we propose a combination of chemical, structural, and biological approaches to reveal the detailed demethylation mechanism and biological functions associated with the AlkB family proteins. We have developed and applied a chemical cross-linking strategy to stabilize labile and transient protein-DNA interactions of the AlkB family proteins. With the use of this strategy we will trap and characterize oxidative repair intermediates which should give clear mechanistic pictures for these as well as related oxygenases. We also propose to characterize protein-DNA interactions in the absence of DNA damage, thus providing further insight into the damage-searching mechanism used by these proteins. Both solution and theoretic methods will be employed to complement the structural study. The cross-linking strategy, proven successful in characterizing AlkB-DNA and ABH2- DNA complexes, will be applied to study other AlkB human homologues, in particular, ABH3, a prostate cancer marker, and FTO, a major factor involved in obesity and energy homeostasis. In addition, we have developed a photoactive unnatural amino acid that can be incorporated site-specifically onto proteins in live mammalian cells with high efficiency and fidelity. Photocross-linking with this new probe will allow us to pull down and identify specific partner proteins or substrates of ABH3 and FTO. Lastly, small molecule inhibitors for FTO will be developed, which can be used to further dissect the cellular function of FTO and serve as potential therapeutic leads to modulate human energy homeostasis. PUBLIC HEALTH RELEVANCE: The AlkB family proteins use a novel oxidative dememthylation mechanism to remove a methyl group from methylated DNA/RNA bases. Human AlkB homologues play key roles in DNA repair, obesity, and various cancers. The proposed research will lead to a better understanding of the demethylation mechanism and biological functions associated with these proteins.