Mechanisms and Transition States for DNA Glycosylases

描述（申请人提供）：DNA碱基切除的第一步 修复途径是受损细胞的糖苷键的水解断裂， 或通过损伤特异性糖基化酶错配碱基。长期目标是 研究的目的是从根本上了解这些酶 特异性识别并切除大量正常碱基对中受损的碱基。 DNA糖基化酶由于其在以下方面的作用而受到健康相关的关注： 监管基因组的诱变前病变，并已被牵连， 调节几种广泛使用的化疗剂的功效， 修饰DNA，如5-氟尿嘧啶和各种烷化剂。具体 该建议的目的是（一）确定和量化施加在 导致受损碱基螺旋外翻转成 酶的活性部位。尿嘧啶DNA糖基化酶（UDG）将被用作范例 系统，以评估诱导螺旋应变和“捏推拉”的作用， 使用NMR和荧光光谱的碱基翻转模型，快速动力学 测量、酶诱变和工程化DNA类似物。(ii)测试角色 在UDG反应中的基态不稳定。最近的水晶 与C-糖苷底物类似物结合的UDG的结构表明， 酶使糖苷键弯曲大约41度， 激活屏障。主要研究者将研究替代方案 用核磁共振、诱变和拉曼光谱解释了这一有趣的机制 光谱学方法。(iii)确定活动站点的功能 UDG的环境，允许形成显着的oxacarbenium 通过测量具有活性的同位素的动力学效应的离子-尿嘧啶阴离子中间体 位点突变体和预期使该中间体不稳定的修饰底物。 (iv)探索阳离子嘌呤的识别和去除机理 通过解析3-甲基腺嘌呤DNA的NMR溶液结构， 糖基化酶I（TAG）结合3-MeA与互补 生物物理学研究这些研究将共同填补我们领域的重大空白 了解DNA修复机制，此外，将有助于解决 关于酶催化性质的几个基本问题。是 预计，详细的化学知识和能量的 这些反应将有助于开发新的小分子， 在体内调节这些酶的活性。这样的抑制剂或活化剂可以 可用作抗病毒剂或抗癌剂。

DESCRIPTION (provided by applicant): The first step in the DNA base excision repair pathway is the hydrolytic cleavage of the glycosidic bond of a damaged or mismatched base by a damage-specific glycosylase. The long-term goal of this research is to obtain a fundamental understanding of how these enzymes specifically recognize and excise damaged bases in a sea of normal base pairs. DNA glycosylases are of health related interest because of their role in policing the genome for premutagnenic lesions, and have been implicated in modulating the efficacy of several widely used chemotherapeutic agents that modify DNA, such as 5-fluorouracil and various alkylating agents. The specific aims of this proposal are to (i) Identify and quantify the forces exerted on the DNA substrate that lead to extrahelical flipping of the damaged base into the enzyme active site. Uracil DNA glycosylase (UDG) will be used as a paradigm system to evaluate the role of induced helix strain and the "pinch-push-pull" model for base flipping using NMR and fluorescence spectroscopy, rapid kinetic measurements, enzyme mutagenesis and engineered DNA analogs. (ii) Test the role of ground state destabilization in the UDG reaction. The recent crystal structure of UDG bound to a C-glycoside substrate analog indicates that the enzyme bends the glycosidic bond by approximately 41 degree possibly to lower the activation barrier. The principal investigator will investigate alternative explanations for this intriguing mechanism using NMR, mutagenesis and Raman spectroscopy approaches. (iii) Determine the features of the active site environment of UDG that allow the formation of a remarkable oxacarbenium ion-uracil anion intermediate by measuring kinetic isotope effects with active site mutants and modified substrates expected to destabilize this intermediate. (iv) Discover the mechanism for recognition and removal of cationic purine bases from DNA by solving the NMR solution structure of 3-methyladenine DNA glycosylase I (TAG) bound to 3-MeA in combination with complementary biophysical studies. Together, these studies will fill significant gaps in our understanding of DNA repair mechanisms, and in addition, will serve to address several fundamental issues concerning the nature of enzymic catalysis. It is anticipated that the detailed knowledge of the chemistry and energetics of these reactions will contribute to the development of novel small molecules to modulate these enzyme activities in vivo. Such inhibitors or activators may find use as anti-viral or anti-cancer agents.