Molecular Mechanisms of Human DNA Polymerase Beta Catalysis, Fidelity and Selective Inhibition

人类 DNA 聚合酶 Beta 催化、保真度和选择性抑制的分子机制

• 批准号: 8918545
• 负责人: MYRON GOODMAN
• 金额: $ 55.96万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8918545
• 关键词: Acids; Active Sites; Address; Area; Binding; Binding Sites; Biological Assay; Cancer cell line; Catalysis; Cations; Cell-Free System; Cells; Chemicals; Chemistry; Complement; DNA Polymerase beta; DNA-Directed DNA Polymerase; Data; Detection; Distal; Electron Nuclear Double Resonance; Electron Spin Resonance Spectroscopy; Electronics; Electrostatics; Evaluation; Family; Free Energy; Grant; Human; Individual; Instruction; Investigation; Ions; Kinetics; Left; Lyase; Malignant Neoplasms; Measurement; Measures; Metals; Mitochondria; Modification; Molecular; Molecular Conformation; Nucleotides; Oxygen; Polymerase; Property; Proteins; Reaction; Relative (related person); Reporting; Research; Role; Series; Site; Specificity; Spin Labels; Stereoisomer; Structure; Translations; Variant; analog; base; bisphosphonate; cancer cell; design; human DNA; inhibitor/antagonist; innovation; insight; member; mutant; novel; novel strategies; prevent; repair enzyme; research study; scaffold; spelling; theories; tool

DNA polymerase mechanisms of base selection and catalysis are explored using a tool-kit of dNTP analogs that have bisphosphonates in place of the ß, γ -bridge oxygen. These analogs are Pol ß substrates that have leaving groups with widely dispirate electronic properties, pKa4 values ranging from 7.8 to 12.3, enabling us to use presteady state kinetic measurements to determine the selection of right and wrong deoxynucleotides occuring at the chemical transition state. Especially important members of the toolkit include all four individually synthesized (R)- and (S)- ß, γ -CHF and ß, γ -CHCI diastereomers. Our recent observation of a pronounced stereoselection for (R)-CHF in Pol ß, involving an electrostatic interaction of F with Arg183, unique to family X pols such as Pol ß, serves as the impetus for a "scaffold" strategy for the selective inhibition of Pol ß relative to the cellular replication Pols δ, ϵ, α, and mitochondrial Pol γ. In Aim 1, Study "a" explores stereoselection in the cellular and mitochondrial pols. Studies "b" and "c" make use of the entire toolkit to explore chemistry vs. conformational change as a rate-limiting step In the transition-state of family X Pol λ, family Y error-prone Pol r\, and for a series of cancer-associated Pol ß variants. In Study "d", newly synthesized bisphosphonate PPI analog leaving groups are used to reverse the polymerase reaction to attain a free energy reaction profile for Pol ß. Study "e" addresses an entirely new area of polymerase mechanistics, namely the use of EPR and ENDOR to elucidate the structure of the metal coordination site and Its role in fidelity. Aim 2, containing three interrelated studies, spells out a detailed scaffold strategy for the design and synthesis of selective Inhibitors of Pol ß and BER by the dual targeting of the dNTP binding site and active site Arg183 (Study "a"). In Study "b", we develop a new approach, using pamoic acid analogs, to inhibit BER by selectively interfering with the Pol ß-assocated lyase. In Study "c", we take an important step along the path toward translation, by evaluating the ability of cell permeabilized scaffold compounds to inhibit cultured cancer cells.

使用DNTP类似物的工具量探索了碱选择和催化剂的DNA聚合酶机制 具有双膦酸盐代替β-γ-桥氧。这些类似物是具有 留下具有广泛驱散电子特性的组，PKA4值范围为7.8至12.3，使我们能够 使用presteady状态动力学测量来确定对与错误的脱氧核苷酸的选择 发生在化学过渡状态。工具包的特别重要成员包括全部四个 单独合成（R） - 和（S） - ß，γ -CHF和ß，γ -CHCI非对映异构体。我们最近对 Polß中（R）-CHF的明显立体选择，涉及F与Arg183的静电相互作用， 家庭X pol的独特 在AIM 1中，研究“ A” 探索细胞和线粒体pol中的立体选择。研究“ B”和“ C”利用了整个 探索化学与构象变化的工具包作为家庭过渡状态的限制步骤 xpolλ，易于错误的pol r \，以及一系列与癌症相关的Polß变体。在研究“ D”中，新的 合成的双膦酸盐PPI类似物离开组用于逆转聚合酶反应 获得Polß的自由能反应曲线。研究“ E”解决了聚合酶的全新领域 机构，即使用EPR和endor来阐明金属协调位点的结构 及其在忠诚中的作用。 AIM 2包含三个相互关联的研究，阐明了一种详细的脚手架策略 通过双重靶向DNTP结合的Polß选择性抑制剂的设计和合成 站点和主动地点ARG183（研究“ A”）。在研究“ B”中，我们使用pamoic酸类似物开发了一种新方法 通过选择性干扰Polß相关的裂解酶来抑制BER。在研究“ C”中，我们采取了重要的 通过评估细胞透化脚手架化合物的能力沿着翻译的路径逐步走 抑制培养的癌细胞。