DYNAMICS OF DNA DAMAGE RECOGNITION BY REPAIR ENZYMES

修复酶识别 DNA 损伤的动力学

• 批准号: 6283581
• 负责人: ROMAN OSMAN
• 金额: $ 38.49万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6283581
• 关键词: DNA directed DNA polymerase; DNA repair; N glycosidase; active sites; adenine; adenosine triphosphate; aminopurine; binding sites; chemical models; computer simulation; endodeoxyribonuclease; enzyme activity; fluorescence resonance energy transfer; lyase; molecular dynamics; nuclear magnetic resonance spectroscopy; pyrimidine dimers; tryptophan analog; ultracentrifugation

DESCRIPTION:The long-term goal of this continuing project is to use a combined theoretical-experimental collaborative approach to develop a molecular understanding of the principles of specific DNA damage and repair. In the proposed new project period, two mechanisms of thymine dimer (TD) repair will be studied. One mechanism is that of the enzyme endonuclease V (endoV) that specifically recognizes the TD and repairs it by excision. The other repair process is accomplished by an error-free translesion synthesis. EndoV specifically recognizes the TD and flips the complementary adenine to the 5'-thymine of the TD into a protein pocket. It appears that this mechanism of base flipping is universal to base excision repair. Thus the investigators will focus this project on gaining an understanding of the molecular, energetic and kinetic elements that differentiate base flipping in damaged DNA from that in undamaged DNA. The specificity of enzymes is determined not only by the recognition event, but also by the catalytic mechanism. The catalytic steps of endoV consist of a glycosylase step followed by a lyase step. The investigators propose to study the mechanism of enzymatic catalysis in endoV as a contribution to selectivity in DNA repair. Translesion synthesis is a newly discovered activity of several polymerases. Polymerase eta (poleta) is a particular example that successfully synthesizes the correct base sequence opposite the TD. Poleta has low processivity, suggesting a tolerant site for DNA and the incoming nucleoside triphosphate (NTP) binding. Low processivity is characteristic of several other polymerases (e.g., polymerase beta) engaged in DNA repair. The investigators propose that critical features of the mechanism of error-free translesion DNA synthesis by poleta are derived from its ability to recognize the special structural and dynamic properties of DNA containing a TD and that the site in the enzyme that preferentially binds deoxy-ATP over other nucleotides is in part formed by the interaction of the protein with the TD-containing template DNA. The investigators propose to identify the dATP binding site on the basis of homology with other low-processive polymerases, construct a model site by molecular modeling techniques and test the modeled predictions by spectroscopic and thermodynamic experiments. The investigators present these aims as a fully integrated collaborative approach in which they combine experimental and theoretical studies in a complementary way. They feel that the results of this integrated approach will lead to a better understanding of the factors that contribute to the specificity of repair enzymes and play an important role in developing a more general understanding of specific protein-DNA interactions.

描述：这个持续项目的长期目标是使用一个组合 理论-实验协作方法来开发分子 理解特定DNA损伤和修复的原理。在 提出新的项目期间，胸腺嘧啶二聚体（TD）修复的两种机制将 被研究。一种机制是内切核酸酶V（endoV）， 专门识别TD并通过切除来修复它。另一个修复 该过程通过无误差的translesion合成来完成。EndoV 特异性识别TD并将互补腺嘌呤翻转为 TD的5 '-胸腺嘧啶进入蛋白质口袋。看来，这一机制 碱基翻转对于碱基切除修复是通用的。因此，研究人员将 这个项目的重点是了解分子，能量和 区分受损DNA中碱基翻转与受损DNA中碱基翻转的动力学元件 完整的DNA酶的特异性不仅取决于 识别事件，但也通过催化机制。催化步骤 endoV由糖基化酶步骤和随后裂解酶步骤组成。调查人员 建议研究endoV中的酶催化机制， 对DNA修复的选择性的贡献。翻译合成是一种新的 发现了几种聚合酶的活性。聚合酶eta（poleta）是一种 成功合成正确碱基序列的特定示例 与TD相对。Poleta的持续合成能力较低，这表明它是一个耐受 DNA和引入的三磷酸核苷（NTP）结合。低持续合成能力是 几种其它聚合酶的特征（例如，聚合酶β）参与 DNA修复研究人员提出，该机制的关键特征 poleta无错误跨损伤DNA合成的能力来自于它的能力， 为了识别DNA的特殊结构和动力学特性， TD和酶中的位点，优先结合脱氧ATP超过 其他核苷酸部分地通过蛋白质与蛋白质的相互作用形成。 含TD的模板DNA。研究人员建议鉴定dATP 结合位点基于与其它低加工性聚合酶的同源性， 通过分子模拟技术构建一个模型站点，并测试模型 通过光谱和热力学实验进行预测。调查人员 将这些目标作为一种完全一体化的协作方法， 联合收割机以互补的方式结合实验和理论研究。他们觉得 这种综合办法的结果将导致更好的 了解有助于修复特异性的因素 酶，并发挥重要作用，在发展更普遍的理解， 特定的蛋白质-DNA相互作用。