Energetics of Lesion Formation, Recognition, and Repair: Biophysical Studies

能量学代表了结构和功能之间缺失的一环。因此，表征损伤对双链体性质和蛋白质识别/结合事件的能量影响对于理解损伤对DNA复制、诱变和修复途径的影响至关重要。这些特征是特别重要的，因为我们已经证明，深刻的病变引起的双链体能量学的改变，甚至可以发生在没有显着的结构变化，提高了有趣的可能性“充满活力的识别”。在申请的资助期间，我们将使用光谱和量热技术的组合来表征对8-oxo-dG双链体性质的影响;脱碱基位点;双链脱碱基病变; Fapy-dG和胸腺嘧啶乙二醇位点，包括其碳环衍生物;模板未对准缺陷（例如凸出结构）;以及源自复制错误的缺陷（例如，凸起、无碱基凸起、单碱基取代），其与癌症和其它疾病相关。我们还将映射的碱基切除修复途径的能量景观特征的修复酶结合其潜在的底物，过渡态，中间态，和产品类似物。研究的特异性修复酶包括两种嘌呤糖基化酶Fpg和Ogg 1（Fpg的哺乳动物功能同源物），以及两种嘧啶糖基化酶E。coli endo VIII（一种称为Nei的Fpg结构同源物）和E. coli Endo Ⅲ.这些研究中的许多将使用不可水解的卡巴类似物进行，以评估在不存在 周转我们将使用等温滴定和停流混合量热法来表征病变对聚合酶结合和模板指导的DNA合成的影响，从而阐明DNA聚合酶保真度的能量来源，包括在translesion合成过程中发生的碱基的错误掺入。结合并行的结构（项目2）和生物学研究（项目1）正在追求作为该计划项目的一部分，我们提出的双链体性质，修复酶识别和模板指导的DNA合成病变的充满活力的表征将使我们能够定义微观/宏观/功能的相关性，没有单一的方法可以单独产生。这种来自多个实验平台的见解的整合将使我们能够更好地理解氧化DNA损伤所涉及的基本过程的机制，包括损伤形成，DNA损伤，DNA损伤和DNA损伤。 修复和诱变。

Energetics represents the missing link between structure and function. As such, characterizing the energetic impacts of lesions on duplex properties and on protein recognition/binding events is crucial for understanding the influences of lesions on DNA replication, mutagenesis, and repair pathways. These characterizations are particularly important since we have demonstrated that profound lesion-induced alterations in duplex energetics can occur even in the absence of significant structural changes, raising the intriguing possibility of "energetic recognition". During the requested funding period, we will use a combination of spectroscopic and calorimetric techniques to characterize the impacts on duplex properties of 8-oxo-dG; abasic sites; bistrand abasic lesions; Fapy-dG and thymine glycol sites, including their carbocyclic derivatives; template misalignment defects (e.g. bulged structures); and defects originating from replication errors (e.g., bulges, abasic bulges, single base substitutions), which have been associated with cancer and other diseases. We also will map the energetic landscape of the base excision repair pathway by characterizing the energetics of repair enzyme binding to their potential substrate, transition state, intermediate state, and product analogs. Specific repair enzymes targeted for study include two purine glycosylases, Fpg and Ogg1 (a mammalian functional homolog of Fpg), as well as two pyrimidine glycosylases, E. coli endo VIII (a structural homolog of Fpg called Nei) and E. coli Endo III. Many of these studies will be conducted using nonhydrolyzable carba analogs to evaluate binding in the absence of turnover. We will use isothermal titration and stopped-flow mixing calorimetry to characterize the impact of lesions on polymerase binding and template-directed DNA synthesis, thereby elucidating the energetic origins of DNA polymerase fidelity, including misincorporation of bases that occurs during translesion synthesis. In conjunction with the parallel structural (Project 2) and biological studies (Project 1) being pursued as part of this program project, our proposed energetic characterizations of lesions on duplex properties, repair enzyme recognition, and template-directed DNA synthesis will allow us to define microscopic/macroscopic/functional correlations that no single approach alone could yield. This integration of insights derived from multiple experimental platforms will enable us to better understand mechanisms underlying fundamental processes involved in oxidative DNA damage, including lesion formation, DNA repair, and mutagenesis.