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

病变形成、识别和修复的能量学：生物物理学研究

• 批准号: 6990361
• 负责人: KENNETH J. BRESLAUER
• 金额: $ 17.86万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-21 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6990361
• 关键词: DNA damage; DNA repair; DNA replication; adduct; bioenergetics; biophysics; calorimetry; enzyme activity; enzyme substrate complex; gene mutation; high throughput technology; neoplasm /cancer genetics; nucleic acid biosynthesis; oxidative stress; thermodynamics

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.

能量学代表了结构和功能之间缺失的一环。因此，表征病变对双链特性和蛋白质识别/结合事件的能量影响对于理解病变对DNA复制、诱变和修复途径的影响至关重要。这些特征尤其重要，因为我们已经证明，即使在没有显著结构变化的情况下，也会发生双相能量学中深刻的损伤引起的改变，从而提出了“能量识别”的有趣可能性。在申请资助期间，我们将结合使用光谱和量热技术来表征对8-氧- dg双相性质的影响；基本的网站;双链基性病变；fpy - dg和胸腺嘧啶乙二醇位点，包括它们的碳环衍生物；模板错位缺陷（如结构凸起）；以及由复制错误引起的缺陷（例如，凸起、碱性凸起、单碱基取代），这些缺陷与癌症和其他疾病有关。我们还将通过表征修复酶与潜在底物、过渡状态、中间状态和产物类似物结合的能量学，绘制碱基切除修复途径的能量景观。研究的特异性修复酶包括两种嘌呤糖基酶Fpg和Ogg1（哺乳动物Fpg的功能同源物），以及两种嘧啶糖基酶E. coli endo VIII （Fpg的结构同源物Nei）和E. coli endo III。许多这些研究将使用非水解碳水化合物类似物来评估在缺乏碳水化合物的情况下的结合