DYNAMICS OF DNA DAMAGE RECOGNITION BY REPAIR ENZYMES

修复酶识别 DNA 损伤的动力学

• 批准号: 6137542
• 负责人: ROMAN OSMAN
• 金额: $ 37.98万
• 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 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6137542
• 关键词: DNA damage; chemical condensation; chemical hydration; computer simulation; endodeoxyribonuclease; enzyme mechanism; enzyme substrate complex; fluorescence spectrometry; hydrogen bond; ionic bond; molecular dynamics; nucleic acid structure; phosphoester ligase; pyrimidine dimers; radiation genetics; thermodynamics; time resolved data

DESCRIPTION: The long term goal of this proposal is to develop a molecular understanding, derived from a combined theoretical-experimental collaborative approach, of the principles of specificity of the recognition of damaged DNA by repair enzymes and the stability of the enzyme-DNA complex. Recent x-ray determination of the structure of several repair enzymes presents an opportunity to investigate in molecular details the mechanisms of selective recognition of DNA damage. The researchers will investigate the recognition of DNA with a thymine dimer lesion that is recognized by the enzyme T4 endonuclease V. The availability of the x-ray structure of the enzyme and the complex with damaged DNA is the impetus for the investigation of the molecular principles of the specificity and stability of the complex. The working hypothesis is that four factors contribute to selective recognition of damaged DNA. These are encoded in 1) changes in the structure of DNA around the damaged area, 2) the ability of DNA to undergo a spontaneous or protein-induced flipping of a base near the damaged site into an extrahelical position, 3) asymmetric distribution of condensed counterions around damaged DNA that produces an electrostatic orientational field, and 4) changes in DNA hydration around the point of damage that influences the thermodynamics of the interaction between the repair enzyme and the damaged DNA. These principles appear to be generally applicable to many repair enzymes recognizing damaged DNA. The aims of this proposal to investigate in detail these factors, cannot be fully accomplished by either an exclusively theoretical or a purely experimental approach and they, therefore, propose a complementary collaborative approach in which they combine computational simulations with time-dependent fluorescence measurements to investigate the factors that determine the specificity of damaged DNA recognition by repair enzymes. Interpretation of experimental results will be supported by the realistic representation of molecular models, their vibrational properties and the aqueous ionic environment derived from molecular simulations. In a complementary way, mechanistic interpretations and predictions from molecular simulations will be tested rigorously by experimental measurements. Such complementary research will lead to a better understanding of the factors that contribute to specificity of repair enzymes and play an important role in specific protein-DNA interactions in general.

描述：本提案的长期目标是开发一种分子生物学方法， 理解，从理论和实验相结合的 合作的方式，识别的具体性原则， 修复酶和酶-DNA的稳定性 复杂. 最近几次修复的结构的X射线测定 酶提供了一个机会，研究分子细节， DNA损伤的选择性识别机制。 研究人员将 研究具有胸腺嘧啶二聚体损伤的DNA的识别， 由酶T4核酸内切酶V识别。X射线的可用性 酶的结构和与受损DNA的复合物是 特异性分子机理的研究， 复合体的稳定性。 工作假设是四个因素 有助于选择性识别受损的DNA。 这些编码在1） 损伤区域周围DNA结构的变化，2） DNA发生自发的或蛋白质诱导的碱基翻转， 受损部位进入螺旋外位置，3） 在受损DNA周围凝聚的抗衡离子， 方向场，和4）在DNA水化周围的点的变化， 破坏，影响热力学之间的相互作用， 修复酶和受损的DNA 这些原则似乎是普遍的。 适用于许多识别受损DNA的修复酶。 其目的是 建议详细调查这些因素，不能完全 通过完全的理论或纯实验来完成 因此，他们提出了一种互补的合作方法， 其中他们将联合收割机计算模拟与时间相关的 荧光测量，以研究决定 修复酶识别受损DNA的特异性。 解释 实验结果将得到以下现实表示的支持： 分子模型，它们的振动性质和水离子 环境源自分子模拟。 以一种互补的方式， 从分子模拟的机制解释和预测将 通过实验测量进行严格测试。 这种互补 研究将有助于更好地了解导致 修复酶的特异性，并在特异性修复中发挥重要作用。 蛋白质与DNA的相互作用