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Solid-State NMR Studies of the Dynamics of Damaged DNA

受损 DNA 动力学的固态核磁共振研究

基本信息

批准号：
7456235
负责人：
Gary A Meints
金额：
$ 17.51万
依托单位：
MISSOURI STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-06-01 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7456235
关键词：
Adenine Nucleotides Aging-Related Process Base Excision Repairs Binding Biological Catalysis Chlorella Cleaved cell Complex Computer Simulation DNA DNA Binding DNA Damage DNA Repair DNA Structure DNA-Protein Interaction Decompression Sickness Deuterium Enzyme Kinetics Enzymes Excision Exhibits Goals Helix (Snails)Knowledge Lesion Localized Lyase Malignant Neoplasms Modeling Molecular Conformation Monitor Nuclear Magnetic Resonance Nucleosides Nucleotides Object Attachment Orphan Paramecium Pathway interactions Play Pliability Positioning Attribute Potential Energy Process Property Proteins Public Health Publishing Pyrimidine Dimers Reaction Research Role Simulate Site Surface Thermodynamics Uracil Vertebral column Virus Work anticancer research base cost design ear helix enzyme activity human disease polymerization prevent repair enzyme repaired solid state tetrahydrofuran

项目摘要

DESCRIPTION (provided by applicant): Damage to DNA has been implicated in numerous human diseases, particularly cancer, and the aging process. Our long term goal is to determine dynamic and structural information from damaged DNA and associated repair enzymes, and apply this information to understanding the fundamental aspects of DNA repair. Our specific hypothesis contains three parts: i) damage to DNA bases alters the local conformational dynamics, ii) these dynamics can be modeled and quantitatively correlated with repair enzyme kinetics and thermodynamics, and iii) the local dynamics play a role in the damage recognition process. This hypothesis is based on an established body of work achieved to determine how DNA is repaired. A process called base excision repair (BER) has evolved using glycosylases to help maintain DNA integrity. Glycosylase activity in BER contains several steps including 1) identifying the DNA damage, 2) forming an active enzyme-DNA complex, 3) removing the damaged base, and 4) removing the abasic site to allow for DNA polymerization. During the complexation and removal steps in BER, the damaged nucleotide is completely rotated out of the DNA helix and stabilized within the binding pocket of the glycosylase before the glycosidic bond is cleaved. This process is often referred to as base flipping and is a common motif in many protein-DNA interactions. The final three steps in BER are well characterized; however, the specific modes by which the repair enzymes identify the DNA damage and flip the damaged nucleotide remain unclear. Deformation of the local DNA structure during the binding and base flipping processes, which often also includes local DNA bending or reciprocal flipping of the nucleotide opposite the lesion, occurs at a significant energy cost that may be partially alleviated by local conformation flexibility (exhibited as large amplitude dynamics) at the lesion site. The proposal herein has two primary specific aims. First, local conformational dynamics in damaged free DNA will be characterized using deuterium solid-state nuclear magnetic resonance (SSNMR), and their biological role evaluated. Second, the local DNA dynamics in complex with a pyrimidine dimer glycosylase will be monitored via deuterium SSNMR. The specific aims are designed to determine fundamental properties of damaged DNA, and the results determined will have implications in cancer research by helping determine essential aspects of DNA repair. PUBLIC HEALTH RELEVANCE Cancer very often originates from damage to DNA, and knowledge of the fundamental aspects of how DNA damage is repaired will allow for better opportunities to find cures. This project proposes two avenues of research: i) to study flexibility of damaged free DNA and ii) study the changes in flexibility in damaged DNA bound to a repair enzyme. The work will determine if the flexibility plays a role in the repair process, and aid in the fundamental understanding of how cancer is prevented.

描述（由申请人提供）：DNA损伤与许多人类疾病，特别是癌症和衰老过程有关。我们的长期目标是从受损的DNA和相关的修复酶中确定动态和结构信息，并将这些信息应用于理解DNA修复的基本方面。我们的具体假设包括三个部分：i）DNA碱基损伤改变了局部构象动力学，ii）这些动力学可以建模并与修复酶动力学和热力学定量相关，iii）局部动力学在损伤识别过程中发挥作用。这一假设是基于确定DNA如何修复的既定工作。一个被称为碱基切除修复（BER）的过程已经进化出使用糖基化酶来帮助维持DNA的完整性。BER中的糖基化酶活性包含几个步骤，包括1）鉴定DNA损伤，2）形成活性酶-DNA复合物，3）去除损伤的碱基，和4）去除脱碱基位点以允许DNA聚合。在BER中的络合和去除步骤期间，受损的核苷酸完全旋转出DNA螺旋，并在糖苷键被切割之前稳定在糖基化酶的结合口袋内。这个过程通常被称为碱基翻转，并且是许多蛋白质-DNA相互作用中的常见基序。BER中的最后三个步骤已经得到了很好的表征;然而，修复酶识别DNA损伤并翻转受损核苷酸的具体模式仍然不清楚。在结合和碱基翻转过程中，局部DNA结构的变形，通常还包括局部DNA弯曲或与病变相对的核苷酸的相互翻转，发生在显著的能量消耗下，其可以通过病变部位的局部构象柔性（表现为大幅度动态）而部分减轻。这里的建议有两个主要的具体目标。首先，在受损的游离DNA的局部构象动力学的特点是使用氘固态核磁共振（SSNMR），其生物学作用进行评估。其次，通过氘SSNMR监测与嘧啶二聚体糖基化酶复合的局部DNA动力学。具体目标是确定受损DNA的基本性质，确定的结果将通过帮助确定DNA修复的基本方面而对癌症研究产生影响。癌症通常源于DNA损伤，了解DNA损伤如何修复的基本方面将为找到治疗方法提供更好的机会。该项目提出了两种研究途径：i）研究受损游离DNA的柔性，ii）研究与修复酶结合的受损DNA的柔性变化。这项工作将确定灵活性是否在修复过程中发挥作用，并有助于从根本上了解癌症是如何预防的。