STRUCTURE OF DNA LESIONS IN SUPERHELICAL DOMAINS

超螺旋域 DNA 损伤的结构

• 批准号: 2910340
• 负责人: STEPHEN D. LEVENE
• 金额: $ 15.63万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2910340
• 关键词: DNA damage; DNA repair; adduct; bacteriophage lambda; chemical models; cis platinum compound; computer simulation; conformation; cryoelectron microscopy; plasmids; psoralens; recombinant DNA; transposon /insertion element

DNA repair systems recognize a multitude of lesions in DNA, including bulge defects, non-homologous regions, cyclobutane dimers, and the presence of DNA-damaging adducts. Efficient recognition and repair of such lesions occurs despite the presence in the cell of a wide range of normal heterogeneities in DNA structure. It has been proposed that these lesions cause significant alterations of DNA structure and dynamics; however, nearly all of what is known about these defects in DNA structure is based on studies that have been carried out on linear DNA molecules in the absence of superhelical stress. Recent work from this laboratory on the structure of intrinsically bent and unbent A-tract containing DNA sequences indicates that the sequence-dependent structure of these tracts in linear DNA fragments cannot be extrapolated to super coiled domains. Dramatic changes in DNA super coiling that occur during transcription raise important questions about the effect of super coiling on defect structure and its role in transcription-couple DNA repair. The objective of this work is to investigate the conformation and dynamics of several DNA-structure defects in superhelical domains as models of DNA lesions recognized by DNA repair systems. The tertiary structure of super coiled molecules containing bulges (insertion/deletion modification), mismatches, and covalent adducts of psoralen and the antineoplastic drug cisplatin will be examined by site-specific recombination and conventional and cryoelectron microscopy in conjunction with computer simulation. The distribution of knots and catenanes generated by bacteriophage lambda integrative (Int) site-specific recombination is a reporter of the tertiary structure of the DNA substrate. Observed distributions of Int- recombination products will be analyzed using Monte Carlo computer simulations of intramolecular recombination in order to quantitate changes in DNA bending distortion and/or bending, flexibility. The conformation of super coiled, lesion-containing DNA molecules deduced from these solution studies will be compared with that observed by independent conventional and cryoelectron microscopy experiments.

DNA修复系统识别DNA中的多种损伤，包括 凸起缺陷，非同源区域，环丁烷二聚体，以及 DNA损伤加合物的存在。有效识别和修复此类 尽管细胞中存在广泛的正常细胞， DNA结构的异质性。有人提出，这些病变 导致DNA结构和动力学的显着改变;然而， 几乎所有关于DNA结构缺陷的知识都是基于 对线性DNA分子进行的研究， 没有超螺旋应力。该实验室最近对 含DNA固有弯曲和非弯曲A束结构 序列表明，这些区块的序列依赖结构 线性DNA片段不能外推到超螺旋结构域。 DNA超螺旋在转录过程中发生的巨大变化 提出了关于超卷曲对缺陷的影响的重要问题 结构及其在转录偶联DNA修复中作用。 本工作的目的是研究构象和动力学 超螺旋结构域中的几种DNA结构缺陷作为DNA的模型 DNA修复系统识别的损伤。超分子的三级结构 含有凸起（插入/缺失修饰）的卷曲分子， 错配，以及补骨脂素和抗抑郁药的共价加合物 顺铂将通过位点特异性重组和常规的 和冷冻电子显微镜结合计算机模拟。的 λ噬菌体产生的结和链烷的分布 整合（Int）位点特异性重组是一种报告基因。 DNA底物的三级结构。观察到的Int- 重组产物将用蒙特卡罗计算机进行分析 分子内重组的模拟，以定量变化 在DNA弯曲变形和/或弯曲，柔性。的构象 从这些溶液中推导出的超螺旋的含有损伤的DNA分子 研究将与独立的常规研究观察到的结果进行比较。 和冷冻电镜实验。