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STRUCTURE OF DNA LESIONS IN SUPERHELICAL DOMAINS

超螺旋域 DNA 损伤的结构

基本信息

批准号：
6386703
负责人：
STEPHEN D. LEVENE
金额：
$ 14.91万
依托单位：
UNIVERSITY OF TEXAS DALLAS
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-05-01 至 2003-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6386703
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

项目摘要

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、环丁烷二聚体和存在破坏DNA的加合物。有效地识别和修复这样的尽管在细胞中存在广泛的正常 DNA结构的异质性。有人提出，这些损伤引起DNA结构和动力学的显著变化；然而，几乎所有关于DNA结构中的这些缺陷的已知信息都是基于关于对线性DNA分子的研究没有超螺旋应力。这个实验室最近的工作是关于含有DNA的固有弯曲和非弯曲A-道的结构层序表明这些区域的层序依赖结构在线性DNA中，片段不能外推到超螺旋结构域。转录过程中发生的DNA超级卷曲的戏剧性变化提出超卷对缺陷影响的重要问题结构及其在转录偶联DNA修复中的作用。这项工作的目的是研究它的构象和动力学作为DNA模型的几种超螺旋结构域中的DNA结构缺陷由DNA修复系统识别的损伤。超级市场的三级结构含有凸起的盘绕分子(插入/删除修饰)，补骨脂素和抗肿瘤药物的错配和共价加合物顺铂将通过定点重组和常规方法进行检测和冷冻电子显微镜与计算机模拟相结合。这个噬菌体Lambda产生的绳结和链烷的分布整合(Int)特定位点重组是The DNA底物的三级结构。观测到的Int-分布重组产物将使用蒙特卡罗计算机进行分析分子内重组的模拟以量化变化在DNA弯曲变形和/或弯曲中，柔韧性。的构象从这些溶液中衍生出超螺旋的、含有病变的DNA分子研究将与独立常规观察到的结果进行比较和冷冻电子显微镜实验。