BIOLOGICAL EFFECTS OF DNA ADDUCTS FORMED BY NITROPYRENES

硝基芘形成的 DNA 加合物的生物学效应

• 批准号: 6150732
• 负责人: Ashis K Basu
• 金额: $ 16.91万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6150732
• 关键词: DNA damage; DNA footprinting; DNA repair; DNA replication; Escherichia coli; adduct; bacteriophage M13; chemical carcinogen; circular dichroism; conformation; fluorescence spectrometry; gel electrophoresis; gel mobility shift assay; gene frequency; gene mutation; nitro compounds; nuclear magnetic resonance spectroscopy; nucleic acid chemical synthesis; nucleic acid sequence; nucleotides; oligonucleotides; pyrenes; radiochemistry; site directed mutagenesis; thermodynamics

DESCRIPTION: The objective of this research is to investigate the mechanism of mutagenesis and DNA repair induced by the nitrated pyrenes that are known environmental pollutants and are suspected to pose threats to human health. To accomplish this goal, the investigator has used the tools of organic synthesis and recombinant DNA technology to construct specific DNA fragments and viral genomes that contained, at preselected sites, the major DNA adduct formed by 1-nitropyrene. A major objective of this application is to employ this technology to construct specific mutagenic hot spot sequences in DNA. Sequence-specificity of mutagenesis will be investigated by introducing these modified viral genomes in Escherichia coli cells, where the DNA replication and repair systems will act upon the single DNA adduct. Progeny DNA molecules will be isolated, and the mutagenic as well a toxic effects of the adduct will be studied both qualitatively and quantitatively. Replication of the genome in hosts with defined defects in DNA repair will elucidate the roles of different repair systems in mutagenesis/genotoxicity induced by these DNA adducts. Another major objective of this proposal is to determine the thermodynamics and kinetics of interaction of the Uvr proteins, and rates of incision by the UvrABC system on site-specific 1-nitropyrene adducts in the same DNA sequence context as employed in the mutagenesis studies. Also, detailed physico-chemical studies will be carried out on the modified oligonucleotides to understand the architectural effects of the 1-nitropyrene-DNA adducts. A primary goal of this work is to define the relationship between the structure and three-dimensional effects of a lesion in DNA and the mutagenicity and DNA repair proteins, that it may induce. Another important aim is to understand the mechanism how the repair proteins, and specifically nucleotide excision repair, protect cells against the toxic and mutagenic properties of these DNA damaging agents.

描述：本研究的目的是调查其机制 已知硝化芘诱导的诱变和 DNA 修复 环境污染物，并被怀疑对人类健康构成威胁。 为了实现这一目标，研究人员使用了有机工具 合成和重组DNA技术构建特定的DNA片段 和在预选位点包含主要 DNA 加合物的病毒基因组 由1-硝基芘形成。 该应用程序的一个主要目标是采用 该技术在 DNA 中构建特定的诱变热点序列。 将通过引入来研究诱变的序列特异性 这些在大肠杆菌细胞中经过修饰的病毒基因组，其中 DNA 复制和修复系统将作用于单个 DNA 加合物。 后代 DNA分子将被分离出来，并且其具有致突变性和毒性作用 将对加合物进行定性和定量研究。 在具有明确 DNA 修复缺陷的宿主中复制基因组将 阐明不同修复系统在诱变/遗传毒性中的作用 由这些DNA加合物诱导。 该提案的另一个主要目标是 确定 Uvr 相互作用的热力学和动力学 蛋白质，以及 UvrABC 系统在位点特异性的切割率 1-硝基芘加合物与中使用的DNA序列背景相同 诱变研究。 此外，详细的物理化学研究将 对修饰的寡核苷酸进行以了解其结构 1-硝基芘-DNA 加合物的影响。 这项工作的主要目标是 定义结构和三维效果之间的关系 DNA 损伤以及致突变性和 DNA 修复蛋白，它可能 诱导。 另一个重要目的是了解修复的机制 蛋白质，特别是核苷酸切除修复，可以保护细胞免受 这些 DNA 损伤剂的毒性和诱变特性。