DNA SEQUENCING OF UV INDUCED CHROMOSOMAL MUTATIONS

紫外线诱导的染色体突变的 DNA 测序

• 批准号: 6519128
• 负责人: Christopher W Lawrence
• 金额: $ 26.32万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519128
• 关键词: DNA damage; DNA directed DNA polymerase; DNA replication; Escherichia coli; bacterial genetics; bacterial proteins; carcinogen testing; high performance liquid chromatography; mutagen testing; mutagens; nuclear magnetic resonance spectroscopy; nucleic acid sequence; operon; radiation carcinogen; radiation genetics; transfection /expression vector; ultraviolet radiation

The long term goal of this work is primarily to understand the molecular mechanisms that produce mutations when mutagen-damaged DNA is replicated in E. coli, and secondarily to investigate other processes that promote tolerance of DNA damage. Although induced mutagenesis, and damage tolerance in general, is regulated very differently in E. coli and eukaryotes, basic enzymological processess are likely to be similar. Information of significance to human genetic diseases, such as cancer, can therefore be gained from studies employing the unrivalled experimental tools available with the bacterium. The aim of this project is to analyze the processes in vivo, by employing the analytical power of experiments in which vectors carrying defined and specifically located lesions are transfected into some of the many well characterized mutant strains that are available with the bacterium. Specific aims include: indentification of the gene products that are responsible for efficient replication past a T-T dimer in cells lacking DNA polymerase II, IV, and V and proofreading activity; identification of the MucB residues responsible for its differences in mutagenic properties comared to pol V; investigation of the RecA-independent recombination mechanism for the error free bypass of the T_T pyrimidine (6-4) pyrimidinone UV-photoproduct; measurement of the deamination rates in vivo of cytosine within a TC cis-syn cyclobutane dimer; and determination of the structure of duplex oligonucleotide molecules that contain a T_C pyrimidine (6-4) pyrimidinone adduct and the dewar isomers of the T-T and T-C (6-4) photoproducts, using nuclear magnetic resonance. The first of these aims is concerned with identifying the DNA polymerase or accessory factor capable of promoting replication past a T-T dimer in the absence of enzymes usually associated with SOS mutagenesis, whereas the second goal is to explore the structural reasons for the markedly different properties of related polymerases. The third aim is designed to explore what appears to be a novel, RecA independent, recombination process, while the last two investigate the reasons for the high mutability and specificity of two important UV photoproducts.

这项工作的长期目标主要是了解诱变剂损伤的DNA在大肠杆菌中复制时产生突变的分子机制。其次，研究促进DNA损伤耐受性的其他过程。 虽然诱导突变和损伤耐受性在大肠杆菌中的调节方式非常不同。大肠杆菌和真核生物的基本酶学过程可能相似。 因此，可以从使用细菌的无与伦比的实验工具进行的研究中获得对人类遗传疾病（如癌症）具有重要意义的信息。 该项目的目的是分析在体内的过程中，通过采用实验的分析能力，其中载体携带定义和具体定位的病变转染到一些许多良好表征的突变株，可与细菌。 具体目标包括：鉴定了在缺乏DNA聚合酶II、IV和V和校正活性的细胞中负责有效复制过T-T二聚体的基因产物，鉴定了与pol V相比具有不同致突变性的MucB残基，研究了T_T嘧啶（6-4）嘧啶酮紫外光产物无错误旁路的RecA非依赖性重组机制，用核磁共振法测定了含有T_C嘧啶（6-4）嘧啶酮加合物和T-T和T-C（6-4）光产物的杜瓦异构体的双链寡核苷酸分子的结构。 第一个目标是确定的DNA聚合酶或辅助因子，能够促进复制过去的T-T二聚体的酶通常与SOS诱变的情况下，而第二个目标是探索相关的聚合酶的显着不同的性能的结构原因。 第三个目的是探索似乎是一种新的，RecA独立的重组过程，而最后两个调查的原因，两个重要的紫外光产物的高突变性和特异性。