DNA DOUBLE STRAND BREAK-INDUCED DAMAGE AND REPAIR

DNA 双链断裂引起的损伤和修复

• 批准号: 2184062
• 负责人: RICHARD A WINEGAR
• 金额: $ 11.24万
• 依托单位: SRI INTERNATIONAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184062
• 关键词: DNA damage; DNA repair; Escherichia coli; chromosome aberrations; enzyme inhibitors; gene mutation; ionizing radiation; nucleic acid sequence; radiation sensitivity; restriction endonucleases; tissue /cell culture; transfection

There is increasing evidence that many of the adverse biological effects of ionizing radiation are the result of DNA double-strand breaks (DSB). However, ionizing radiation produces such a broad spectrum of DNA lesions that it has previously been impossible to relate these lesions to biologically significant endpoints such as mutations and chromosomal aberrations. Recent investigations demonstrate that restriction enzymes introduced into mammalian cells permit the induction of very specific DNA lesions (DSB at short, defined sequences). This makes it possible to determine directly the types of genetic alterations which arise from specific types of DSB. We have constructed an EBV-based shuttle vector (pHAZE) for analyzing restriction enzyme-, X-ray and radon-induced mutations. This vector is stably maintained in human lymphoblastoid cells as an episomal element and contains the entire 3.1 kb E. coli lacZ gene as a target for mutagenesis. Restriction enzymes introduced into cells containing the vector induce a wide variety of mutations including frameshifts, deletions, insertions, and inversions. The specific types of mutations, however, are related to the type of the initial DSB (i.e., blunt, 5' overhang, 3' overhang). These differences appear to be a consequence of the specific repair processes to which each type of DSB is subject. The objectives of this proposal are to better understand the biological consequences of DSB, the role of specific DNA repair processes in the production of DSB-induced chromosome aberrations and mutation, and the nature of the defects underlying the DNA repair deficiency syndromes ataxia telangiectasia (AT) and Bloom's syndrome (BS). The specific aims are to: 1) analyze and compare the spectrum of mutations induced by restriction enzyme-produced DSB in normal and DNA repair-deficient cell lines; 2) compare the sensitivity of normal and DNA repair-deficient cell lines to restriction enzyme-induced chromosome aberrations; 3) study the effect of specific DNA repair-inhibiting compounds on the spectrum of mutations induced by restriction enzymes and 4) study the effect of specific DNA repair-inhibiting compounds on restriction enzyme-induced chromosome aberration production. Restriction enzyme-induced mutations will be analyzed in the shuttle vector pHAZE stably maintained in normal, AT, and BS lymphoblastoid cell lines. Restriction enzymes that produce specific types of DSB will be introduced into these cells by electroporation. After allowing 24 h for repair, the vector will be rescued from the mammalian cells, transformed into E. coli for screening. Mutant copies of pHAZE will be analyzed by restriction digest mapping and DNA sequencing. The clastogenic potential of specific types of DSB will be analyzed in normal, AT, and BS cell by scoring chromosome aberrations from cells treated with various restriction enzymes. Our increased knowledge of the processes involved in DSB-induced mutations and chromosome aberrations, will further our understanding how ionizing radiations produce adverse biological consequences.

越来越多的证据表明，许多不良生物学作用 电离辐射是DNA双链断裂（DSB）的结果。 但是，电离辐射会产生如此广泛的DNA病变 以前无法将这些病变与 生物学上具有重要意义的终点，例如突变和染色体 畸变。 最近的研究表明限制酶 引入哺乳动物细胞允许诱导非常具体的DNA 病变（简短定义的序列DSB）。 这使得 直接确定由 特定类型的DSB。 我们已经构建了一个基于EBV的班车向量 （phaze）用于分析限制酶 - ，X射线和ra诱导的 突变。 该载体稳定地保持在人淋巴母细胞中 作为偶发元素，并包含整个3.1 kb大肠杆菌lacz基因 诱变的目标。 引入细胞的限制酶 包含矢量会引起多种突变 移码，删除，插入和反转。 特定类型的 但是，突变与初始DSB的类型有关（即 Blunt，5'Overhang，3'悬垂）。 这些差异似乎是 每种DSB所在的特定维修过程的结果 主题。 该提案的目标是更好地了解 DSB的生物学后果，特定DNA修复过程的作用 在生产DSB诱导的染色体畸变和突变，以及 DNA修复缺乏综合征的基础缺陷的性质 山texia telangictia（AT）和Bloom综合征（BS）。 具体目标 为：1）分析和比较由 在正常和DNA修复细胞中限制酶产生的DSB 线； 2）比较正常和DNA修复细胞的灵敏度 限制酶诱导的染色体畸变的线； 3）研究 特定DNA修复抑制化合物对光谱的影响 由限制酶引起的突变，4）研究 特定的DNA修复抑制限制酶诱导的化合物 染色体畸变产生。 限制酶诱导的突变 将在稳定维持在正常的航天飞机矢量中进行分析 AT和BS淋巴细胞细胞系。 产生的限制酶 特定类型的DSB将通过 电穿孔。 允许24小时维修后，向量将是 从哺乳动物细胞中获救，转化为大肠杆菌进行筛查。 Phaze的突变副本将通过限制摘要映射和 DNA测序。 特定类型的DSB的层生成潜力将是 通过得分从正常，AT和BS细胞中分析 用各种限制酶处理的细胞。 我们对 DSB诱导的突变和染色体畸变所涉及的过程， 将进一步理解电离辐射如何产生不利 生物后果。