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，许多不良生物效应 电离辐射是 DNA 双链断裂 (DSB) 的结果。 然而，电离辐射会产生如此广泛的 DNA 损伤 以前不可能将这些病变与 具有生物学意义的终点，例如突变和染色体 像差。 最近的研究表明，限制性内切酶 引入哺乳动物细胞可以诱导非常特异性的 DNA 损伤（DSB 在短的、明确的序列上）。 这使得可以 直接确定遗传改变的类型 DSB 的特定类型。 我们构建了基于 EBV 的穿梭载体 (pHAZE) 用于分析限制酶、X 射线和氡诱导的 突变。 该载体在人淋巴母细胞中稳定维持 作为附加型元件，包含整个 3.1 kb 大肠杆菌 lacZ 基因 诱变的目标。 引入细胞的限制性内切酶 含有该载体的载体会诱导多种突变，包括 移码、删除、插入和倒位。 具体类型有 然而，突变与初始 DSB 的类型有关（即 钝的，5' 突出，3' 突出）。 这些差异似乎是 每种 DSB 所执行的特定修复过程的结果 主题。 该提案的目的是为了更好地理解 DSB 的生物学后果，特定 DNA 修复过程的作用 DSB 诱导的染色体畸变和突变的产生，以及 DNA 修复缺陷综合征潜在缺陷的本质 毛细血管扩张性共济失调 (AT) 和布卢姆综合征 (BS)。 具体目标 的目的是：1）分析和比较由 正常细胞和 DNA 修复缺陷细胞中限制性内切酶产生的 DSB 线路； 2) 比较正常细胞和DNA修复缺陷细胞的敏感性 限制酶诱导的染色体畸变系； 3）研究 特定 DNA 修复抑制化合物对光谱的影响 限制性内切酶诱导的突变，4) 研究 特定的 DNA 修复抑制化合物对限制性内切酶诱导的 染色体畸变的产生。 限制性内切酶诱导的突变 将在穿梭载体中分析PHAZE稳定维持在正常， AT 和 BS 淋巴母细胞系。 产生的限制性内切酶 特定类型的 DSB 将被引入到这些细胞中 电穿孔。 允许 24 小时修复后，向量将是 从哺乳动物细胞中解救出来，转化到大肠杆菌中进行筛选。 pHAZE 的突变拷贝将通过限制性消化图谱进行分析，并 DNA 测序。 特定类型 DSB 的断裂潜力为 通过对正常细胞、AT 和 BS 细胞中的染色体畸变进行评分来进行分析 用各种限制性酶处理的细胞。 我们增加的知识 DSB 诱导的突变和染色体畸变所涉及的过程， 将进一步我们了解电离辐射如何产生不利影响 生物学后果。