CELLULAR AND MOLECULAR RESPONSE TO DNA REPAIR DEFICIENCY

DNA 修复缺陷的细胞和分子反应

• 批准号: 2545805
• 负责人: ROGER A. PEDERSEN
• 金额: $ 22.43万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2545805
• 关键词: CHO cells; DNA damage; DNA repair; alkylating agents; carcinogen testing; carcinogenesis; developmental genetics; genetically modified animals; ionizing radiation; laboratory mouse; mammalian embryology; mutagen testing; neoplasm /cancer genetics; CHO cells; DNA damage; DNA repair; alkylating agents; carcinogen testing; carcinogenesis; developmental genetics; genetically modified animals; ionizing radiation; laboratory mouse; mammalian embryology; mutagen testing; neoplasm /cancer genetics

DESCRIPTION: This research team has recently created a mouse that is heterozygous for a null mutation in the DNA repair gene XRCC1 (X-ray repair cross-complementing). The presence of this and other members of the XRCC class of DNA repair genes protects cells against the lethal effects of ionizing radiation and alkylating agents. The XRCC1-complemented CHO cell line, EM9, is hypersensitive to many mutagenic agents, and demonstrates a 10-fold increase in baseline levels of sister chromatid exchange over wild type cells. In addition, EM9 cells are deficient in rejoining DNA single-strand breaks. Homozygous knockout embryos generated by intercrossing mice heterozygous for a novel, null XRCC1 mutation die between embryonic day (E) 7.5 and 9.5. Therefore, XRCC1 may be essential for embryonic development in the mouse. As such, this mutation acts at the earliest time of any known mammalian DNA repair deficiencies. This proposal is to further characterize the XRCC1 knockout mutants and to determine the developmentally essential role of XRCC1 in repairing DNA damage during mammalian embryogenesis. Because of the important role XRCC1 plays in modulating the damaging effects of various mutagens, the characterization of the knockout mice and cells derived from them will improve understanding of the mechanisms employed by cells to protect against DNA damage, particularly DNA base damage induced by exposure to radiation and radio-mimetic chemicals. Accordingly, specific aim 1 will be to determine the cellular basis for the lineage-specific abnormal phenotype of the XRCC1 null mutant embryo. The hypothesis underlying this aim is that the lethal null mutant phenotype results from the accumulation of spontaneous damage, which is occurring in each cell generation, to critical threshold levels during early embryogenesis. The principal investigator predicts that both spontaneous base damage and its repair by XRCC1 are ubiquitous but that the embryonic and extraembryonic lineages differ in their tolerance for unrepaired damage. The experimental approach to test this prediction will involve some descriptive observations and also the use of chimeric arrangements with normal embryonic cells to look for the possibility of rescue and further development of XRCC1 -/- embryonic cells along embryonic cell lineages as well as the extraembryonic cell lineages. Specific aim 2 will be to analyze the molecular mechanisms of the response to DNA damage in XRCC1-deficient cells. The hypothesis underlying this aim is that p53 mediates the molecular responses to DNA damage, including apoptosis, in XRCC1 null mutant embryos. This hypothesis predicts that the p53-/- null mutation will rescue XRCC1 null mutant embryos to a later stage of development and diminish their apoptotic phenotype. The experimental approach to test this hypothesis will involve interbreeding XRCC1 and p53 mutants. Specific aim 3 will be to analyze the genetic consequences of the XRCC1 null mutant phenotype in vitro and in vivo. The hypothesis underlying this aim is that XRCC1 function is essential for DNA strand break repair in all cell types and developmental stages owing to its role in base pair excision repair. This hypothesis predicts that the homozygous null mutants will exhibit elevated mutation rates, genetic instability and unrepaired chromosomal damage and that developing individuals would have impaired meiosis as a result of germ cell-specific XRCC1 dysfunction. The experimental approach to test this hypothesis will involve generating a conditional knockout for XRCC1 using Cre/lox technology. An overall rationale for the planned studies is that understanding the role of DNA repair genes during natural development, when it appears that spontaneous breaks do occur and that failure to repair them is embryo-lethal, could establish an essential role for these DNA repair genes during development and that there is a heretofore unknown, functionally relevant, incidence of DNA strand breaks that occurs during normal development. In addition, identifying tissue-specific and/or stage-specific role(s) for XRCC1 and similar gene products during development might lead to the identification of functionally significant polymorphisms in these genes that increase the risk of carriers to diseases resulting from unrepaired DNA damage.

描述：该研究团队最近创建了一只鼠标 DNA修复基因XRCC1中无效突变的杂合子（X射线修复 交叉汇编）。 XRCC和其他成员的存在 一类DNA修复基因可保护细胞免受致命作用 电离辐射和烷基化剂。 XRCC1融合的CHO单元 EM9线对许多诱变剂都过敏，并证明了 野生姐妹染色单体交换的基线水平增加了10倍 类型单元格。 另外，EM9细胞不足以重新加入DNA 单链断裂。 由 杂合的杂合子的杂合无效，无效的XRCC1突变死亡 胚胎日（E）7.5和9.5。 因此，XRCC1对于 小鼠中的胚胎发育。 因此，这个突变作用于 任何已知的哺乳动物DNA修复缺陷的最早时间。 这个建议 是为了进一步表征XRCC1敲除突变体并确定 XRCC1在修复DNA损伤过程中的发展上至关重要的作用 哺乳动物的胚胎发生。 由于XRCC1在调节破坏效果中起着重要作用 在各种诱变剂中，敲除小鼠和细胞的表征 从它们中得出的将提高对所采用机制的理解 细胞以防止DNA损伤，尤其是由DNA碱基损伤引起的 暴露于辐射和无线电模拟化学物质。 因此，具体目标1将是确定的细胞基础 XRCC1 NULL突变体胚胎的谱系特异性异常表型。 这 该目标的基本假设是致命的无效突变表型 自发损害的积累结果，发生在 每个细胞生成，在早期达到临界阈值水平 胚胎发生。 主要研究者预测两者都是自发的 XRCC1的基本损坏及其修复无处不在，但胚胎是 胚外谱系的容忍度有所不同。 测试该预测的实验方法将涉及一些 描述性观察以及使用嵌合安排的使用 正常的胚胎细胞以寻找营救的可能性 沿胚胎细胞谱系的XRCC1 - / - 胚胎细胞的开发为 以及胚外细胞谱系。 特定目标2将是分析 XRCC1缺陷中对DNA损伤响应的分子机制 细胞。 该目标的基础假设是p53介导 XRCC1无效突变体中对DNA损伤的分子反应，包括凋亡 胚胎。 该假设预测p53 - / - 无效突变将营救 XRCC1无效突变胚胎到后来的发展阶段，并减少了它们 凋亡表型。 检验该假设的实验方法将 涉及杂交XRCC1和P53突变体。 特定目标3将是 在体外分析XRCC1 NULL突变表型的遗传后果 和体内。 这个目的的基本假设是XRCC1函数是 所有细胞类型的DNA链断裂修复至关重要 由于其在碱基对切除修复中的作用而阶段。 这个假设 预测纯合无零突变体将表现出升高的突变 速率，遗传不稳定和未修复的染色体损害， 发育中的个体会因细菌而损害减数分裂 细胞特异性XRCC1功能障碍。 测试这一点的实验方法 假设将涉及使用XRCC1的条件敲除 CRE/LOX技术。 计划研究的总体理由是 了解DNA修复基因在自然发育中的作用， 看来确实发生了自发休息，并且无法修复它们 是胚胎致死的，可以为这些DNA修复确定重要作用 发展过程中的基因，迄今为止有一个未知的基因， 功能相关的DNA链断裂的发生率发生在 正常发展。 另外，确定组织特异性和/或 XRCC1和类似基因产物的阶段特异性作用 开发可能导致识别功能意义 这些基因中的多态性增加了疾病携带者的风险 由未修复的DNA损伤产生。