CELLULAR AND MOLECULAR RESPONSE TO DNA REPAIR DEFICIENCY

描述：这个研究团队最近创造了一种鼠标，它是 DNA修复基因XRCC1(X射线修复)中零突变的杂合性 交叉互补)。XRCC的这个成员和其他成员的存在 一类DNA修复基因保护细胞免受 电离辐射和烷化剂。XRCC1互补的CHO细胞 品系EM9对许多诱变剂过敏，并显示出一种 姐妹染色单体交换的基线水平比野生的增加了10倍 键入单元格。此外，EM9细胞在重新连接DNA方面存在缺陷 单链断裂。产生的纯合子基因敲除胚胎 一种新的XRCC1零突变杂合子的交叉小鼠在 胚胎期(E)7.5天和9.5天。因此，XRCC1可能对 小鼠的胚胎发育。因此，这种突变作用于 已知的哺乳动物DNA修复缺陷的最早时间。这项建议 是进一步鉴定XRCC1基因敲除突变体，并确定 XRCC1在DNA损伤修复中的发育关键作用 哺乳动物胚胎发生。 由于XRCC1在调节损伤效应中起着重要作用 在各种诱变剂中，基因敲除小鼠和细胞的特征 从它们派生出来的将提高对 保护细胞免受DNA损伤，特别是由 暴露在辐射和模拟辐射的化学物质中。 因此，具体目标1将是确定 XRCC1缺失突变胚胎的谱系特异性异常表型。这个 这一目的背后的假设是致命的零突变表型 结果是自发性损害的积累，这发生在 每一代细胞，在早期达到临界阈值水平 胚胎发生。首席调查员预测，两者都是自发的 XRCC1的碱基损伤及其修复是普遍存在的，但胚胎 而胚外血统对无法修复的损伤的耐受性也不同。 验证这一预测的实验方法将涉及到一些 描述性观察以及嵌合排列的使用 寻找拯救正常胚胎细胞的可能性并进一步 XRCC1-/-胚胎细胞沿着胚胎细胞谱系的发育 以及胚外细胞谱系。具体目标2将是分析 XRCC1基因缺失对DNA损伤应答的分子机制 细胞。这一目标背后的假设是，p53介导了 XRCC1缺失突变体对DNA损伤的分子反应，包括细胞凋亡 胚胎。这一假设预测，P53-/-零突变将挽救 XRCC1缺失突变胚胎到发育后期，并减少其 细胞凋亡表型。检验这一假说的实验方法将 涉及XRCC1和P53突变体的杂交。具体目标3将是 XRCC1缺失突变表型的遗传后果分析 在活体内。这个目标背后的假设是XRCC1函数是 对所有细胞类型和发育过程中的DNA链断裂修复是必不可少的 由于它在碱基对切除修复中的作用，它可以分为三个阶段。这一假设 预测纯合子零突变体将表现出高突变 发病率、遗传不稳定性和未修复的染色体损伤 由于细菌的存在，发育中的个体会损害减数分裂 细胞特异性XRCC1功能障碍。测试这一点的实验方法 假设将涉及使用以下命令生成XRCC1的条件敲除 CRE/LOX技术。计划中的研究的总体理由是 了解DNA修复基因在自然发育过程中的作用 看起来，自发的断裂确实会发生，而修复它们的失败 对胚胎是致命的，可以确定这些DNA修复的关键作用 基因在发育过程中，有一种迄今为止未知的， 与功能相关的，发生DNA链断裂的事件 正常发育。此外，识别组织特异性和/或 XRCC1及类似基因产物的阶段特异性作用(S) 发展可能导致识别功能上的重大意义 这些基因的多态会增加携带者患病的风险 由未修复的DNA损伤造成。

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.