CELLULAR AND MOLECULAR RESPONSE TO DNA REPAIR DEFICIENCY

描述：这个研究团队最近创造了一种小鼠， DNA修复基因XRCC 1（X射线修复）中无效突变的杂合子 交叉互补）。 这位和XRCC的其他成员的出席 一类DNA修复基因可以保护细胞免受 电离辐射和烷基化剂。 XRCC 1补充CHO细胞 细胞系EM 9对许多诱变剂高度敏感， 10-与野生型相比，姐妹染色单体交换的基线水平增加倍数 类型细胞。 此外，EM 9细胞缺乏DNA重新连接， 单链断裂 纯合子敲除胚胎产生的 一种新的XRCC 1无效突变的杂合子小鼠在 胚龄（E）7.5和9.5天。 因此，XRCC 1可能是必不可少的， 小鼠的胚胎发育 因此，这种突变作用于 已知哺乳动物DNA修复缺陷的最早时间。 这项建议 进一步表征XRCC 1敲除突变体，并确定 XRCC 1在修复DNA损伤中的重要作用 哺乳动物胚胎发生 由于XRCC 1在调节损伤效应中起重要作用， 各种诱变剂，敲除小鼠和细胞的表征 从它们中获得的信息将有助于理解 细胞以保护免受DNA损伤，特别是DNA碱基损伤， 暴露于辐射和放射性模拟化学品。 因此，具体目标1将是确定用于本发明的细胞基础。 XRCC 1无效突变胚胎的谱系特异性异常表型。 的 这一目标的基础假设是，致死无效突变表型 结果是自发损害的积累，这是发生在 每一代细胞，在早期 胚胎发生 首席研究员预测， 碱基损伤及其修复XRCC 1是普遍存在的，但胚胎 而胚外谱系对未修复损伤的耐受性不同。 测试这一预测的实验方法将涉及一些 描述性的观察，也使用嵌合安排， 正常的胚胎细胞，以寻找拯救的可能性，并进一步 XRCC 1-/-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将分析 XRCC 1基因缺陷型细胞对DNA损伤反应的分子机制 细胞 这一目标的假设是，p53介导了 XRCC 1无效突变体对DNA损伤的分子反应，包括细胞凋亡 胚胎 这一假设预测p53-/-无效突变将挽救 XRCC 1无效突变胚胎发育到后期阶段，并减少其 凋亡表型 检验这一假设的实验方法将 涉及XRCC 1和p53突变体的杂交。 具体目标3将是 体外分析XRCC 1无效突变表型的遗传后果 和体内。 这一目标背后的假设是，XRCC 1功能是 在所有细胞类型和发育中对DNA链断裂修复至关重要 阶段，由于其在碱基对切除修复中的作用。 这一假设 预测纯合无效突变体将表现出升高的突变 率，遗传不稳定性和未修复的染色体损伤， 发育中的个体会由于细菌感染而导致减数分裂受损， 细胞特异性XRCC 1功能障碍。 测试这一点的实验方法 假设将涉及使用以下方法产生XRCC 1的条件性敲除 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过程中的作用， 似乎自发性断裂确实会发生， 是胚胎致命的，可以建立这些DNA修复的重要作用， 基因在发育过程中，有一个迄今为止未知的， 功能相关，发生DNA链断裂， 正常发展。 此外，识别组织特异性和/或 XRCC 1和类似基因产物的阶段特异性作用 发展可能导致识别功能上重要的 这些基因的多态性增加了携带者患病的风险 是由未修复的DNA损伤造成的

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.