CELLULAR AND MOLECULAR RESPONSE TO DNA REPAIR DEFICIENCY
DNA 修复缺陷的细胞和分子反应
基本信息
- 批准号:2545805
- 负责人:
- 金额:$ 22.43万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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ROGER A. PEDERSEN其他文献
ROGER A. PEDERSEN的其他文献
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{{ truncateString('ROGER A. PEDERSEN', 18)}}的其他基金
PLURIPOTENT CELL MODELS FOR POLARITY AND EPIGENESIS
极性和表观发生的多能细胞模型
- 批准号:
6995087 - 财政年份:2005
- 资助金额:
$ 22.43万 - 项目类别:
DIFFERENTIATION AND MORPHOGENESIS OF EXTRAEMBRYONIC MESODERM
胚外中胚层的分化和形态发生
- 批准号:
6108552 - 财政年份:1999
- 资助金额:
$ 22.43万 - 项目类别:
DIFFERENTIATION AND MORPHOGENESIS OF EXTRAEMBRYONIC MESODERM
胚外中胚层的分化和形态发生
- 批准号:
6272168 - 财政年份:1998
- 资助金额:
$ 22.43万 - 项目类别:
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