Molecular Genetic Study of Repair of Radiation Damage
Molecular Genetic Study of Repair of Radiation Damage
批准号:
6331584
负责人:
THOMAS Dominic STAMATO
金额:
$24.78万
依托单位国家:
美国
项目类别:
财政年份:
2001
资助国家:
美国
项目状态:
已结题
起止时间:
2001-04-06 至 2004-03-31
关键词:
DNA damage DNA repair HeLa cells antisense nucleic acid complementary DNA enzyme activity immunoprecipitation molecular cloning molecular genetics monoclonal antibody nuclear matrix nucleic acid sequence polymerase chain reaction protein kinase radiation genetics recombinant proteins transfection /expression vector western blottings
中文摘要
描述:使用改良的迁移率变化测定,我们发现
DNA 末端将优先与含有基质的环状质粒 DNA 结合
附着区 (MAR) DNA 序列,并且这种相互作用需要
DNA 末端结合蛋白 Ku,DNA 依赖性蛋白激酶催化亚基
(DNA-PKcs),以及核提取物中的其他未知因子。生化筛查
鉴定出含有 55 kD 主条带和另一个条带的蛋白质片段
条带强度至少 10 倍,刺激质粒 DNA 末端结合
当添加纯化的 Ku/DNA-PKcs 时。该蛋白质的氨基酸测序
鉴定了一个人类 cDNA 序列,该序列编码与类似的 55 kD 蛋白质
酿酒酵母 DNA 修复基因 PSO4。
我们假设酵母 PSO4 蛋白的人类同源物 bPSO4,
具有修复双链 DNA 断裂的功能并与
DNA-PKcs/Ku 指导 DNA 末端与 MAR DNA 序列的关联
核基质。目标 1 是调查人类 PSO4 的参与
DNA-PKcS/Ku 中的蛋白质介导 DNA 末端与 MAR DNA 序列的关联
并鉴定可能与 PSO4 蛋白相关的其他蛋白
协会。纯化的重组 hPSO4 蛋白刺激的能力
将通过纯化的 DNA-PKcs/Ku 检测 DNA 末端与 MAR 质粒 DNA 的结合。
将制备并使用针对 hPSO4 蛋白的多克隆和单克隆抗体
免疫共沉淀并从核中鉴定 hPSO4 相关蛋白
从 DNA 损伤和未损伤的细胞中制备的提取物。单克隆HPSO4
抗体将用于确定蛋白质的分布是否
DNA 损伤反应的变化以及 hPSO4 蛋白是否共定位
与其他 DSB 修复蛋白(DNA-PK/Ku、XRCC4、连接酶 IV)一起使用。在目标 2 中,我们将
探索获得缺乏该基因的细胞系的可能性
PSO4 蛋白的表达。靶向基因破坏和/或表达
反义 hPSO4 mRNA 将用于生成 hPSO4 缺陷的细胞系
蛋白质表达。将检测这些细胞系的敏感性是否增加
DNA 损伤剂及其表型与酵母 pso4-1 突变体的比较。我们
相信了解人类 P04 同源物的修复功能将
揭示人类双链DNA断裂修复机制
细胞并帮助我们了解这种修复途径在遗传中的作用
癌症等疾病。
英文摘要
DESCRIPTION: Using a modified mobility shift assay, we have found find that
DNA-ends will bind preferentially to circular plasmid DNA containing matrix
attachment region (MAR) DNA sequences and that this interaction requires the
DNA-end-binding protein Ku, DNA dependent protein kinase catalytic subunit
(DNA-PKcs), and other unknown factors in nuclear extract. A biochemical screen
identified a protein fraction containing a major band of 55 kD and one other
band of at least 10 fold intensity which stimulated plasmid DNA-end binding
when added with purified Ku/DNA-PKcs. Amino acid sequencing of this protein
identified a human cDNA sequence which codes for a 55 kD protein that similar
to the yeast Saccharomyces cerevisiae DNA repair gene PSO4.
We hypothesize that the human homolog of the yeast PSO4 protein, bPSO4,
functions in the repair of double strand DNA breaks and interacts with
DNA-PKcs/Ku to direct the association of DNA ends with MAR DNA sequences on the
nuclear matrix. Aim 1 is to Investigate the involvement of the human PSO4
protein in DNA-PKcS/Ku mediated association of DNA ends with MAR DNA sequences
and identify other proteins that may be involved with PSO4 protein in this
association. The ability of purified recombinant hPSO4 protein to stimulate
binding of DNA-ends to MAR plasmid DNA by purified DNA-PKcs/Ku will be assayed.
Poly- and monoclonal antibodies to the hPSO4 protein will be prepared and used
to co-immunoprecipitate and identify hPSO4-associated proteins from nuclear
extracts prepared from DNA-damaged and non-damaged cells. Monoclonal HPSO4
antibody will be used to determine whether the distribution of the protein
changes in response to DNA damage and whether the hPSO4 protein co-localizes
with other DSB repair proteins (DNA-PK/Ku, XRCC4, ligase IV). In Aim 2 we will
explore the possibility of obtaining cell lines that are deficient in the
expression of PSO4 protein. Targeted gene disruption and/or expression of
antisense hPSO4 mRNA will be used generate cell lines defective in hPSO4
protein expression. These lines will be assayed for increased sensitivity to
DNA damaging agents and their phenotype compared to the yeast pso4-1 mutant. We
believe that understanding the repair function of the human P04 homolog will
shed new light on the mechanism of double strand DNA break repair in human
cells and help us understand the role of this repair pathway in genetic
diseases like cancer.
期刊论文(0)
专著(0)
科研奖励(0)
会议论文
CHARACTERIZATION OF A MAMMALIAN REPAIR GENE
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批准号:6628203
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项目类别:
-
资助金额:$29.38万
-
财政年份:2001
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
Molecular Genetic Study of Repair of Radiation Damage
-
批准号:6514637
-
项目类别:
-
资助金额:$24.9万
-
财政年份:2001
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
Molecular Genetic Study of Repair of Radiation Damage
-
批准号:6633781
-
项目类别:
-
资助金额:$24.9万
-
财政年份:2001
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
CHARACTERIZATION OF A MAMMALIAN REPAIR GENE
-
批准号:6497565
-
项目类别:
-
资助金额:$28.52万
-
财政年份:2001
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
CHARACTERIZATION OF A MAMMALIAN REPAIR GENE
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批准号:6261185
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项目类别:
-
资助金额:$29.33万
-
财政年份:2001
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
GENETIC STUDY OF RADIATION RESISTANCE
-
批准号:2098996
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项目类别:
-
资助金额:$26.38万
-
财政年份:1994
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
GENETIC STUDY OF RADIATION RESISTANCE
-
批准号:2008136
-
项目类别:
-
资助金额:$24.64万
-
财政年份:1994
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
GENETIC STUDY OF RADIATION RESISTANCE
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批准号:2098995
-
项目类别:
-
资助金额:$26.08万
-
财政年份:1994
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负责人:THOMAS Dominic STAMATO
-
依托单位:
POLY-ADP-RIBOSE AND REPAIR OF RADIATION INDUCED DAMAGE
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批准号:3192532
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项目类别:
-
资助金额:$8.72万
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财政年份:1990
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负责人:THOMAS Dominic STAMATO
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依托单位:
POLY (ADP-RIBOSE) AND REPAIR OF RADIATION INDUCED DAMAGE
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批准号:3192526
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项目类别:
-
资助金额:$21.48万
-
财政年份:1988
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
POLY (ADP-RIBOSE) AND REPAIR OF RADIATION INDUCED DAMAGE
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批准号:2093004
-
项目类别:
-
资助金额:$22.87万
-
财政年份:1988
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
POLY (ADP-RIBOSE) AND REPAIR OF RADIATION INDUCED DAMAGE
-
批准号:3192531
-
项目类别:
-
资助金额:$22.19万
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财政年份:1988
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
POLY-ADP-RIBOSE AND REPAIR OF RADIATION INDUCED DAMAGE
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批准号:3192525
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项目类别:
-
资助金额:$9.79万
-
财政年份:1988
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
POLY-ADP-RIBOSE AND REPAIR OF RADIATION INDUCED DAMAGE
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批准号:3192529
-
项目类别:
-
资助金额:$9.31万
-
财政年份:1988
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
POLY-ADP-RIBOSE AND REPAIR OF RADIATION INDUCED DAMAGE
-
批准号:3192530
-
项目类别:
-
资助金额:$1.6万
-
财政年份:1988
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负责人:THOMAS Dominic STAMATO
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依托单位:
ISOLATION OF RADIATION SENSITIVE MAMMALIAN CELL MUTANTS
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批准号:2091813
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项目类别:
-
资助金额:$25.86万
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财政年份:1987
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负责人:THOMAS Dominic STAMATO
-
依托单位:
ISOLATION OF RADIATION SENSITIVE MAMMALIAN CELL MUTANTS
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批准号:2091814
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项目类别:
-
资助金额:$23.4万
-
财政年份:1987
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负责人:THOMAS Dominic STAMATO
-
依托单位:
ISOLATION OF RADIATION SENSITIVE MAMMALIAN CELL
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批准号:3188347
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项目类别:
-
资助金额:$18.8万
-
财政年份:1987
-
负责人:THOMAS Dominic STAMATO
-
依托单位:
ISOLATION OF RADIATION SENSITIVE MAMMALIAN CELL
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批准号:3188352
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项目类别:
-
资助金额:$19.32万
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财政年份:1987
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负责人:THOMAS Dominic STAMATO
-
依托单位:
ISOLATION OF RADIATION SENSITIVE MAMMALIAN CELL MUTANTS
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批准号:2712608
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项目类别:
-
资助金额:$25.05万
-
财政年份:1987
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负责人:THOMAS Dominic STAMATO
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依托单位:
海外基金