Molecular Genetic Study of Repair of Radiation Damage

辐射损伤修复的分子遗传学研究

• 批准号: 6331584
• 负责人: THOMAS Dominic STAMATO
• 金额: $ 24.78万
• 依托单位: LANKENAU INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-06 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6331584
• 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

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.

描述：使用修改的移动性转移测定法，我们发现 DNA末端将优先结合含有基质的圆形质粒DNA 附着区域（MAR）DNA序列，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-PKC）和核提取物中的其他未知因素。生化屏幕 鉴定出一个含有55 kd和另一个主要带的蛋白质分数 刺激质粒DNA末端结合的至少10倍强度的条带 当添加纯化的ku/dna-pkcs时。该蛋白的氨基酸测序 确定了人类cDNA序列，该序列编码为55 kD蛋白，相似 酿酒酵母DNA修复基因PSO4。 我们假设酵母PSO4蛋白BPSO4的人类同源物， 在修复双链DNA断裂并与之相互作用的功能中 DNA-PKCS/KU指导DNA的关联与MAR DNA序列结束 核基质。目标1是研究人类PSO4的参与 DNA-PKCS/KU中介导的DNA与MAR DNA序列结束的蛋白质 并确定可能与PSO4蛋白有关的其他蛋白质 协会。纯化的重组HPSO4蛋白刺激的能力 通过纯化的DNA-PKCS/KU，将分析DNA末端与MAR质粒DNA的结合。 将制备对HPSO4蛋白的聚和单克隆抗体 从核中共免疫沉淀并鉴定与HPSO4相关的蛋白 根据DNA受损和未受损的细胞制备的提取物。单克隆HPSO4 抗体将用于确定蛋白质的分布是否 响应DNA损伤以及HPSO4蛋白是否共定位的变化 与其他DSB修复蛋白（DNA-PK/KU，XRCC4，连接酶IV）。在目标2中，我们将 探索获得不足的细胞系的可能性 PSO4蛋白的表达。靶向基因破坏和/或表达 反义HPSO4 mRNA将用于HPSO4中有缺陷的细胞系 蛋白表达。这些线将被分析以提高对 与酵母PSO4-1突变体相比，DNA破坏剂及其表型。我们 相信了解人P04同源物的维修功能将 为人类双链DNA断裂修复机制的新灯 细胞并帮助我们了解该修复途径在遗传中的作用 癌症等疾病。