Molecular Genetic Study of Repair of Radiation Damage

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。

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.