Molecular Genetic Study of Repair of Radiation Damage

描述：使用改进的迁移率变化分析，我们发现 DNA末端将优先与含有基质的环状DNA结合 附着区(MAR)DNA序列，这种相互作用需要 DNA末端结合蛋白Ku，DNA依赖蛋白激酶催化亚基 (DNA-PKcs)，以及核提取液中其他未知因素。生化屏幕 鉴定出一个含有55kD主带和另一条带的蛋白质组分 刺激质粒DNA末端结合的至少10倍强度的条带 加入纯化的Ku/DNA-PKcs。该蛋白的氨基酸序列分析 发现了一个编码55kD蛋白的人cDNA序列，该蛋白与 与酿酒酵母DNA修复基因PSO4结合。 我们假设酵母PSO4蛋白的人类同源物，bPSO4， 双链DNA断裂修复中的功能及其相互作用 DNA-PKcs/Ku引导DNA末端与MAR DNA序列在 核矩阵。目的1是研究人类PSO4的参与 DNA中的蛋白质-PKcs/Ku介导的DNA末端与MAR DNA序列的结合 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蛋白刺激活性的研究 用纯化的DNA-PKcs/Ku检测DNA末端与MAR质粒DNA的结合。 将制备和使用针对hPSO4蛋白的多克隆抗体和单抗 免疫共沉淀法从细胞核中鉴定hPSO4相关蛋白 从DNA损伤和未损伤的细胞中提取的提取物。单克隆性HPSO4 抗体将被用来确定蛋白质的分布 对DNA损伤的反应变化以及hPSO4蛋白是否共定位 与其他DSB修复蛋白(DNA-PK/Ku、XRCC4、连接酶IV)结合。在《目标2》中我们将 探索获得缺陷细胞系的可能性 PSO4蛋白的表达。靶向基因中断和/或表达 反义hPSO4将被用来产生hPSO4缺陷的细胞系 蛋白质表达。将对这些品系进行检测，以增加对 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同源基因的修复功能将 对人类双链DNA断裂修复机制的新认识 细胞，并帮助我们了解这一修复途径在遗传 像癌症这样的疾病。

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.