Molecular Genetic Study of Repair of Radiation Damage

描述：使用改进的迁移率变动分析，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，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）和核提取物中的其他未知因子。一个生化检测 鉴定了含有55 kD主带和另一个主带的蛋白质级分， 刺激质粒DNA末端结合的至少10倍强度的条带 当加入纯化的Ku/DNA-PKcs时。该蛋白的氨基酸序列 鉴定了编码55 kD蛋白质的人cDNA序列， 酵母酿酒酵母DNA修复基因PSO 4。 我们假设，人类同源的酵母PSO 4蛋白，bPSO 4， 在修复双链DNA断裂中起作用，并与 DNA-PKcs/Ku指导DNA末端与DNA末端上的MAR DNA序列的结合。 核基质目的1是研究人PSO 4参与 DNA-PKcS/Ku介导的DNA末端与MAR DNA序列结合的蛋白质 并鉴定可能与PSO 4蛋白有关的其他蛋白质， 协会纯化的重组hPSO 4蛋白刺激细胞增殖的能力 通过纯化的DNA-PKcs/Ku分析DNA末端与MAR质粒DNA的结合。 将制备并使用针对hPSO 4蛋白的多克隆抗体和单克隆抗体 共免疫沉淀和鉴定hPSO 4相关蛋白， 从DNA损伤和未损伤细胞制备的提取物。单克隆HPSO 4 抗体将被用来确定是否分布的蛋白质 对DNA损伤的反应变化以及hPSO 4蛋白是否共定位 与其他DSB修复蛋白（DNA-PK/Ku，XRCC 4，连接酶IV）。在目标2中， 探索获得缺乏细胞系的可能性， PSO 4蛋白的表达。靶向基因破坏和/或表达 反义hPSO 4 mRNA将用于产生hPSO 4缺陷的细胞系 蛋白质表达将测定这些细胞系对以下物质的敏感性增加： 与酵母pso 4 -1突变体相比的DNA损伤剂及其表型。我们 相信了解人类P04同源物的修复功能， 为人类双链DNA断裂修复机制提供了新的思路 帮助我们理解这种修复途径在遗传学中的作用。 比如癌症

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.