ISOLATION OF RADIATION SENSITIVE MAMMALIAN CELL MUTANTS

描述：基于以前的工作表明，一个“ku样”复杂的 哺乳动物细胞中的蛋白质与双链断裂结合， 缺乏修复缺陷的中国仓鼠突变体，xrs，长 一个学期的目标是检验这一假设，即这种“库样”复杂的发挥作用， 在DNA损伤后调节基因转录、保护 DNA末端从核酸酶消化，影响DNA修复， 解旋酶、核酸外切酶激酶和拓扑异构酶活性，以及 从而具有将单链DNA插入双链DNA的能力。 这一假设，这是制定来解释生化基础 细胞抵抗电离辐射杀死细胞的能力， 通过几个特定的目的进行测试：（a）鉴定相关蛋白质 与推定的DNA修复复合物;和（B）研究 复合体的生物学功能。 为了鉴定蛋白质 与复合物相关的蛋白质将被纯化，通过 使用抗体的2D对角凝胶和蛋白质印迹的组合 已知或怀疑与肿瘤相关的已知蛋白质 复杂的假设功能。 部分序列分析将 对未用已知抗体鉴定的蛋白质、cDNA克隆 通过用从所述细胞中产生的序列筛选文库来分离。 未知肽将被测序并与已知序列比较。 Ku抗体也将用于免疫沉淀相关的 CHO和人类细胞中的蛋白质。 此外，修改后的移动性 转移试验，一系列修复缺陷仓鼠突变体将被 检查“Ku样“复合体的异常。 电子 将使用显微镜来确定DNA的端对端缔合 这种复杂的发生。

DESCRIPTION: Based on previous work showing that a "ku-like" complex of proteins in mammalian cells binds to double strand breaks, but is absent in a repair-deficient Chinese hamster mutant, xrs, the long term goal is to test the hypothesis that this "ku-like" complex plays some role in regulating gene transcription after DNA damage, protect DNA ends from nuclease digestion, affect DNA repair and have helicase, exonuclease kinase and topoisomerase activities, as well as to have the ability to insert single strand DNA into duplex DNA. This hypothesis, which is formulated to explain the biochemical basis of cellular resistance to cell killing by ionizing radiation, will be tested via several specific aims: (a) to identify proteins associated with a putative DNA repair complex; and (b) to investigate the biological function of the complex. To identify the proteins associated with the complex, proteins will be purified, analyzed by a combination of 2D diagonal gels and Western blotting using antibodies to known proteins known or suspected to be associated with the hypothesized function of the complex. Partial sequence analysis will be done on proteins not identified with known antibodies, cDNA clones isolated by screening libraries with sequences generated from the unknown peptides will be sequenced and compared to known sequences. Ku antibodies will also be used to immunoprecipitate associated proteins in both CHO and human cells. In addition a modified mobility shift assays, a series of repair deficient hamster mutants will be examined for abnormalities in the "ku-like "complex. Electron microscopy will be used to determine if end-to- end association of DNA by this complex occurs.