ISOLATION OF RADIATION SENSITIVE MAMMALIAN CELL MUTANTS

描述：基于之前的工作，显示了一个类似于ku的复合体 哺乳动物细胞中的蛋白质结合到双链断裂，但 缺乏修复缺陷的中国仓鼠突变体XRS，Long 学期目标是测试这样一种假设，即这种类似于ku的复杂结构 在DNA损伤后调节基因转录的一些作用，保护 DNA末端来自核酸酶消化，影响DNA修复并具有 解旋酶、核酸外切酶和拓扑异构酶活性 具有将单链DNA插入双链DNA的能力。 这一假说是用来解释生物化学基础的 细胞对电离辐射杀死细胞的抵抗力将是 通过几个具体目标进行测试：(A)确定相关蛋白质 与一个假定的DNA修复复合体；以及(B)研究 复合体的生物学功能。为了鉴定这些蛋白质 与该复合体相关的蛋白质将被提纯，通过 利用抗体结合2D斜线凝胶和Western blotting 与已知或怀疑与此相关的已知蛋白质结合 该复合体的假想功能。部分序列分析将 在未与已知抗体、cDNA克隆鉴定的蛋白质上进行 通过筛选文库和从 未知的多肽将被测序，并与已知的序列进行比较。 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，改进后的机动性 移位分析，一系列修复缺陷的仓鼠突变体 检查“酷似”复合体中的异常现象。电子 显微镜将被用来确定DNA的端到端关联 由这种情结发生。

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.