Isolation of genes for radiation sensitivity

描述（由申请人提供）：在哺乳动物细胞中，蛋白质Mre11， RadSO和nibrin形成MRN复合物，其作用既可以感知DNA双链断裂（DSB）的存在，也可以促进ATM向下游效应物发出信号，从而激活细胞周期检查点和修复机制。该复合物的三种成分中的任何一种发生突变都会导致以对电离辐射（IR）过敏、癌症发病率增加、免疫缺陷和一组重叠的生长发育缺陷为特征的综合征。Mre11和RadSO是必不可少的基因，它们编码的蛋白质具有高度的进化保守性，可以根据其他更易实验处理的生物中同源物的数据推断它们的功能和结构。然而，Nibrin的保守性要差得多，因此它的功能也就不太为人所知。我们最初通过NBS家族的遗传连锁研究确定了编码nibrin的NBS1基因。随后，我们利用人nibrin的位点特异性突变和转染NBS患者细胞系的方法来识别功能域并评估它们在细胞对IR暴露的反应中的作用。这种方法使我们能够识别和表征在哺乳动物DNA损伤反应中起作用的nibrin的几个不同结构域，以及等待表征的新功能和新的相互作用伙伴。然而，这种方法受到所有已知人类NBS突变的半形性质的限制，这些突变导致NBS细胞系中合成的截断蛋白提供部分nibrin功能。同样的效应也限制了nibrin在发育中作用的研究的信息量，因为小鼠中敲除等位基因的纯合性会导致胚胎致死。我们现在已经开发了基于条件基因失活的细胞系和整个动物模型，这将使我们能够在零背景下研究nibrin在细胞DNA损伤反应和哺乳动物发育中的作用。我们建议利用这些系统来探索nibrin在感知DNA损伤和激活DNA损伤反应、在损伤反应过程中保护端粒、在修复完成后下调损伤反应途径以及在免疫系统发育中的作用。

DESCRIPTION (provided by applicant): In mammalian cells, the proteins Mre11, RadSO and nibrin form the MRN complex which acts both in sensing the presence of DNA double-strand breaks (DSB) and in facilitating signaling by ATM to downstream effectors that activate cellular cycle checkpoint and repair mechanisms. Mutations in any of the three components of this complex result in syndromes characterized by hypersensitivity to ionizing radiation (IR), an increased incidence of cancer, immunodeficiency, and an overlapping set of growth and developmental defects. Mre11 and RadSO are essential genes, and the high degree of evolutionary conservation in the proteins they encode allow inferences regarding their function and structure based on data from homologues in other more experimentally tractable organisms. Nibrin, however, is considerably less well conserved and its function is, accordingly, less well understood. We originally identified the NBS1 gene that encodes nibrin through genetic linkage studies in NBS families. Subsequently, we have utilized the approach of site-specific mutagenesis of human nibrin and transfection into NBS patient cell lines to identify functional domains and evaluate their roles in the cellular response to IR exposure. This approach has allowed us to identify and characterize several distinct domains of nibrin that function in the mammalian DNA damage response, as well as novel functions and new interacting partners that await characterization. However, this approach is limited by the hypomorphic nature of all known human NBS mutations that results in the synthesis of a truncated protein providing partial nibrin function in NBS cell lines. This same effect also limits the informativeness of studies of the role of nibrin in development since homozygosity for knockout alleles in mice results in embryonic lethality. We have now developed cell line and whole animal models based on conditional gene inactivation that will allow us to study the role of nibrin in cellular DNA damage responses and in mammalian development in a null background. We propose to use these systems to explore the role of nibrin in sensing DNA damage and activating DNA damage responses, in protecting telomeres during the course of damage responses, in downregulating damage response pathways after completion of repair and in the development of the immune system.