Isolation of Genes for Radiation Sensitivity

培养的人细胞对电离辐射(IR)致死效应的敏感性是一个复杂的特性，它是由多个基因及其产物在参与DNA损伤感知和反应的不同途径中发挥作用的。在这笔赠款的支持下，我们研究的重点一直是确定人类群体中导致辐射敏感性的基因，长期目标是(1)了解暴露于环境诱变剂(IR)与癌症发生之间的关系，以及(2)开发辐射敏感性的标记，使我们能够更好地根据患者的个体敏感性定制放射治疗方案。这种方法使我们研究了在人类群体中代表极端辐射敏感性的患者，遗传性疾病共济失调-毛细血管扩张症(A-T)和奈梅根破裂综合征(NBS)患者，其中辐射敏感表型作为隐性性状遗传，允许定位和定位克隆负责基因。在之前的资助期间，我们成功地鉴定了NBS及其蛋白产物Nibrin的基因，并通过证明Nibrin是ATM激酶的底物，阐明了A-T和NBS之间的生化联系。在本申请中，我们建议用两种一般方法来扩展我们对A-T和NBS的研究。首先，我们将重点介绍Nibrin的功能。我们将通过定点突变来绘制蛋白质上的功能结构域，通过跟踪完成的酵母双杂交筛选的线索来确定与其相互作用的其他蛋白质，并开发一个小鼠模型来探索Nibrin在组织中和人类患者无法获得的发育阶段的功能。在第二种方法中，我们将在DNA损伤反应途径中存在特定缺陷的细胞系中使用微阵列进行转录谱分析，以识别这些途径中的新分子，并开发与辐射敏感性相关的转录表达的“指纹”。最后，我们将在我们已经确定的NBS1基因缺乏突变的NBS家族中寻找与辐射敏感性有关的其他基因，这表明NBS是一种异质性疾病。这些拟议的研究将使我们能够将从识别导致罕见疾病的基因中获得的洞察力扩展到更详细地理解人体细胞对电离辐射的反应所激活的生化途径。

The sensitivity of cultured human cells to the lethal effects of ionizing radiation (IR) is a complex trait that is contributed to by multiple genes and their products functioning within various pathways involved in DNA damage sensing and response. The focus of our studies supported by this grant has been to identify genes in human populations that contribute to radiation sensitivity with the long-term goals of (1) understanding the relationship between exposure to an environmental mutagen (IR) and cancer development, and (2) developing markers for radiation sensitivity that would allow us to better tailor radiation therapy protocols to individual patient sensitivities. This approach has led us to study patients that represent the extremes of radiation sensitivity in human populations, patients with the inherited disorders Ataxia- telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) where the radiation sensitive phenotype is inherited as a recessive trait, allowing the mapping and positional cloning of the responsible genes. In the previous funding period, we successfully identified the gene for NBS and its protein product, nibrin, and elucidated the biochemical connection between A-T and NBS by demonstrating that nibrin is a substrate for the ATM kinase. In this application we propose to extend our studies of A-T and NBS with 2 general approaches. In the first, we will focus on the function of nibrin. We will map functional domains on the protein by site-specific mutagenesis, identify other proteins with which it interacts by following leads from a completed yeast two-hybrid screen, and develop a mouse model to explore the function of nibrin in tissues and at developmental stages not accessible in human patients. In the second approach, we will use transcript profiling with microarrays in cell lines with specific defects in DNA damage response pathways to identify new molecules in these pathways and to develop a "fingerprint" of transcript expression associated with radiation sensitivity. Finally, we will search for additional genes involved in radiation sensitivity in a collection of NBS families we have identified that lack mutations in the NBS1 gene, indicating that NBS is a heterogeneous disorder. These proposed studies should allow us to extend the insights gained from the identification of genes responsible for rare disorders characterized by radiation sensitivity into a more detailed understanding of the biochemical pathways activated in human cells in response to ionizing radiation.