Isolation of Genes for Radiation Sensitivity

培养的人类细胞对电离辐射（IR）致死效应的敏感性是一个复杂的特征，这是由多个基因及其产物在参与DNA损伤感知和反应的各种途径中发挥作用所促成的。 我们研究的重点是确定人群中有助于辐射敏感性的基因，其长期目标是（1）了解暴露于环境诱变剂（IR）和癌症发展之间的关系，以及（2）开发辐射敏感性标记物，使我们能够更好地根据个体患者的敏感性定制放射治疗方案。 这种方法使我们研究了代表人群中极端辐射敏感性的患者，患有遗传性疾病共济失调-毛细血管扩张症（A-T）和奈梅亨断裂综合征（NBS）的患者，其中辐射敏感表型作为隐性性状遗传，从而允许对负责基因进行定位和定位克隆。 在上一个资助期，我们成功地鉴定了NBS及其蛋白产物nitrobin的基因，并通过证明nitrobin是ATM激酶的底物来阐明A-T和NBS之间的生物化学联系。 在这个应用中，我们提出了扩展我们的研究A-T和NBS与2个一般的方法。 首先，我们将重点介绍nitrile的功能。 我们将通过位点特异性诱变来绘制蛋白质的功能结构域，通过从完成的酵母双杂交筛选中获得的线索来识别与之相互作用的其他蛋白质，并开发小鼠模型来探索nitrobin在组织中的功能，以及在人类患者中不可及的发育阶段。 在第二种方法中，我们将在DNA损伤反应途径中具有特定缺陷的细胞系中使用微阵列的转录谱分析，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NBS家族集合中寻找与辐射敏感性相关的其他基因，这些NBS家族缺乏NBS 1基因突变，表明NBS是一种异质性疾病。 这些拟议中的研究应该使我们能够扩展的见解，从识别基因负责罕见的疾病，其特征是辐射敏感性到一个更详细的了解在人体细胞中激活的生物化学途径电离辐射。

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.