GENETIC VARIATION IN HUMAN NHEJ DNA REPAIR GENES

描述：（申请人的描述）DNA 双链断裂 (DSB) 发生 自发性的，是由于暴露于各种环境损害而引起的 包括电离辐射、氧化应激、金属以及自然和 人造遗传毒性物质。 DSB 的精确修复对于细胞至关重要 生存并防止基因组重排可能导致 癌症。人类体细胞有多种修复 DSB 的机制，但 最常用的机制似乎是容易出错的非同源机制 末端连接途径（NHEJ）。该途径包括至少五种蛋白质 DNA-PK 的三个组成部分似乎是 NHEJ 机制所独有的 （DNA-PKcs、Ku70、Ku80），双链 DNA 激活蛋白激酶，以及 DNA 连接酶 IV 和 XRCC4。其他 NHEJ 成分（例如 MRE11、聚合酶 b、 核酸外切酶 FEN-1）可以与其他修复系统共享，毫无疑问 其他 NHEJ 成分仍有待发现。这五个基因加在一起 独特的 NHEJ 组件必须协同工作，跨越大约一兆碱基 人类基因组，其中 20,145 bp 编码氨基酸。自然遗传 变异预计会在编码内产生约20个单核苷酸变异 该基因组的区域，其中大约一半可能会改变氨基酸 酸序列。其中一半将发生在非常大的 DNA-PK 催化区域内 亚基，DNA-PKcs。我们建议使用高性能变性液体 对 PCR 产物进行色谱分析和直接核苷酸测序，以发现 五个 NHEJ 基因全部（约 160 个）外显子的共同多态性 来自大约 90 个不同的参考个体。然后我们将检查该模式 患有健康状况的患者中的多态性可能表明 这些基因中的一个或多个的活性缺陷。最后，我们将 表征细胞系中 NHEJ 通路内的个体反应 具有不同 NHEJ 多态性的个体，以确定效率或 人类 NHEJ 的准确性受到自然遗传变异的影响 这些基因可能会导致人类疾病。这项研究还 将提供一组中等密集的映射多态性标记 五个已知的 NHEJ 基因用于后续连锁研究，可以揭示微妙的 效应和更复杂的等位基因相互作用。

DESCRIPTION: (Applicant's Description) DNA double-strand breaks (DSBs) occur spontaneously and are caused by exposures to a variety of environmental insults including ionizing radiation, oxidative stress, metals, and natural and man-made genotoxic substances. Accurate repair of DSBs is essential for cell survival and for the prevention of genome rearrangements that may lead to cancer. Human somatic cells have several mechanisms for repairing DSBs, but the most commonly used mechanism appears to be the error prone non-homologous end-joining pathway (NHEJ). This pathway includes at least five proteins that appear to be unique to the NHEJ mechanism, the three components of DNA-PK (DNA-PKcs, Ku70, Ku80), the double-stranded DNA activated protein kinase, and DNA ligase IV and XRCC4. Additional NHEJ components (e.g. MRE11, polymerase b, exonuclease FEN-1) may be shared with other repair systems, and undoubtedly other NHEJ components remain to be discovered. Together the genes for the five unique NHEJ components, which must work in concert, span about one megabase of the human genome, 20,145 bp of which code for amino acids. Natural genetic variation is expected to yield about20 single nucleotide variants within coding regions of this gene set, of which about half might be expected to alter amino acid sequences. Half of these will occur within the very large DNA-PK catalytic subunit, DNA-PKcs. We propose to use denaturing high-performance liquid chromatography and direct nucleotide sequencing of PCR products to discover the common polymorphisms in all ( about160) of the exons of the five NHEJ genes from about 90 different reference individuals. Then we will examine the pattern of polymorphisms in patients with medical conditions that might suggest a deficiency in the activity of one or more of these genes. Finally, we will characterize individual reactions within the NHEJ pathway in cell lines from individuals with different NHEJ polymorphims to determine if the efficiency or accuracy of NHEJ in humans is affected by the natural genetic variations within these genes in a way that might contribute to human disease. This study also will provide a moderately dense set of mapped polymorphic markers within the five know NHEJ genes for subsequence linkage studies that could reveal subtle effects and more complex allele interactions.