GENETIC VARIATION IN HUMAN NHEJ DNA REPAIR GENES

描述：（申请人描述）DNA双链断裂（DSB）发生 自发地并且由暴露于各种环境损害引起 包括电离辐射，氧化应激，金属，天然和 人造遗传毒性物质。DSB的准确修复对于细胞的生长至关重要。 生存和预防基因组重排，可能导致 癌人类体细胞有几种修复DSB的机制，但 最常用的机制似乎是容易出错的非同源 末端连接途径（NHEJ）。该途径包括至少五种蛋白质， 似乎是NHEJ机制所独有的，DNA-PK的三个组分 （DNA-PKcs，Ku 70，Ku 80），双链DNA活化蛋白激酶，和 DNA连接酶IV和XRCC 4。另外的NHEJ组分（例如MRE 11，聚合酶B， 外切核酸酶FEN-1）可能与其他修复系统共享，毫无疑问， 其他NHEJ成分仍有待发现。将五种基因结合在一起 独特的NHEJ组件，必须协同工作，跨越约1兆字节， 人类基因组，其中20,145 bp编码氨基酸。天然遗传 变异预计产生约20个编码内的单核苷酸变异 这一基因组的区域，其中约一半可能会改变氨基 酸性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。我们建议使用变性高性能液体 通过PCR产物的层析和直接核苷酸测序来发现 在5个NHEJ基因的所有（约160个）外显子中存在共同多态性 来自90个不同的参考个体然后我们将研究 多态性的患者的医疗条件，可能表明 这些基因中的一个或多个的活性缺乏。最后我们将 表征细胞系中NHEJ途径内的单个反应， 具有不同NHEJ多晶型的个体，以确定效率或 NHEJ在人类中的准确性受到体内天然遗传变异的影响， 这些基因在某种程度上可能会导致人类疾病。本研究还 将提供一组中等密度的映射多态性标记， 五个已知的NHEJ基因的子序列连锁研究，可以揭示微妙的 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.