INTERACTION BETWEEN GENE EXPRESSION AND DNA DAMAGE DUE TO IONIZING RADIATION

电离辐射在癌症治疗中被广泛应用。虽然有些人 癌症已经得到了成功的治疗，这是不可接受的比例 治疗仍然失败。随之而来的复发和继发性肿瘤 通常比原始版本更具攻击性，可能是因为 治疗导致的突变。这些突变没有被很好地定义 由于各种类型的DNA损伤电离辐射， 损害的随机性，以及它在 基因组。核酸化学的最新进展使之成为可能 合成在DNA中检测到的多种类型的损伤 受到电离辐射的。这些修饰的核苷酸可以是 通过标准分子整合到克隆基因的特定位置 生物学技术。拟议的工作将综合利用这一点 DNA损伤部位类型、电离特性的探讨 在克隆的DNA片段中的指定位置的辐射。我们会 然后确定这种损伤如何影响真核基因的表达 和基因的完整性。DNA损伤将被定位在合成的 与转录因子结合位点相对应的寡核苷酸。 将利用凝胶移动分析来确定结合和 含有正常或非正常转录复合体的稳定性 受损的DNA元素。损害也将被纳入到 合成DNA模板中的特定位点并在体外转录 用核提取液。特定类型的DNA损伤的影响 基因转录和保真度的大小将被确定。相关 实验还将确定转录是否会将两个 相反的单链断裂变成更致命的双链断裂。 最后，我们将测量双工的相对稳定性 含碱基损伤位点的寡核苷酸的竞争性 迁移率变化分析，并确定稳定性是否与 从In获得的聚合酶旁路或致突变结果 体外转录研究。

Ionizing radiation is widely utilized in cancer therapy. While some cancers have been treated successfully, an unacceptable fraction of treatments still fail. The ensuing recurrences and secondary tumors are often more aggressive than the original, possibly because of treatment-induced mutations. These mutations are not well defined due to the varied types of DNA damage ionizing radiation, the randomness of the damage, and its relative infrequency in the genome. Recent advances in nucleic acid chemistry make it possible to synthesize many of the types of damage detected in DNA subjected to ionizing radiation. These modified nucleotides can be incorporated into specific sites in cloned genes by standard molecular biology techniques. The proposed work will utilize this combined approach to place types of DNA damage, characteristic of ionizing radiation, at designated sites in a cloned DNA fragment. We will then determine how such damage affects eukaryotic gene expression and gene integrity. DNA damage will be positioned in synthetic oligonucleotides corresponding to transcription factor binding sites. A gel shift assay will be utilized to determine the binding and stability of transcription complexes containing either normal or damaged DNA elements. Damage will also be incorporated into specific sites in a synthetic DNA template and transcribed in vitro with a nuclear extract. The effect of specific types of DNA damage of gene transcription and fidelity will be determined. Related experiments will also determine if transcription transforms two opposed single-strand breaks into a more lethal double-strand break. Finally, we will measure the relative stability of duplex oligonucleotides containing base damage sites using the competitive mobility shift assay and determine whether a stability correlates with the polymerase by-pass or mutagenicity results obtained from the in vitro transcription studies.