INTERACTION BETWEEN GENE EXPRESSION AND DNA DAMAGE DUE TO IONIZING RADIATION

电离辐射广泛用于癌症治疗。 虽然一些 癌症得到了成功的治疗，这是不可接受的一部分， 治疗仍然失败。 随后的复发和继发性肿瘤 通常比原来的更具侵略性，可能是因为 治疗诱导的突变。 这些突变并没有很好的定义 由于电离辐射对DNA损伤的类型多种多样， 破坏的随机性，以及它在 基因组 核酸化学的最新进展使得 来合成DNA中检测到的许多类型的损伤 遭受电离辐射。 这些修饰的核苷酸可以是 通过标准分子标记插入克隆基因的特定位点 生物技术拟议的工作将利用这一结合 DNA损伤类型的定位方法， 辐射，在克隆的DNA片段的指定位点。 我们将 然后确定这种损伤如何影响真核基因表达 和基因完整性。 DNA损伤将定位于合成 对应于转录因子结合位点的寡核苷酸。 将使用凝胶位移测定来确定结合和 转录复合物的稳定性， 受损的DNA 损害也将纳入 合成DNA模板中的特定位点并在体外转录 核提取物 特定类型的DNA损伤的影响 基因转录和保真度将被确定。相关 实验还将确定转录是否将两个 相反的单链断裂变成更致命的双链断裂。 最后，我们将测量双链体的相对稳定性 含有碱基损伤位点的寡核苷酸， 迁移率变动分析并确定稳定性是否与 聚合酶旁路或致突变性结果来自于 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.