REPAIR OF DNA DAMAGED BY MODEL ENVIRONMENTAL CHEMICALS

本研究的目的是了解转录的影响和作用， 基因扩增对基因和转录链特异性修复的影响 紫外线（UV）和化学致癌物引起的DNA损伤- N-乙酰氧基-2-乙酰氨基芴（NAAAF）和苯并（a）芘二醇环氧化物 （BPDE）。已经发现哺乳动物细胞优先修复 环丁烷嘧啶二聚体（CPD）在转录链的活性 然而，我们发现BPDE-DNA加合物的修复显示， 很少，如果有的话，基因特异性和链特异性修复， NAAAF-DNA加合物的结果两者都不显示。我们假设1）紫外线照射 和NAAAF或BPDE处理对基因活性的影响非常不同， 2)转录可以特异性地修饰CPD以成为更好的底物 对于切除修复，（可能通过引入二聚体内磷酸二酯 键断裂），以及3）基因扩增对优先配对的任何影响 在很大程度上反映了大多数放大的 基因是转录活性的。 为了确定转录对CPD修饰的影响，我们 建议检查二聚体内磷酸二酯键的发生 转录链与非转录链的断裂， 与二氢叶酸还原酶（DHFR）基因的非编码区相比， 正常人成纤维细胞和修复缺陷型着色性干皮病组 A和D细胞。我们还建议构建一个环状寡核苷酸， 含有带有二聚体内磷酸二酯键的定点CPD 并测试其对T4内切酶V和UVRABC的敏感性 核酸酶切口。 我们推测NAAAF和BPDE可能更严重地阻碍转录 比紫外线照射更有效。为了验证这一理论，我们将研究 这些试剂通过核径流转录抑制转录 分析并确定其与优先修复程度的关系 在DHFR基因中。为了研究DNA损伤的优先修复是否 暂时与转录结合，我们将检查 高原地区中国仓鼠卵巢细胞c-fos和c-myc基因的研究 诱导这些基因转录后的阶段与血小板- 衍生生长因子。为了研究基因扩增的影响， 关于致癌物-DNA加合物的修复，我们建议比较 DNA加合物在二倍体次黄嘌呤磷酸核糖转移酶基因 基因座与扩增的DHFR基因在同一细胞中的基因座，并相关 这两个基因的转录修复效率 活动 我们已经发现ERCC 1CHO突变体及其亲本细胞修复 NAAAF-DNA加合物具有相同的低效动力学，然而， 亲本细胞更有效地去除DHFR基因中的加合物。我们 假设CHO细胞可能具有修复NAAAF-DNA的能力 活性基因的加合物，而ERCC 1突变细胞缺乏这种加合物。 能力;其他类别的ERCC突变体可能在不同的步骤缺陷 修复。我们将通过分析NAAAF的去除来测试这一假设- 基因组DNA以及ERCC突变体中基因水平的DNA加合物， 野生型CHO细胞。

This research aims to understand the effects of transcription and the role of gene amplification on the gene-and transcribed-strand-specific repair of DNA damage induced by ultraviolet light (Uv) and chemical carcinogens- N-acetoxy-2-acetylaminofluorene (NAAAF) and benzo(a)pyrene diol epoxide (BPDE). It has been found that mammalian cells preferentially repair cyclobutane pyrimidine dimers (CPD) in the transcribed strand of active genes, however, we have found that the repair of BPDE-DNA adducts shows little, if any, gene-specific and strand-specific repair, and the repair of NAAAF-DNA adducts shows neither. We hypothesize that 1) UV irradiation and NAAAF or BPDE treatment have very different effects on gene activity, 2) transcription may specifically modify CPD to become better substrates for excision repair, (perhaps by introducing an intradimer phosphodiester bond break), and 3) any effects of gene amplification on preferential pair are largely a reflection of whether or not the majority of the amplified genes are transcriptionaliy active. To determine the effect of transcription on the modification of CPD we propose to examine the occurrence of intradimer phosphodiester bond breakage in the transcribed versus nontranscribed strand, and coding versus noncoding regions of the dihydrofolate reductase (DHFR) gene in normal human fibroblasts and repair deficient xeroderma pigmentosum groups A and D cells. We also propose to construct a circularized oligonucleotide containing a site-directed CPD with an intradimer phosphodiester bond break and to test its susceptibility to T4 endonuclease V and UVRABC nuclease incisions. We hypothesize that NAAAF and BPDE may hinder transcription more severely than UV irradiation does. To test this theory we will examine transcription inhibition by these agents by a nuclear runoff transcription assay and determine its relationship to the degree of preferential repair in DHFR gene. To investigate whether the preferential repair of DNA damage is temporarily coupled with transcription, we will examine repair of the c-fos and c-myc genes of Chinese hamster ovary (CHO) cells in plateau phase after induction of transcription of these genes with platelet- derived growth factors. To investigate the effects of gene amplification on the repair of carcinogen-DNA adducts, we propose to compare the repair of DNA adducts at the diploid hypoxanthine phosphoribosyl transferase gene locus with that for amplified DHFR genes in the same cells, and correlate the repair efficiency of these two genes with their transcription activities. We have found that the ERCC1CHO mutant and its parental cells repair NAAAF-DNA adducts with the same inefficient kinetics, however, the parental cells remove the adducts more efficiently in the DHFR gene. We hypothesize that CHO cells may have the capacity to repair NAAAF-DNA adducts from active genes, while ERCC1 mutant cells are deficient in this capability; other classes of ERCC mutants may deficient in different steps of repair. We will test this hypothesis by analyzing the removal of NAAAF- DNA adducts at gene level in genomic DNA as well as in ERCC mutants and wild type CHO cells.