REPAIR OF DNA DAMAGED BY MODEL ENVIRONMENTAL CHEMICALS

本研究旨在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 紫外线和化学致癌物引起的DNA损伤- N-乙酰氧基-2-乙酰氨基荧烷和苯并(A)芘二醇环氧化物 (BPDE)。已发现哺乳动物细胞优先修复 环丁烷嘧啶二聚体(CPD)在活性转录链中的作用 然而，我们已经发现，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，以及修复 NAAAF-DNA加合物显示两者都没有。我们假设紫外线照射 NAAAF和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，使其成为更好的底物 用于切除修复，(可能通过引入一种内消旋磷酸二酯 键断裂)，以及3)基因扩增对优先配对的影响 在很大程度上反映了大多数被放大的 基因具有转录活性。 为了确定转录对CPD修饰的影响，我们 建议检查传统分子内磷酸二酯键的发生 转录的链与非转录的链的断裂，以及编码 二氢叶酸还原酶(DHFR)基因非编码区的比较 正常人成纤维细胞和修复缺陷性色素性干皮病 A细胞和D细胞。我们还建议构建一种环状寡核苷酸 含有具有分子内磷酸二酯键的定点CPD 断裂并检测其对T4内切酶V和UVRABC的敏感性 核酸酶切面。 我们推测NAAAF和BPDE可能会更严重地阻碍转录 比紫外线辐射的影响更大。为了验证这一理论，我们将检验 这些试剂通过核径流转录抑制转录 检测并确定其与优先修复程度的关系 在DHFR基因中。调查DNA损伤的优先修复是否 是临时配合转录的，我们会检查一下修复情况 高原条件下中国仓鼠卵巢细胞的c-fos和c-myc基因 用血小板诱导这些基因转录后的阶段- 衍生的生长因子。研究基因扩增的效果 关于致癌物-DNA加合物的修复，我们建议将修复 二倍体次黄嘌呤磷酸核糖转移酶基因的DNA加合物 与扩增的dhfr基因在同一细胞中的位置，并相互关联。 这两个基因的修复效率及其转录 活动。 我们发现ERCC1CHO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ERCC1突变细胞在这方面是缺陷的 能力；其他类别的ERCC突变体可能在不同的步骤中存在缺陷 修理的问题。我们将通过分析NAAAF的移除来验证这一假设- DNA加合物在基因组DNA以及ERCC突变体和 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.