DNA REPAIR IN A HORMONE RESPONSIVE GENE

这项提案的主要目标是了解分子 转录偶联DNA修复的细节， 抗DNA损伤诱导的表型变化的机制 哺乳动物细胞 这种侮辱是由各种各样的环境因素造成的。 紫外线（UV）辐射和化学致癌物等物质。 由于DNA中的大体积化学加合物和紫外光产物的修复发生在 通过相同的机制（切除修复），紫外线辐射被用作 我们大多数研究的原型环境代理人。 我们将研究 DNA修复效率、转录活性 和哺乳动物基因中两种不同类别的染色质结构， 酵母中的一种诱导基因。 修复与RNA聚合酶II（polII）表达的关系将在下文中详细描述。 在含有单纯疱疹病毒胸苷的小鼠细胞中检测 小鼠乳腺肿瘤病毒长末端融合蛋白激酶（TK）基因 重复（LTR）。 这一“LTL”建筑稳定地融入了 这些细胞的基因组和TK基因的表达需要 糖皮质激素 我们已经证明， tk基因对紫外线非常敏感， 在这种结构中进行有效修复后。 由于紫外光产物形成 优选在LTR区域，转录可被UV阻断 破坏启动子元件。 我们将使用连接介导的PCR来检测 LTR中特定位点的UV光产物的产量和修复 启动pol II所需的区域。 我们还将研究 的紫外线损伤的结合激素受体的LTR使用“凝胶- 偏移”测定。 将检查核糖体RNA基因（rDNA）中N-甲基嘌呤的修复 在小鼠Friend红白血病细胞中。 我们分离了 将这些细胞转化为转录活性和非活性形式的染色质， 使用peptide交联，并发现EcoRI消化细胞核， 仅释放活性部分。 与pol II基因不同， 活性和非活性rDNA两条链中的光产物是低效的 并且可能是由于“庞大的”切除修复复合物被 核仁区室 我们将确定是否有效的修复活动 rDNA也存在于不同的损伤“类别”（N-甲基嘌呤）， 这些基因组序列被小得多的 碱基切除修复蛋白。 最后，我们使用一个简单的酵母质粒，含有一个诱导基因， 和组成型表达的基因，作为模型染色质底物， 研究转录偶联修复在修复熟练（wt）和修复 缺陷（rad-）酵母细胞。 我们将检查修复在特定的网站 在WT/RAD-细胞的“等基因组”中， RAD基因是转录偶联修复所必需的。 因此，我们将研究基因表达的影响和局部的变化， 染色质结构对DNA损伤效率的影响。 由于这些损伤 可能会改变特定基因的表达，这些基因是建立 肿瘤表型，这些研究应该提供有价值的见解， 细胞抵抗肿瘤转化的防御机制， 环境致癌物。

The broad objective of this proposal is to understand the molecular details of transcription-coupled DNA repair, an important defense mechanism against phenotypic changes induced by insults to DNA in mammalian cells. Such insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. Since repair of bulky chemical adducts and UV photoproducts in DNA occurs via the same mechanism (excision repair), UV radiation is used as a prototype environmental agent for most of our studies. We will examine the relationship between DNA repair efficiency, transcriptional activity and chromatin structure of two different classes in mammalian genes, and an inducible gene in yeast. The relationship of repair to RNA polymerase II (polII) expression will be examined in mouse cells containing the herpes simplex virus thymidine kinase (tk) gene fused to the mouse mammary tumor virus long terminal repeat (LTR). This "LTL" construction is stably integrated into the genome of these cells and expression of the tk gene requires glucocorticoid hormone. We have shown that hormone-induced transcription of the tk gene is acutely sensitive to UV radiation and returns rapidly following efficient repair in this construct. Since UV photoproducts form preferentially in the LTR region, transcription may be blocked by UV damage to promoter elements. We will use ligation-mediated PCR to examine the yield and repair of UV photoproducts at specific sites in the LTR region required for initiation of pol II. We will also examine the effect of UV damage on the binding of hormone receptor to the LTR using a "gel- shift" assay. Repair of N-methyl purines in ribosomal RNA genes (rDNA) will be examined in mouse Friend erythroleukemia cells. We have fractionated the rDNA of these cells into transcriptionally active and inactive forms of chromatin, using psoralen crosslinking, and find that EcoRI digestion of nuclei releases only the active fraction. Unlike pol II genes, repair of UV photoproducts in both strands of active and inactive rDNA is inefficient and may result from blockage of "bulky" excision repair complexes by the nucleolar compartment. We will determine if inefficient repair of active rDNA also occurs for a different "class" of lesions (N-methyl purines), which are rapidly removed from other genomic sequences by the much smaller base-excision repair proteins. Finally, we are using a simple yeast plasmid, containing an inducible gene and a constitutively expressed gene, as a model chromatin substrate to study transcription-coupled repair in repair proficient (wt) and repair deficient (rad-) yeast cells. We will examine repair at specific sites of this plasmid in "isogenic sets" of wt/rad-cells to determine if specific RAD genes are required for transcription-coupled repair. Thus, we will examine the effects of gene expression and changes in local chromatin structure on the efficiency of DNA lesions. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide valuable insight into the cell's defense mechanism for resisting neoplastic transformation by environmental carcinogens.