DNA REPAIR IN A HORMONE RESPONSIVE GENE

这项提议的广泛目标是理解分子 转录偶联DNA修复的细节，一种重要的防御 抗DNA损伤所致细胞表型改变的机制 哺乳动物细胞。这样的侮辱是由各种各样的环境造成的 例如紫外线(UV)辐射和化学致癌物。 因为DNA中笨重的化学加合物和紫外光产物发生了修复 通过相同的机制(切除修复)，紫外线辐射被用作 我们大部分研究的环境制剂原型。我们将研究 DNA修复效率与转录活性的关系 以及哺乳动物基因中两个不同类别的染色质结构，以及 酵母中的一种可诱导基因。 修复与RNA聚合酶II(PolII)表达的关系将是 在含有单纯疱疹病毒胸苷的小鼠细胞中进行检测 激酶(Tk)基因与小鼠乳腺肿瘤病毒长末端的融合 重复(Ltr)。这个“ltl”结构被稳定地集成到 这些细胞的基因组和tk基因的表达需要 糖皮质激素。我们已经证明荷尔蒙诱导的转录 Tk基因的一部分对紫外线辐射非常敏感，恢复得很快。 在此结构中进行了有效的修复。因为紫外光产品形成了 优先在Ltr区域，转录可能被紫外线阻断 对启动子元件的破坏。我们将使用连接介导的聚合酶链式反应来检测 LTR中特定部位紫外光产物的产额和修复 启动POL II所需的区域。我们还将检查其影响 紫外线损伤对激素受体与LTR结合的影响 Shift“化验。 将研究核糖体RNA基因(RDNA)中N-甲基嘌呤的修复 在小鼠友红白血病细胞中。我们已经对rDNA进行了分离 这些细胞转化为转录活性和非活性形式的染色质， 使用补骨脂素交联，并发现EcoRI消化的细胞核 仅释放有效部分。与polII基因不同，紫外线的修复 活性和非活性rDNA链中的光产物效率都很低。 这可能是由于“庞大的”切除修复复合体被 核仁室。我们将确定是否有效的修复无效 RDNA也发生在另一类病变(N-甲基嘌呤)中， 它们被小得多的基因迅速从其他基因组序列中移除 碱基切除修复蛋白。 最后，我们使用了一种简单的酵母质粒，它含有一个可诱导的基因。 和一个结构性表达的基因，作为模型染色质底物 转录偶联修复在修复专家(Wt)和修复中的研究 缺乏(rad-)酵母细胞。我们将检查特定地点的维修情况 此质粒在wt/rad-细胞的“等基因组”中确定是否具有特异性 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和局部变化的影响。 染色质结构对DNA损伤效率的影响。因为这些损伤 可能会改变特定基因的表达，以建立 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 环境致癌物质。

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.