DNA REPAIR IN A HORMONE RESPONSIVE GENE

这项提案的主要目标是了解分子 DNA修复的细节，一种重要的防御机制， 在染色质中的DNA包装和基因转录的背景下。 这种损害是由各种各样的环境因素引起的，例如 紫外线辐射和化学致癌物。UV辐射和 硫酸二甲酯将被用作原型环境剂的研究， 核苷酸切除修复（NER）和碱基切除修复（BER）。 我们将研究DNA修复，转录和 明确核小体中不同类型基因的染色质结构 (or转录因子）复合物，以及在完整的酵母中， 哺乳动物细胞RNA pol II基因启动子的修复将在 小鼠病毒基因启动子（LTR），其在与A结合后被诱导 受体。紫外线对DNA损伤的主要形式（csCTD）是 合成，并将纳入LTR的特定位点进行研究 csCTD对受体结合的影响。这个序列也将是 由核小体定位元件包围并包装成核小体， 研究了核小体中csCTD取向对NER效率的影响， 爪蟾提取物。G：：U错配也将被合成到LTR中， 使用纯化的人抗体检测蛋白结合对BER效率的影响。 以及BER蛋白和哺乳动物细胞提取物。最后，修复CTD， 将在酵母核糖体RNA基因（rDNA）中检查N-甲基嘌呤（NMP）。 与pol II基因不同，活性和非活性rDNA中UV光产物的修复是 在哺乳动物细胞中效率低，但在酵母中快速。我们将诱导不同的 部分转录活性核糖体染色质在酵母中，通过改变 生长条件，并检查每个级分中CTD的修复。我们将 确定活性rDNA的有效修复是否也发生在NMPs上， 通过较小的BER蛋白快速从其他基因组序列中移除。因此 我们将研究基因表达和局部染色质变化的影响， DNA修复的效率。由于这些病变可能会改变 表达建立肿瘤所需的特定基因 表型，这些研究应该提供深入了解细胞的防御 抵抗环境致癌物转化的机制。

The broad objective of this proposal is to understand the molecular details of DNA repair, an important defense mechanism against damage to DNA, in the context of DNA packaging and gene transcription in chromatin. Such insults result from a wide variety of environmental agents, such as ultraviolet (UV) radiation and chemical carcinogens. UV radiation and dimethylsulfate will be used as prototype environmental agents for studies on nucleotide excision repair (NER) and base excision repair (BER), respectively. We will examine the relationship between DNA repair, transcription and chromatin structure of different classes of genes in well-defined nucleosome (or transcription factor) complexes in vitro, as well as in intact yeast and mammalian cells. Repair of an RNA pol II gene promoter will be examined in a mouse viral gene promoter (LTR), which is induced upon binding of a hormone-receptor. The major form of UV damage in DNA (csCTD) has been synthesized and will be incorporated into specific sites of the LTR for studies on the effect of csCTDs on receptor binding. This sequence will also be bracketed by nucleosome positioning elements and packaged into a nucleosome for studies on the effect of csCTD orientation in a nucleosome on NER efficiency in Xenopus extracts. G::U mismatches will also be synthesized into the LTR to examine the effect of protein binding on BER efficiency, using purified human and BER proteins and mammalian cell extracts. Finally, repair of CTDs and N-methyl purines (NMP) will be examined in yeast ribosomal RNA genes (rDNA). Unlike pol II genes, repair of UV photoproducts in active and inactive rDNA is inefficient in mammalian cells, but is rapid in yeast. We will induce different fractions of transcriptionally active ribosomal chromatin in yeast, by varying growth conditions, and examine repair of CTDs in each fraction. We will determine if efficient repair of active rDNA also occurs for NMPs, which are rapidly removed from other genomic sequences by the smaller BER proteins. Thus we will examine the effects of gene expression and changes in local chromatin structure on the efficiency of DNA repair. Since these lesions may alter the expression of specific genes required for establishing the neoplastic phenotype, these studies should provide insight into the cell's defense mechanism for resisting transformation by environmental carcinogens.