DNA REPAIR IN A HORMONE RESPONSIVE GENE

这项提议的广泛目标是理解分子 DNA修复的细节，一种重要的损伤防御机制 DNA，在DNA包装和染色质基因转录的背景下。 这种侮辱是由各种各样的环境因素造成的，例如 紫外线辐射和化学致癌物。紫外线辐射和 将使用二甲基硫酸酯作为原型环境试剂进行研究 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别基因的染色质结构 (或转录因子)复合体，以及在完整酵母和 哺乳动物细胞。RNA polII基因启动子的修复将在 小鼠病毒基因启动子(LTR)，它是由一个 激素受体。紫外线损伤DNA(Csctd)的主要形式是 人工合成的，并将被合并到LTR的特定位置进行研究 关于csCTDS对受体结合的影响。这一序列也将是 被核小体定位元件包围并包装成核小体 核小体中csCTD定位对NER效率影响的研究 非洲爪哇提取物。G：：U不匹配也将被合成到LTR中以 用纯化的人检测蛋白质结合对误码率效率的影响 以及BER蛋白和哺乳动物细胞提取物。最后，CTD的修复和 将在酵母核糖体RNA基因(RDNA)中检测N-甲基嘌呤(NMP)。 与polII基因不同，活性和非活性rDNA中紫外光产物的修复是 在哺乳动物细胞中效率低下，但在酵母中迅速。我们将推出不同的 酵母中转录活性核糖体染色质的不同组分 生长条件，并检查每个组分中CTDS的修复。我们会 确定NMP是否也会有效修复活性rDNA，它们是 被较小的BER蛋白迅速从其他基因组序列中删除。因此， 我们将研究基因表达和局部染色质变化的影响 结构对DNA修复效率的影响。因为这些损伤可能会改变 建立肿瘤所需的特定基因的表达 表型，这些研究应该提供对细胞防御的洞察力 抗环境致癌物质转化的机制。

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.