DNA DAMAGE EFFECT ON GENE EXPRESSION

DNA 损伤对基因表达的影响

• 批准号: 2608517
• 负责人: DAVID L MITCHELL
• 金额: $ 15.55万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2608517
• 关键词: DNA binding protein; DNA damage; DNA footprinting; chemical stability; gel mobility shift assay; gene expression; gene frequency; genetic promoter element; genetic regulation; genetic transcription; histones; molecular site; nucleic acid sequence; nucleosomes; pyrimidine dimers; radiation genetics; transcription factor; ultraviolet radiation

The goal of this proposal is to answer fairly straightforward questions about how DNA damage and DNA binding proteins are effected by one another. Two systems have been selected for study, one involving interactions between transcription factors and their binding sites and another between nucleosome core proteins and DNA. For each system we will determine the distribution of UV damage in bound and free substrates using sequencing gel analysis of DNA incised at sites of UV photoproducts. We will test the hypothesis that, in addition to their direct inhibitory effects on transcription, UV photoproducts may effect gene expression through regulatory pathways as well. Initially, we will quantify the capacity of transcription factors to bind promoters in which either a cyclobutane pyrimidine dimer or (6-4) photoproduct has been positioned at a defined location. DNA substrates for these studies are purified using immunoseparation techniques. By using different types of pyrimidine dimers, the effects of varying degrees of helical distortion and unwinding on transcription factor binding can be ascertained. Should differences in transcription factor binding be observed in the UV-damaged substrates, we will use these substrates to determine the effects of promoter damage on the initiation and progression of transcription. Our second experimental approach is designed to determine the ability of core histones to reassemble on a damaged DNA template. Phased nucleosome substrates containing specific photoproducts at selected sites will be reconstituted in vitro. DNA protein interactions will be analyzed by EMSA, micrococcal nuclease digestion, and DNase I footprinting. The effect of alterations in nucleosome stability or positioning on gene expression will be tested using in vitro transcription. Data from these studies will increase our understanding of the role DNA damage and its effect on protein-DNA interactions play in regulating transcription.

这个提议的目的是回答相当直接的问题 DNA损伤和DNA结合蛋白是如何受到 另 选择了两个系统进行研究，其中一个涉及 转录因子与其结合位点之间的相互作用， 另一个在核小体核心蛋白和DNA之间。对于每个系统，我们 将决定紫外线损伤在结合和自由中的分布 使用UV位点切割的DNA的测序凝胶分析底物 照片产品我们将测试的假设，除了他们的 对转录的直接抑制作用，UV光产物可能影响 基因表达也是通过调控途径。首先，我们将 定量转录因子结合启动子的能力， 环丁烷嘧啶二聚体或（6-4）光产物具有 被放置在一个确定的位置。这些研究的DNA底物 使用免疫分离技术进行纯化。通过使用不同 类型的嘧啶二聚体，不同程度的螺旋的影响， 转录因子结合上的扭曲和解旋， 确定。转录因子结合的差异是否应该 观察到的紫外线损坏的基板，我们将使用这些基板， 确定启动子损伤对启动的影响， 转录的进展。我们的第二个实验方法是 旨在确定核心组蛋白在一个 受损的DNA模板 含有特异性的定相核小体底物 将在体外重建选定部位的光产物。DNA 蛋白质相互作用将通过EMSA，微球菌核酸酶 消化和DNA酶I足迹。改变的影响 将测试核小体稳定性或基因表达定位 使用体外转录。这些研究的数据将增加我们的 理解DNA损伤的作用及其对蛋白质-DNA的影响 相互作用在调节转录中起作用。