DNA DAMAGE EFFECT ON GENE EXPRESSION

这项提议的目标是回答相当直白的问题 关于DNA损伤和DNA结合蛋白是如何由一种 又一个。选择了两个系统进行研究，其中一个涉及 转录因子及其结合部位之间的相互作用和 核小体核心蛋白和DNA之间的另一种。对于每个系统，我们 将决定紫外线损伤在束缚和自由状态下的分布 紫外光切割DNA的测序凝胶分析底物 摄影产品。我们将检验这一假设，除了他们的 直接抑制转录，紫外光产物可能会影响 基因表达也通过调控途径进行。最初，我们将 量化转录因子结合启动子的能力 环丁烷嘧啶二聚体或(6-4)光产物具有的 被放置在指定的位置。用于这些研究的DNA底物 使用免疫分离技术进行纯化。通过使用不同的 不同类型的嘧啶二聚体对不同程度螺旋的影响 转录因子结合的扭曲和解离可以是 已经确定了。转录因子结合的差异应该是 在紫外线损坏的衬底中观察到的，我们将使用这些衬底来 确定启动子损伤对启动和启动的影响 转录的进程。我们的第二个实验方法是 旨在确定核心组蛋白在 DNA模板已损坏。含特定核小体的相变底物 选定地点的照相产品将在体外进行重组。脱氧核糖核酸 蛋白质的相互作用将通过EMSA，微球菌核酸酶进行分析 消化和DNase I足迹。发生变化的影响 将测试核小体对基因表达的稳定性或定位 使用体外转录。来自这些研究的数据将增加我们的 对DNA损伤的作用及其对蛋白质-DNA影响的认识 相互作用在调节转录方面发挥作用。

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.