UV light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes.
UV light as a footprinting agent: modulation of UV-induced DNA damage by transcription factors bound at the promoters of three human genes.
复制标题
紫外线作为足迹剂：通过结合在三个人类基因启动子上的转录因子调节紫外线诱导的 DNA 损伤。
DOI：
10.1006/jmbi.1995.0331
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Pfeifer,GP
中科院分区：
文献类型：
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作者：
Tornaletti,S;Pfeifer,GP
哺乳动物细胞中蛋白质-DNA 相互作用可以通过基因组测序技术在核苷酸水平上进行分析。最灵敏的基因组测序方法使用连接介导的聚合酶链式反应 (LMPCR) 进行信号放大，以检测 DNA 修饰或链断裂的位置。多种探测方法均与 LMPCR 兼容，但最常用的是硫酸二甲酯足迹法。在这里，我们检查了紫外线 (UV) 光作为体内足迹试剂检测各种蛋白质-DNA 接触的适用性。已经沿着三个人类基因的启动子序列检查了两种主要类型的紫外线诱导的 DNA 光产物（环丁烷嘧啶二聚体和（6-4）光产物）的分布。紫外线照射的裸 DNA 和紫外线照射的细胞的比较揭示了两种光产品的紫外线损伤光谱的差异。这些差异可能是光产物频率的降低或急剧增加。在 c-jun 基因的启动子处，这些差异（“照片足迹”）与两个 AP-1 样转录因子、CCAAT 盒结合蛋白、SP-1 序列、NF-jun 序列、与血清反应因子 (RSRF) 相关的结合位点和未知因子结合的序列共定位。在编码增殖细胞核抗原（PCNA）的基因启动子中，在两个 SP-1 样序列和两个 CCAAT 盒处观察到光足迹。 c-fospromoter 的特征在于血清反应元件 (SRE)、三元复合因子 (TCF) 的相邻结合位点、AP-1 位点和生长因子诱导蛋白 (SIF) 的结合位点处的光足迹。照片足迹可能是特定转录因子或相关因子家族的特征，因为我们注意到，当在不同基因中分析同一位点时，在几个常见因子结合位点看到的照片足迹是相似或相同的。在远离转录因子结合位点的序列处没有看到光足迹。我们的紫外光足迹数据与使用其他探测策略的实验数据的比较表明，只要因子结合位点内的任一 DNA 链上存在二嘧啶序列，紫外光就有可能揭示所有蛋白质-DNA 相互作用。使用这种探测剂的简单性及其检测多种不同因素的特异性应该使紫外光成为体内足迹研究普遍有用的工具。f2
Protein-DNA interactions in mammalian cells can be analyzed at the nucleotide level of resolution by genomic sequencing techniques. The most sensitive genomic sequencing method uses the ligation-mediated polymerase chain reaction (LMPCR) for signal amplification to detect the position of DNA modifications or strand breaks. Various probing methods are compatible with LMPCR, but dimethyl sulfate footprinting has most commonly been used. Here, we have examined the suitability of ultraviolet (UV) light as anin vivofootprinting agent to detect a wide variety of protein-DNA contacts. The distribution of the two major types of UV-induced DNA photoproducts (cyclobutane pyrimidine dimers and (6-4) photo-products) has been examined along the promoter sequences of three human genes. A comparison of UV-irradiated naked DNA and UV-irradiated cells reveals differences in the UV damage spectrum for both types of photoproducts. These differences can be either decreases or dramatic increases of photoproduct frequency. At the promoter of the c-jungene, these differences ("photofootprints") co-localize with binding sites for two AP-1-like transcription factors, a CCAAT box binding protein, an SP-1 sequence, an NF-jun sequence, a related to serum response factor (RSRF) binding site and a sequence bound by an unknown factor. In the promoter of the gene coding for proliferating cell nuclear antigen (PCNA), photofootprints were seen at two SP-1 like sequences and two CCAAT boxes. The c-fospromoter is characterized by photofootprints at the serum response element (SRE), at the adjacent binding site for ternary complex factor (TCF), at an AP-1 site and at a binding site for a growth factor inducible protein (SIF). Photofootprints may be signatures of specific transcription factors or families of related factors since we noticed that the photofootprints seen at several common factor binding sites were similar or identical when the same site was analyzed in different genes. Photofootprints were not seen at sequences distant from transcription factor binding sites. A comparison of our UV photofootprinting data with data from experiments using other probing strategies shows that UV light has the potential to reveal all protein-DNA interactions provided there is a dipyrimidine sequence on either DNA strand within a factor binding site. The simplicity of using this probing agent together with its specificity for detecting a large variety of different factors should make UV light a generally useful tool forin vivofootprinting studies.f2