DNA Deformations and Interactions with Transcription Fac

DNA 变形及其与转录因子的相互作用

• 批准号: 7291749
• 负责人: Victor Zhurkin
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7291749
• 关键词: DNA; Deformations; Interactions; Transcription; Fac

We report on several specific projects that are related to the DNA sequence-dependent structural characteristics, important for interactions with proteins (including human p53 and bacterial gal-repressor), higher order self-organization of genomic DNA (formations of nucleosomes and DNA loops), and gene regulation. p53-induced activation and repression of transcription. In response to DNA damage and other types of cellular stress, the p53 protein becomes activated and binds DNA sequence-specifically, functioning as a transcriptional factor. p53 is unique in regulating a wide spectrum of genes: thousands of human genes are either activated or repressed by p53. Normally, the p53 tetramer binds to DNA response elements, consisting of two decamers RRRCWWGYYY (half-sites) separated by a spacer. (The length of the spacer, S, varies from 0 to 14 bp in the known functional binding sites, but in most cases S=0 or 1.) How many putative p53 binding sites, consistent with this scheme, are there in the human genome? What is the distribution of the spacer lengths? Recent sequencing of the human genome allows us to answer these questions. The distribution of spacers proves to be extremely nonuniform in all human chromosomes, with strong peaks in the profile, exceeding the average background 3-4 fold. The peaks at S=0 and 10 bp, and the gap at 4-5 bp are consistent with our earlier computer modeling and electrophoresis measurements, indicating the lateral positioning of the p53 core domains on the outer side of the DNA loop. In general, these data agree with the idea that the p53 tetramer can bind DNA wrapped in nucleosomes in the course of transcriptional activation of the chromatin-assembled genes. Currently, we are exploring localization of the putative p53 sites with respect to the starts of transcription (in collaboration with C. Harris, NCI). Our database includes more than 1500 human genes, proven to be up- and down-regulated by p53 under various conditions. The results of computer analysis indicate statistically significance difference between the up- and down-regulated genes in terms of distribution of the p53 sites in the vicinity of genes. The up-regulated genes are characterized by a high occurrence of the p53 sites with spacer S=0. On the other hand, the down-regulated genes reveal a higher fraction of the p53 sites with "unusual" spacer S=3 bp, shown earlier to repress transcription of several genes. Based on these observations, we suggested a three-dimensional model accounting for the functional difference between the tetrameric p53 sites with spacers S=0 and 3 base pairs (the model is currently being tested). If confirmed experimentally, this novel concept opens exciting prospects for predicting the p53-activated and repressed genes. DNA packaging and distribution of the A- and G-tracts in pro- and eukaryotes Periodic positioning of the A- and G-tracts in DNA causes DNA curvature in solution and facilitates its bending in the complexes with proteins. Here, we analyzed distribution of these sequences in the pro- and eukaryotic genomes. Distribution of the strongly bent A-tracts (4-7 bp) in the prokaryotic genomes reveals a remarkable periodicity of 10-11 bp. Such a periodicity may reflect the intrinsic propensity of prokaryotic DNA for bending and forming the loop-shaped structures. Based on these data and by analogy with the "gene repression" gal- and lac-loops in E. coli, we hypothesize that the loop folds with the structural period of 100 bp may be elementary units of the prokaryotic nucleoid packaging. This hypothesis was tested by the micrococcal nuclease digestion of bacterial nucleoids (in collaboration with S. Adhya, NCI). The results show that the 100 bp DNA fragments are highly overrepresented in digestion products, thereby implying a highly specific nucleoid packaging, with the DNA structural period of 100 bp.

我们报告了几个与DNA序列依赖性结构特征相关的具体项目，这些结构特征对于与蛋白质（包括人p53和细菌gal-阻遏物）的相互作用，基因组DNA的高阶自组织（核小体和DNA环的形成）和基因调控至关重要。 p53诱导的转录激活和抑制。 在DNA损伤和其他类型的细胞应激反应中，p53蛋白被激活并特异性结合DNA序列，作为转录因子发挥作用。p53在调节广泛的基因方面是独特的：成千上万的人类基因被p53激活或抑制。正常情况下，p53四聚体与DNA反应元件结合，DNA反应元件由两个十聚体RRRCWWGYYY（半位点）组成，由间隔区隔开。(The间隔区S的长度在已知的功能结合位点中从0到14 bp变化，但在大多数情况下S=0或1。在人类基因组中，有多少个假定的p53结合位点与这个方案一致？垫片长度的分布情况如何？ 最近人类基因组测序使我们能够回答这些问题。间隔区的分布证明在所有人类染色体中是极不均匀的，在谱中具有强峰，超过平均背景3-4倍。在S=0和10 bp处的峰以及在4-5 bp处的差距与我们早期的计算机建模和电泳测量一致，表明p53核心结构域在DNA环外侧的横向定位。一般来说，这些数据同意的想法，p53四聚体可以结合DNA包裹在核小体的过程中的转录激活的染色质组装的基因。 目前，我们正在探索与转录起始相关的p53位点的定位（与C. Harris，NCI）。我们的数据库包括1500多个人类基因，这些基因被证明在各种条件下被p53上调和下调。计算机分析的结果表明，在基因附近的p53位点的分布方面，上调和下调基因之间的差异具有统计学意义。上调基因的特征在于具有间隔子S=0的p53位点的高发生率。另一方面，下调的基因揭示了具有“不寻常”间隔子S= 3bp的p53位点的较高分数，所述间隔子S= 3bp较早地显示抑制几个基因的转录。基于这些观察结果，我们提出了一个三维模型，解释了间隔区S=0和3个碱基对的四聚体p53位点之间的功能差异（该模型目前正在测试中）。 如果实验证实，这一新的概念为预测p53激活和抑制基因开辟了令人兴奋的前景。 原核生物和真核生物中A区和G区的DNA包装和分布 DNA中A区和G区的周期性定位会导致溶液中的DNA弯曲，并促进其在与蛋白质的复合物中弯曲。在这里，我们分析了这些序列在原核和真核基因组中的分布。 在原核生物基因组中，强烈弯曲的A-束（4-7 bp）的分布揭示了10-11 bp的显著周期性。这种周期性可能反映了原核DNA弯曲和形成环状结构的内在倾向。根据这些数据，并通过与大肠杆菌中的“基因阻遏”半乳糖环和乳糖环的类比，大肠杆菌中，我们推测100 bp结构周期的环状折叠可能是原核生物类核包装的基本单位。这一假设通过细菌类核的微球菌核酸酶消化进行了验证（与S. Adhya，NCI）。结果表明，100 bp的DNA片段在消化产物中高度过量，从而意味着高度特异性的类核包装，DNA结构周期为100 bp。