DNA Deformations and Interactions in Complexes with Proteins

DNA 变形以及与蛋白质复合物的相互作用

• 批准号: 6433091
• 负责人: ROBERT L JERNIGAN
• 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/6433091
• 关键词: DNA; DNA binding protein; X ray crystallography; chemical models; chemical structure function; chromatin; computer simulation; conformation; intermolecular interaction; nucleic acid sequence; nucleic acid structure; nucleosomes; oncoprotein p21; protein sequence; pyrimidine dimers; transcription factor; tumor suppressor genes; tumor suppressor proteins

Based on the results of computer simulations and high-resolution crystal structures, we have elucidated the extents of DNA sequence-dependent bending, twisting and stretching deformability. With these knowledge-based elasticity functions for DNA, we built a stereochemically feasible model for the tetrameric p53-DNA complex. The predicted directionality and magnitude of the DNA bending and twisting were subsequently confirmed by gel electrophoresis experiments. Now we are entering the second phase of this study, when the precise localization of the DNA bends is to be determined, and the complexes are to be compared for various p53 mutants. For this aim, we are using an original method, iodine-125 radioprobing, recently developed at NIH. Preliminary results for the wild type p53 are consistent with the predicted increase in DNA twisting and major groove bending in the consensus CATG motifs in the p53 binding sites. As a next step, we will analyze the DNA distortions in the complexes between various p53 mutants and the DNA response elements regulating the p53-related genes. New areas are: (1) DNA looping and transcription regulation; analysis of the multimeric complex with GalR repressor and HU protein. We will apply our mechanistic "rules" to DNA containing ~100 base pairs, in order to interpret the available data on the GalR-HU binding to DNA, and to suggest new experiments, to elucidate the complicated 3D structure of this transcription regulation complex. (2) DNA binding to mutant hSRY protein (human testis determining factor). When mutant hSRY binds to cognate DNA, the degree of DNA bending differs from the wild type case. Based on the knowledge of the sequence-dependent properties of DNA, we will analyze how the atomic interactions at the DNA-protein interface lead to an increase or decrease in the level of the DNA deformation in the complex, which, in turn, is involved in regulation of transcription. Z01 BC 08371-17

基于计算机模拟的结果和高分辨率的晶体结构，我们已经阐明了DNA序列依赖的弯曲，扭曲和拉伸变形的程度。 利用这些基于知识的DNA弹性函数，我们建立了四聚体p53-DNA复合物的立体化学可行模型。 随后通过凝胶电泳实验证实了预测的DNA弯曲和扭曲的方向性和幅度。 现在我们进入了这项研究的第二阶段，当DNA弯曲的精确定位被确定时，复合物将被用于各种p53突变体的比较。 为此，我们正在使用一种新颖的方法，碘-125放射性探测，最近在NIH开发。 野生型p53的初步结果与p53结合位点中共有CATG基序中DNA扭曲和大沟弯曲的预测增加一致。 作为下一步，我们将分析各种p53突变体和调节p53相关基因的DNA反应元件之间的复合物中的DNA畸变。新的研究领域是：（1）DNA成环和转录调控;分析GalR阻遏物和HU蛋白的多聚体复合物。 我们将把我们的机械“规则”应用于含有~100个碱基对的DNA，以解释GalR-HU与DNA结合的现有数据，并提出新的实验，以阐明这种转录调控复合物的复杂3D结构。(2)DNA结合突变hSRY蛋白（人睾丸决定因子）。当突变体hSRY与同源DNA结合时，DNA弯曲程度与野生型情况不同。 基于DNA序列依赖性的知识，我们将分析DNA-蛋白质界面上的原子相互作用如何导致复合物中DNA变形水平的增加或减少，这反过来又参与了转录的调控。Z01 BC 08371-17