DNA Deformations and Interactions in Complexes with Proteins

基于计算机模拟结果和高分辨晶体结构，我们阐明了DNA序列依赖的弯曲、扭转和拉伸变形能力的程度。利用这些基于知识的DNA弹性函数，我们为四聚体P53-DNA复合体建立了一个立体化学可行的模型。随后，凝胶电泳实验证实了所预测的DNA弯曲和扭曲的方向性和大小。现在我们正在进入这项研究的第二阶段，届时将确定DNA弯曲的精确定位，并将比较这些复合体与不同的p53突变体。为了达到这个目的，我们正在使用一种独创的方法，即NIH最近开发的碘-125放射性探测方法。野生型P53的初步结果与预测的P53结合位点上共识CATG基序中DNA扭曲和主要沟槽弯曲的增加一致。下一步，我们将分析不同的p53突变体与调控p53相关基因的DNA反应元件之间的复合体中的DNA扭曲。新的领域包括：(1)DNA环和转录调控；与GalR阻遏物和Hu蛋白的多聚体复合体的分析。我们将把我们的机制“规则”应用于含有约100个碱基对的DNA，以解释现有的关于GalR-Hu与DNA结合的数据，并提出新的实验，以阐明这种转录调控复合体的复杂三维结构。(2)DNA与突变的hSRY蛋白(人睾丸决定因子)的结合。当突变体hSRY与同源DNA结合时，DNA的弯曲程度与野生型不同。基于对DNA序列依赖性质的了解，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体中DNA变形水平的增加或减少，这反过来又参与转录调控。Z01公元前08371-17

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