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: 6289199
• 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/6289199
• 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

The ultimate goals are: (1) to deduce the rules governing the sequence-dependent mechanical properties of the DNA duplex in complexes with proteins, (2) to apply these rules to specific cases of fundamental and practical interest. These goals can be achieved only through direct comparison between theory and experiment. Earlier, we developed a new approach to elucidate the DNA bending and twisting elasticities from the DNA variability in crystals. To complete these studies, in 1998-99 we analyzed the longitudinal deformations in DNA -- its stretching and compression. This kind of DNA motion is important for recombination, where the DNA extension serves more efficient recognition of the sister chromosomes, and is also advantageous for strand exchange. Our calculations provided the first detailed stereochemical model for the cooperative DNA stretching observed recently in micromanipulation experiments with single DNA molecules. In particular, we showed that extreme DNA stretching is accompanied by the B-A inter-family transition, similar to that observed for the complex with RecA protein. Importantly, we demonstrated that longitudinal deformations in DNA occur also in the complexes with TBP (TATA box binding proteins). This opens the possibilities for applying this kind of mechanistic model to other nucleoprotein systems, such as HIV reverse transcriptase and RNA polymerase bound to DNA and RNA strands. This work is in progress.Our theoretical analysis of the DNA complex with tumor suppressor protein p53 led to the gel electrophoresis experiments in which the predicted directionality and magnitude of DNA bending were confirmed (in collaboration with Dr. E. Appella, NCI). 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. To this aim, we are using an original method, iodine-125 radioprobing, developed recently at NIH. This approach has allowed us to detect the localized kinks in the DNA- CRP (CAP) complex in solution (in collaboration with Dr. R. Neumann, Clinical Center). Based on our calculations, we designed the necessary sequences, which will be used in the radioprobing experiments to visualize the local deformations in the p53-bound DNA.New subjects include:(i) DNA looping and transcription regulation; analysis of the multimeric complex with GalR repressor and HU protein (in collaboration with Dr. S. Adhya, NCI). We will apply our mechanistic rules for DNA to the mini-plasmid containing several hundred base pairs, in order to interpret the available data on the GalR-HU binding to DNA, and to suggest new experiments, that could elucidate the complicated 3D structure of this transcription regulation complex.(ii) DNA binding to mutant hSRY protein (human testis determining factor; in collaboration with Dr. M. Clore, NIDDK). When mutant hSRY binds to cognate DNA, the degree of DNA bending differs from the wild type case. Based on 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 08371-16 - databases, DNA binding protein, DNA folding, Gene regulation, molecular models, transcriptional control, p, Tumor Suppressor,

最终目标是：（1）推导出 规则 控制与蛋白质复合的DNA双链体的序列依赖性机械性质，（2）将这些规则应用于具有基本和实际意义的特定情况。这些目标只有通过理论和实验的直接比较才能实现。早些时候，我们开发了一种新的方法来阐明DNA的弯曲和扭转弹性的DNA在晶体中的可变性。为了完成这些研究，在1998-99年，我们分析了DNA的纵向变形--它的拉伸和压缩。这种DNA运动对于重组是重要的，其中DNA延伸更有效地识别姐妹染色体，并且也有利于链交换。我们的计算提供了第一个详细的立体化学模型的合作DNA拉伸最近在显微操作实验中观察到的单DNA分子。特别是，我们发现，极端的DNA拉伸伴随着B-A家族间的过渡，类似于观察到的复合物与RecA蛋白。重要的是，我们证明了DNA的纵向变形也发生在与TBP（TATA盒结合蛋白）的复合物中。这为将这种机制模型应用于其他核蛋白系统，如HIV逆转录酶和RNA聚合酶结合到DNA和RNA链上提供了可能性。我们对DNA与抑癌蛋白p53复合物的理论分析导致了凝胶电泳实验，在该实验中，DNA弯曲的预测方向和幅度得到了证实（与E. Appella，NCI）。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。这种方法使我们能够检测溶液中DNA-CRP（CAP）复合物中的局部扭结（与R. Neumann，临床中心）。基于我们的计算，我们设计了必要的序列，这些序列将用于放射探测实验，以可视化p53结合DNA中的局部变形。新的主题包括：（i）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（与S. Adhya，NCI）。我们将应用我们的机械 规则 为了解释GalR-HU与DNA结合的现有数据，并提出新的实验，可以阐明这种转录调控复合物的复杂的3D结构。(ii)DNA与突变hSRY蛋白（人睾丸决定因子）的结合; Clore，NIDDK）。当突变体hSRY与同源DNA结合时，DNA弯曲的程度不同于野生型情况。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，这反过来又涉及转录的调控。Z 01 BC 08371-16 -数据库，DNA结合蛋白，DNA折叠，基因调控，分子模型，转录控制，p，肿瘤抑制剂，