DNA Deformations and Interactions in Complexes with Prot

DNA 变形和与 Prot 复合物的相互作用

• 批准号: 6559001
• 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/6559001
• 关键词: DNA; DNA binding protein; X ray crystallography; chemical structure function; computer simulation; conformation; intermolecular interaction; nucleic acid sequence; nucleic acid structure; p53 gene /protein; protein folding; protein sequence; pyrimidine dimers; structural biology; transcription factor

Nucleic Acids The expansion of structural databases for nucleic acids, proteins and nucleo-protein complexes, as well as continued progress in elucidating the sequence-dependent structural properties of DNA and RNA, will improve the accuracy with which large nucleo-protein complexes can be characterized. X-ray and NMR data will be used for better understanding the sequence dependence of different forms of DNA and also binding motifs of proteins in the major and minor grooves. 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. Additional subjects include: (1)Gal Repressosome. Genetic data were used to determine the overall orientation of the gal repressors in a tetrameric structure. Knowledge-based computer modeling was employed to determine the trajectory of the DNA loop. According to our data, a similar (antiparallel) DNA loop is formed upon binding of the lac repressors to DNA. These findings imply that the antiparallel DNA looping may be a general feature of the condensed bacterial nucleoid, as opposed to the parallel DNA 'wrapping' around histones in eukaryotic chromatin. (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. (3) Protein-Nucleic Acid Interactions. This is a genome analysis of protein binding sites for p53 sites in the human genome with a subsequent characterization of the conformational features of the adjacent DNA. Location of tandem p53 sites (putative storage pools), particularly on human chromosomes 21 and 22. (4) Iodine-125 Radioprobing - a novel method for tracing the DNA and RNA trajectories in nucleoprotein complexes. Radioprobing has been successfully applied to the CRP-DNA complex and to the T7 transcription elongation complex. In the latter case we were able to locate for the first time the DNA-RNA heteroduplex outside of the polymerase. This finding opens exciting prospects for the field of transcription-coupled mismatch repair. (5) Tetrameric p53-DNA complex. Iodine-125 radioprobing data indicate that upon binding to p53, DNA is severely bent and overtwisted at the CATG sequences, consistent with our computations and gel electrophoresis results. Thus, the apparent role of the sequence-specific DNA deformation is to control the p53-p53 interactions in order to form an interface on the surface of the p53 tetramer, suitable for the binding of other proteins, such as Mdm2. The next step is to analyze the effects of mutations at the p53 binding site, and of the p53 mutants, on stabilization of the tetrameric p53-DNA assemblage. (6) p53 and DNA helicases. Building models of their interactions and understanding how phophorylation controls interactions. Z01 BC 08371-17

核酸核酸、蛋白质和核蛋白复合物的结构数据库的扩展，以及在阐明DNA和RNA的序列依赖性结构特性方面的持续进展，将提高表征大型核蛋白复合物的准确性。X射线和核磁共振数据将用于更好地理解不同形式的DNA的序列依赖性，以及在大沟和小沟中蛋白质的结合基序。 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。 其他科目包括： （1）Gal阻遏体。 遗传数据用于确定gal阻遏物在四聚体结构中的总体方向。采用基于知识的计算机建模来确定DNA环的轨迹。根据我们的数据，一个类似的（反平行的）DNA环形成后，结合的乳糖阻遏DNA。这些发现意味着，反平行的DNA循环可能是浓缩的细菌类核的一般特征，而不是平行的DNA“包裹”在真核染色质中的组蛋白周围。 (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，这反过来又参与了转录的调控。 (3)蛋白质-核酸相互作用。这是对人类基因组中p53位点的蛋白质结合位点的基因组分析，随后对相邻DNA的构象特征进行表征。串联p53位点（假定的储存池）的位置，特别是在人类染色体21和22上。 (4)碘-125放射性探测-一种追踪核蛋白复合物中DNA和RNA轨迹的新方法。放射探测已成功地应用于CRP-DNA复合物和T7转录延伸复合物。在后一种情况下，我们首次能够在聚合酶之外定位DNA-RNA异源双链体。这一发现为转录偶联错配修复领域开辟了令人兴奋的前景。 (5)四聚体p53-DNA复合物。碘-125放射性探测数据表明，结合p53后，DNA是严重弯曲和过度扭曲的CATG序列，与我们的计算和凝胶电泳结果一致。因此，序列特异性DNA变形的明显作用是控制p53-p53相互作用，以便在p53四聚体的表面上形成适合于结合其他蛋白质（如Mdm 2）的界面。下一步是分析p53结合位点突变和p53突变体对四聚体p53-DNA组合稳定性的影响。 (6)p53和DNA解旋酶。建立它们相互作用的模型，并了解磷酸化如何控制相互作用。 Z01 BC 08371-17