Characterization of Protein-DNA Complexes

蛋白质-DNA 复合物的表征

• 批准号: 6487117
• 负责人: DONALD C ZAPIEN
• 金额: $ 3.1万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-04 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6487117
• 关键词: DNA binding protein; DNA footprinting; SDS polyacrylamide gel electrophoresis; X ray crystallography; binding sites; biotechnology; chromosomes; gel mobility shift assay; genetic manipulation; nonhistone nucleoprotein; nucleoproteins; peptide chemical synthesis; polymerase chain reaction; protein engineering; protein sequence; protein structure; transcription factor; ultracentrifugation

DESCRIPTION: (provided by applicant) Chromosomal proteins bind to DNA in a nonsequence-specific fashion, but the mechanism by which these proteins recognize DNA is poorly understood. The DNA complexes of chromosomal proteins are involved in mediating cellular processes. Therefore, a better understanding of the binding interactions of these complexes better enable clinicians to address disorders whose origin may be related to these interactions. It has been suggested that the reasons for the non-specificity lies in the interaction of two principal loci of the protein with DNA. Proposed here is a study to test this hypothesis through three specific aims. The first aim is to design and produce a sequence-specific protein from a nonsequence-specific protein scaffold. The second is to test the specificity of the designed protein to DNA. Thirdly, we propose to examine the interactions that are important for specificity by determining the three-dimensional structure of the designed protein in complex with DNA. The DNA scaffold for HMG-D, a nonsequence-specific protein, can be genetically engineered so that sequence-neutral residues will be replaced by residues which recognize specific-sequences, resulting in the putative protein, SPEC. The binding site will be evaluated by a electrophoretic mobility shift assay (EMSA) using oligomers of random sequence. Competitive band shift experiments employing a long chain probe DNA and competitor oligomers will be used to ascertain the binding site sequence and length. DNase I footprinting assays will be used to further evaluate the sequence specificity by SPEC. The three-dimensional structure of the complex will be determined by first co-crystallizing SPEC and duplex DNA, followed by collection and analysis of X-ray diffraction data.

描述：（由申请人提供）染色体蛋白质与DNA结合， 非序列特异性的方式，但这些蛋白质 对DNA的认识很少。染色体蛋白质的DNA复合物 参与调节细胞过程。因此，更好地理解 这些复合物的结合相互作用更好地使临床医生， 解决其起源可能与这些相互作用有关的疾病。它有 有人认为，非特异性的原因在于相互作用 蛋白质的两个主要位点与DNA的结合。这里提出的是一项研究，以测试 这一假设通过三个具体目标。第一个目标是设计和 从非序列特异性蛋白质产生序列特异性蛋白质 脚手架二是测试设计的蛋白质对DNA的特异性。 第三，我们建议审查对以下方面很重要的相互作用： 通过确定设计的三维结构， 与DNA复合的蛋白质。HMG-D的DNA支架，一种非序列特异性 蛋白质，可以被基因工程改造，使得序列中性残基将 被识别特定序列的残基取代，导致 结合位点将通过电泳分析来评估。 使用随机序列的寡聚体的迁移率变动测定（EMSA）。竞争 使用长链探针DNA和竞争对手的带移实验 寡聚体将用于确定结合位点序列和长度。dna酶 I足迹分析将用于进一步评价序列特异性 复合物的三维结构将由 首先使SPEC和双链体DNA共结晶，然后收集和分析 X射线衍射数据。