DYNAMIC FLUORESCENCE STUDIES OF DNA--PROTEIN COMPLEXES

DNA-蛋白质复合物的动态荧光研究

• 批准号: 3303231
• 负责人: David P MILLAR
• 金额: $ 20.76万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-15 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303231
• 关键词: DNA; DNA binding protein; DNA directed DNA polymerase; DNA repair; active sites; chemical binding; chemical structure function; deoxyribonucleoside triphosphate; divalent metal; fluorescence spectrometry; fluorescent dye /probe; nucleic acid sequence; polymerization; protein sequence; site directed mutagenesis; solutions; spleen exonuclease; synthetic nucleic acid

Interactions between proteins and nucleic acids are of fundamental importance in replication, transcription, recombination and other processes involved in the regulation of gene activity. The broad, long term objective of this proposal is to develop a new approach for studying the structure and dynamics of DNA:protein complexes in solution. This approach is specifically directed at a large and important class of DNA-binding proteins that do not exhibit a strong sequence-specificity in their interaction with DNA, and whose complexes with DNA are difficult to study by conventional structural methods. The proposal will initially focus on DNA:polymerase complexes as models of inherently dynamic complexes. The specific aims are: (1) Define the binding modes of DNA polymerase bound to DNA in solution during polymerization and editing of mispaired bases. (2) Estimate the separation between the polymerase and 3'-5' exonuclease (editing) active sites in the solution complex. (3) Study how the presence of mispaired bases in the DNA substrate influences the distribution of the two binding modes of the complex. (4) Determine whether there are local changes in the base-pairing of the bound DNA substrate during editing. (5) Determine how dNTP's and divalent metal ions affect the distribution. (6) Define which amino acid residues of the polymerase affect the mode of DNA binding and the dynamics of the complex. (7) Elucidate the common structural and dynamic features of a series of different DNA polymerases. The information obtained from this study is strongly health-related since it will provide insight into the molecular mechanisms used to suppress mutations during DNA replication. The experimental design is to characterize the structure and dynamics of DNA:polymerase complexes in solution using site-specific fluorescent probes attached to synthetic DNA oligomers and picosecond time-resolved fluorescence spectroscopy. Steady-state fluorescence spectroscopy will be used to measure binding properties. The DNA substrate and the protein structure will be systematically modified by oligonucleotide synthesis and site-directed mutagenesis, respectively, and the effect on the structure, dynamics and binding affinity of the complex will be determined. The approach developed here will be applied in future studies to other DNA-binding proteins.

蛋白质和核酸之间的相互作用是重要的 在复制、转录、重组和其它方面重要性 参与基因活动调节的过程。 宽，长 这项建议的长期目标是发展一种新的研究方法， DNA：蛋白质复合物在溶液中的结构和动力学。 这 这种方法专门针对一大类重要的 DNA结合蛋白，不表现出很强的序列特异性， 它们与DNA的相互作用，以及它们与DNA的复合物很难与DNA结合。 用传统的结构方法进行研究。 该提案最初将 专注于DNA：聚合酶复合物作为固有动力学模型 配合物 具体目标是：（1）确定DNA的结合模式 聚合酶在聚合和编辑过程中与溶液中的DNA结合， 错配碱基(2)估计聚合酶和 溶液复合物中的3 '-5'核酸外切酶（编辑）活性位点。（三） 研究DNA底物中错配碱基的存在如何影响 复合物的两种结合模式的分布。(4)确定 结合DNA的碱基配对是否有局部变化 在编辑过程中。(5)确定dNTP和二价金属 离子影响分布。(6)定义哪些氨基酸残基 聚合酶影响DNA结合的模式和动力学的 复杂. (7)阐述了一个通用的结构和动态特性， 一系列不同的DNA聚合酶 从这获得的信息 这项研究与健康密切相关，因为它将深入了解 在DNA复制过程中抑制突变的分子机制。 实验设计是为了表征结构和动力学， 使用位点特异性荧光检测溶液中的DNA：聚合酶复合物 探针连接到合成的DNA寡聚体和皮秒时间分辨 荧光光谱法 稳态荧光光谱将 用于测量结合性能。 DNA底物和蛋白质 结构将通过寡核苷酸合成进行系统修饰 和定点突变，以及对 结构，动力学和结合亲和力的复合物将是 测定 本文提出的方法将在今后的工作中得到应用 其他DNA结合蛋白的研究。