DYNAMIC FLUORESCENCE STUDIES OF DNA-PROTEIN COMPLEXES

DNA-蛋白质复合物的动态荧光研究

• 批准号: 6385991
• 负责人: David P MILLAR
• 金额: $ 29.68万
• 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 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385991
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primers; DNA replication; active sites; aminopurine; bioenergetics; chemical binding; chemical kinetics; conformation; enzyme mechanism; enzyme structure; enzyme substrate; fluorescence spectrometry; fluorescent dye /probe; nucleic acid sequence; protein sequence; protein structure function; purine analog; site directed mutagenesis; spleen exonuclease; stop flow technique; time resolved data

DESCRIPTION: Accurate replication of DNA is an essential requirement of all living organisms, and errors made in copying genetic material can result in a wide range of disorders. DNA polymerases achieve high DNA replication fidelity through the concerted action of base selection during polymerization, coupled with preferential removal of misincorporated nucleotides at a remote 3' -5' exonuclease site. Although crystal structures of DNA substrates bound to a few DNA polymerases have been solved, it is not understood how polymerization and exonucleolysis are coordinated to achieve efficient and accurate replication of DNA. In addition, the mechanistic roles of the amino acid residues that interact with the DNA substrate at the polymerase and 3' - 5' exonuclease sites are generally unknown. The broad, long term objective of this proposal is to understand the physical basis for high DNA polymerase replication fidelity. The proposal will focus on the Klenow fragment of DNA polymerase I from E. coli, which has served as a model for describing the molecular basis of template-directed DNA synthesis. The specific aims are: 1. Investigate the mechanisms responsible for melting duplex DNA and straining the resulting single-stranded DNA substrate at the 3' -5' exonuclease site. 2. Dissect the energetics of duplex DNA binding and distortion within the polymerase domain and characterize conformational changes of the enzyme-DNA complex induced by nucleotide binding. 3. Elucidate the mechanism by which the DNA primer terminus is transferred from the polymerase site to the 3' -5' exonuclease site. Time-resolved fluorescence anisotropy decay will be used to measure the distribution of dansyl-labeled DNA primer-templates between the polymerase and 3' -5' exonuclease sites. The enzyme will be judiciously modified by site-directed mutagenesis techniques in order to test specific hypotheses concerning the nature of the critical DNA-protein interactions at the polymerase and 3' -5' exonuclease site. The fluorescent DNA substrates will also be used to monitor the rate of movement of the primer terminus between the two active sites. Data on melting of specific base-pairs within critical regions of the enzyme-bound DNA will be obtained using a novel fluorescent probe. The information obtained from this study will provide insight into the molecular mechanisms used to suppress mutations during DNA replication.

描述：DNA的准确复制是所有生物的基本要求。 生物体，在复制遗传物质时所犯的错误可能导致 各种各样的疾病 DNA聚合酶实现高DNA复制 通过碱基选择的协同作用， 聚合，再加上优先除去错误掺入的 在远程3' -5'核酸外切酶位点处的核苷酸。 虽然水晶 结合到一些DNA聚合酶的DNA底物的结构已经被 解决，不理解聚合和核酸外切是如何 协调以实现DNA的高效和准确复制。 在 此外，相互作用的氨基酸残基的机械作用 在聚合酶和3'-5'核酸外切酶位点处的DNA底物， 一般未知。 这项建议的长远目标是 理解高DNA聚合酶复制保真度的物理基础。 该计划将集中在DNA聚合酶I的Klenow片段，从大肠杆菌。 大肠杆菌，它已作为一个模型，用于描述的分子基础， 模板指导的DNA合成。 具体目标是：1. 探讨 负责解链双链体DNA和拉紧 在3 ′-5 ′核酸外切酶位点产生单链DNA底物。 2. 剖析双链体DNA结合和扭曲的能量学， 聚合酶结构域和表征构象变化的酶-DNA 由核苷酸结合诱导的复合物。 3. 阐明了 DNA引物末端从聚合酶位点转移到3 ′端， -5'核酸外切酶位点。 时间分辨荧光各向异性衰减将是 用于测量丹磺酰标记的DNA引物-模板的分布 在聚合酶和3 ′-5 ′核酸外切酶位点之间。 这种酶将 通过定点诱变技术明智地修饰， 检验关于关键DNA-蛋白质性质的特定假设 在聚合酶和3' -5'核酸外切酶位点的相互作用。 荧光 DNA底物也将用于监测细胞的移动速率。 两个活性位点之间的引物末端。 特定的熔解数据 将获得酶结合DNA的关键区域内的碱基对 使用一种新的荧光探针。 从这项研究中获得的信息 将有助于深入了解用于抑制 DNA复制过程中的突变