DYNAMIC FLUORESCENCE STUDIES OF DNA-PROTEIN COMPLEXES

描述：准确复制DNA是所有 生物体，以及复制遗传物质的错误可能会导致 一系列的疾病。DNA聚合酶实现DNA高复制 通过基地选择的协同行动实现保真度 聚合，再加上优先去除未并入的 位于遥远的3‘-5’核酸外切酶部位的核苷酸。虽然水晶 与几种DNA聚合酶结合的DNA底物的结构已经被 解决了，还不知道聚合和核外溶解是如何 协调以实现高效和准确的DNA复制。在……里面 此外，相互作用的氨基酸残基的机械作用 DNA底物位于聚合酶和3‘-5’核酸外切酶部位 一般不为人所知。这项提议的广泛、长期目标是 了解高DNA聚合酶复制保真度的物理基础。 该提案将重点放在来自E. Coli，它已经成为描述病毒分子基础的模型 模板引导的DNA合成。具体目标是：1.调查 导致双链DNA融化和应变的机制 在3‘-5’核酸外切酶部位形成单链DNA底物。2. 剖析细胞内双链DNA结合和扭曲的能量学 聚合酶结构域与酶-DNA构象变化的表征 由核苷酸结合引起的复合体。3.阐明其致病机制 DNA引物末端从聚合酶位点转移到3‘端 -5‘外切酶切位点。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’核酸外切酶之间。这种酶将会是 通过定点突变技术进行了明智的修改，以便 测试关于关键DNA-蛋白质性质的特定假设 聚合酶和3‘-5’核酸外切酶位点的相互作用。《荧光报》 DNA底物也将被用来监测 两个活性位点之间的引物末端。关于特定的熔化的数据 将获得酶结合DNA关键区域内的碱基对 使用一种新型的荧光探针。从这项研究中获得的信息 将提供对分子机制的洞察用于抑制 DNA复制过程中的突变。

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.