DYNAMIC FLUORESCENCE STUDIES OF DNA/PROTEIN COMPLEXES

DNA的精确复制是所有生物的基本要求 生物体，以及在复制遗传物质方面的错误可能导致 范围广泛的疾病。虽然DNA是在体内合成的 DNA聚合酶的极高保真度，目前还不清楚 这种保真度是如何在分子水平上实现的。广泛的、长期的 这项建议的目的是了解碱基错配是如何导致 来自聚合酶的错误被识别并选择性地去除酶。 该提案最初将专注于DNA聚合酶的Klenow片段 我来自大肠杆菌，它已经成为描述分子的模型 DNA复制保真度的基础，并将随后扩展到 来自其他生物的聚合酶。通过以下方式移除未合并的碱基 Klenow片段似乎涉及3‘端的熔化和移位 将DNA复制到酶的单独编辑结构域中，然后 错配碱基的核外裂解移除和随后的DNA返回 到聚合域。在上一次支持期间， 利用新的溶液光谱方法进行了直接测量。 结合到聚合结构域和编辑结构域的DNA。它是 建议使用这种方法来研究两者的结构特征 能够识别不匹配的碱基对的DNA和酶。这个 具体目标是：1.检验两个或两个以上碱基熔化的假设 在将引物末端转移到编辑过程中需要配对 Klenow片段的结构域。2.判断是否优先分区 不匹配碱基对进入编辑结构域的数量增加是由于 DNA的融化能力，不利于DNA与 聚合域，或这些效应的组合。3.识别 参与DNA结合和错配识别的氨基酸残基 本垒打。4.测量引物末端在不同物种之间的转移率 聚合区和编辑区，并鉴定氨基酸 促进转移的残留物。5.确定Klenow碎片 通过拒绝内部错位序列来抑制移码突变 聚合域。6.描述其他网站的编辑功能 DNA聚合酶m顺序检验原理的通用性 为Klenow碎片建立了不匹配识别。时间分辨率 丹磺酰标记DNA的荧光各向异性衰变将用于 测量与聚合或聚合结合的DNA末端的分布 编辑Klenow片段的结构域。DNA和酶将被明智地 利用寡核苷酸合成和定点突变进行修饰 技术，以及对DNA在体内分布的影响 每个域都将被确定。描述结合的热力学数据 与酶的每个结构域不匹配的DNA，以及不匹配的融化 DNA序列，也将获得。停流荧光技术 将被用来测量DNA在结构域之间的转移速度。这个 从这项研究中获得的信息将为深入了解 用于抑制DNA复制过程中突变的分子机制。

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. Although DNA is synthesized in vivo with extremely high fidelity by DNA polymerases, it is not clearly understood how this fidelity is achieved on a molecular level. The broad, long term objective of this proposal is to understand how mismatched bases resulting from polymerase errors are recognized and selectively removed the enzyme. The proposal will initially focus on the Klenow fragment of DNA Polymerase I from E. coli, which has served as a model for describing the molecular basis of DNA replication fidelity, and will subsequently be extended to polymerases from other organisms. Removal of misincorporated bases by Klenow fragment appears to involve melting and translocation of the 3' end of the DNA into a separate editing domain of the enzyme, followed by exonucleolytic removal of the mismatched base and subsequent return of DNA to the polymerization domain. During the previous period of support, a novel solution spectroscopic method was used to make a direct measurement of DNA bound to the polymerization domain and to the editing domain. It is proposed to use this method to investigate the structural features of both the DNA and enzyme that enable mismatched base pairs to be recognized. The specific aims are: 1. Test the hypothesis that melting of two or more base pairs is required for transfer of the primer terminus into the editing domain of Klenow fragment. 2. Determine whether preferential partitioning of mismatched base pairs into the editing domain is due to increased melting capacity of the DNA, disfavored binding of DNA to the polymerization domain, or a combination of these effects. 3. Identify amino acid residues involved in DNA binding and recognition of mismatched bases. 4. Measure the rate of transfer of the primer terminus between the polymerization domain and the editing domain and identify amino acid residues that facilitate transfer. 5. Determine whether Klenow fragment suppresses frameshift mutations by rejecting misaligned sequences within the polymerization domain. 6. Characterize the editing function of other DNA polymerases m order to test the generality of the principles of mismatch recognition established for Klenow fragment. Time-resolved fluorescence anisotropy decay of dansyl-labeled DNA will be used to measure the distribution of DNA termini bound to the polymerization or editing domains of Klenow fragment. The DNA and enzyme will be judiciously modified using oligonucleotide synthesis and site-directed mutagenesis techniques, respectively, and the effect on the distribution of DNA in each domain will be determined. Thermodynamic data describing binding of mismatched DNAs to each domain of the enzyme, and melting of mismatched DNA sequences, will also be obtained. Stopped-flow fluorescence techniques will be used to measure the rate of transfer of DNA between domains. The information obtained from this study will provide insight into the molecular mechanisms used to suppress mutations during DNA replication.