Mechanisms of Fidelity and Mutagenesis

描述：（由申请人提供）本项目的长期目标是 了解控制DNA复制的化学相互作用。的 本研究建议的短期目标是确定 聚合酶和DNA小沟之间的氢键 DNA复制的保真度。关键的相互作用，控制 DNA复制的保真度发生在几个过渡态中， 包括DNA合成的各个步骤。过渡状态将是 通过构效关系探讨。反应物的结构将 通过DNA的原子取代和氨基酸的取代来改变 聚合酶，并且反应的进展将用 前稳态动力学将研究两种聚合酶， E.大肠杆菌和哺乳动物DNA聚合酶β。DNA的生化机制 这些蛋白质的合成已经得到了很好的研究，从而提供了一个坚定的 结构功能分析的基础。此外，由于酶显示 由于结构上的差异，它们可能利用不同的保真度机制 控制将检验以下具体假设。(1)氢键 聚合酶和DNA小沟之间的相互作用对催化作用至关重要， DNA复制的保真度特别地，聚合酶β的Arg 283使得 与模板碱基的嘌呤的N3-位的关键氢键。（二） Arg 668为E.大肠杆菌DNA聚合酶I作为一个传感器的正确几何形状， 末端碱基对通过与鸟嘌呤的N3和环氧相互作用 dNTP的传入。(3)聚合酶与小分子之间的相互作用 DNA的沟取决于DNA的序列。(4)的相互作用 聚合酶与dTTP和dCTP的O2-位置之间的相互作用是重要的， 核苷酸和蛋白质之间的空间相互作用。(5)低 保真度旁路聚合酶使用小沟相互作用来复制DNA。的 这些实验的结果将提供非常具体的机理信息 聚合酶是如何高保真复制DNA的这些知识将有助于 DNA复制的基础知识

DESCRIPTION: (PROVIDED BY APPLICANT) The long-term goal of this project is to understand the chemical interactions that govern replication of DNA. The short-term object of this research proposal is to determine the role that hydrogen bonding, between the polymerase and the minor groove of the DNA, plays in the fidelity of DNA replication. The critical interactions that control the fidelity of DNA replication occur in the transition states of several of the individual steps that comprise DNA synthesis. The transition states will be probed by structure activity relationships. The structure of the reactants will be altered by atomic substitution of the DNA and amino acid substitution of the polymerases, and the progress of the reaction will be measured with pre-steady-state kinetics. Two polymerases will be studied, DNA polymerase I of E. coli and mammalian DNA polymerase beta. The biochemical mechanisms of DNA synthesis by these proteins have been well studied, thus providing a firm foundation for structure-function analyses. Moreover, as the enzymes display structural differences, they may utilize different mechanisms for fidelity control. The following specific hypotheses will be tested. (1) Hydrogen bonds between polymerase and the minor groove of DNA are crucial to catalysis and fidelity of DNA replication. In particular, Arg283 of polymerase beta makes a crucial hydrogen bond to the N3-position of a purine of the template base. (2) Arg668 of E. coli DNA polymerase I acts as a sensor for the correct geometry at the terminal base pair through interactions with N3 of guanine and ring oxygen of the incoming dNTP. (3) The interactions between polymerases and the minor groove of DNA are dependent on the sequence of the DNA. (4) The interactions between polymerases and the O2-positions of dTTP and dCTP are important due to steric interactions between the nucleotide and the protein. (5) The low fidelity bypass polymerases use minor groove interactions to replicate DNA. The results of these experiments will provide very specific mechanistic information of how polymerases replicate DNA with high fidelity. This knowledge will add to our fundamental knowledge of DNA replication.