Mechanisms of Fidelity and Mutagenesis

描述：(申请人提供)本项目的长期目标是 了解支配DNA复制的化学相互作用。这个 这项研究建议的短期目标是确定 聚合酶和DNA的小凹槽之间的氢键起作用 DNA复制的保真度。关键的交互作用控制着 DNA复制的保真度发生在几个 组成DNA合成的各个步骤。过渡态将是 通过结构活动关系进行了探讨。反应物的结构将 通过DNA的原子取代和氨基酸的取代 聚合酶，反应的进展将用 稳态前动力学。将研究两种聚合酶，DNA聚合酶I 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被很好地研究，从而提供了一种坚实的 为结构功能分析奠定基础。此外，正如酶显示的那样 结构差异，它们可能使用不同的保真度机制 控制力。以下具体假设将得到检验。(1)氢键 聚合酶和DNA的细沟之间对催化和 DNA复制的保真度。特别是，聚合酶β的Arg283使 与模板碱基的嘌呤的N3-位形成关键氢键。(2) 大肠杆菌DNA聚合酶I的Arg668作为正确几何构型的传感器 末端碱基对通过与鸟嘌呤和环氧的N3相互作用 传入的dNTP的。(3)聚合酶与辅酶的相互作用 DNA的凹槽取决于DNA的序列。(4)互动 在聚合酶和dTTP和dCTP的O2-位置之间是重要的，因为 核苷酸和蛋白质之间的空间相互作用。(5)低谷 Fidelity旁路聚合酶使用微小的沟槽相互作用来复制DNA。这个 这些实验的结果将提供非常具体的机械信息 聚合酶如何高保真地复制DNA。这一知识将增加 我们对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.