Structural bases of high fidelity of DNA polymerase delta

描述（由申请人提供）：准确的DNA复制对于维持基因组稳定性以及抑制诱变和致癌作用至关重要。DNA聚合酶d（Pod）是一种高保真聚合酶，在从酵母到人类的复制中起着不可或缺的作用。从酵母S.酿酒酵母由三个亚基Pol3、Pol31和Pol32组成。Pol3是全酶的催化亚基，编码聚合酶和3'至5'核酸外切酶校对功能。Pol3的聚合酶或核酸外切酶结构域的突变降低了Pod的保真度，导致小鼠和人类癌症。例如，已经在癌细胞系和散发性结肠癌中鉴定了映射到Pol3的突变。在这里，我们提出了结构，生物化学和遗传学研究酵母Pod是至关重要的了解这种高保真聚合酶的作用机制。我们将：1）确定Pol3在聚合和编辑模式下的晶体结构。这些结构将提供对Pol3对正确核苷酸的高选择性的基础的机械理解，并将深入了解其保真度的构象转变。2)我们将使用结构信息进行突变，a）改变DNA合成的保真度，和B）影响向编辑模式的转变。总之，这些突变将测试从结构推断的特定假设，以形成核苷酸选择的基础并有助于活性位点转换。我们将通过生物化学和遗传学手段研究这些突变对Pod功能的影响。3)为了了解Pol31和Pol32亚基对Pod结构和功能的贡献，我们将对Pod全酶进行生化和结构研究。将确定Pol31和Pol32对DNA结合能力和Pold合成DNA的持续合成能力的影响，并进行预稳态动力学分析，以确定聚合反应的不同步骤对Pold高保真度的贡献。此外，我们还将测定Pold全酶与DNA和dNTP三元复合物的结构。Pol3和Pod全酶的三元结构的比较对于破译Pol31和Pol32对Pod功能的贡献将是非常宝贵的。结合结构，生物化学和遗传的方法，这里提出的将是重要的定义的行动机制，波德的聚合和校对功能，并划定其高保真度的结构基础。

DESCRIPTION (provided by applicant): Accurate DNA replication is crucial for the maintenance of genomic stability and for the suppression of mutagenesis and carcinogenesis. DNA polymerase d (Pold) is a high fidelity polymerase that plays an indispensable role in replication from yeast to humans. Pold from the yeast S. cerevisiae is comprised of three subunits, Pol3, Pol31, and Pol32. Pol3 is the catalytic subunit of the holoenzyme, encoding both the polymerase and the 3' to 5' exonuclease proofreading functions. Mutations in either the polymerase or the exonuclease domain of Pol3 that lower the fidelity of Pold cause cancers in mice and humans. For example, mutations that map to Pol3 have been identified in cancer cell lines and in sporadic colon cancers. Here, we propose structural, biochemical, and genetic studies on yeast Pold that are crucial for understanding the action mechanisms of this high fidelity polymerase. We will: 1) Determine the crystal structures of Pol3 in the polymerizing and editing modes. The structures will provide a mechanistic understanding of the basis for the high selectivity of Pol3 for the correct nucleotide, and will yield insights into the conformational transitions that underlie its fidelity. 2) We will use the structural information to make mutations that a) alter the fidelity of DNA synthesis, and b) affect the transition to the editing mode. Together, these mutations will test specific hypotheses that are inferred from the structures to form the basis of nucleotide selection and to contribute to active site switching. We will examine the effects of these mutations on Pold function by both biochemical and genetic means. 3) To understand the contributions that the Pol31 and Pol32 subunits make to Pold structure and function, we will carry out biochemical and structural studies on the Pold holoenzyme. The effects of Pol31 and Pol32 on the DNA binding proficiency and on the processivity of DNA synthesis by Pold will be determined, and pre-steady state kinetic analyses will be carried out to identify the contributions that different steps of the polymerization reaction make to the high fidelity of Pold. In addition, we will determine the structure of Pold holoenzyme in ternary complex with DNA and dNTP. A comparison of the ternary structures of Pol3 and Pold holoenzyme will be invaluable for deciphering the contributions of Pol31 and Pol32 to Pold function. The combined structural, biochemical, and genetic approaches proposed here will be important for defining the action mechanisms of Pold's polymerizing and proofreading functions and for delineating the structural bases of its high fidelity.