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和抑制突变和致癌至关重要。DNA聚合酶d （Pold）是一种高保真度的聚合酶，在酵母向人类的复制过程中起着不可缺少的作用。酿酒酵母的Pold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。在小鼠和人类中，降低Pol3保真度的聚合酶或外切酶结构域的突变会导致癌症。例如，在癌细胞系和散发性结肠癌中已经发现了指向Pol3的突变。在这里，我们提出了结构，生化和遗传学的研究酵母原是理解这种高保真聚合酶的作用机制至关重要。我们将：1)确定Pol3在聚合和编辑模式下的晶体结构。这些结构将为Pol3对正确核苷酸的高选择性提供机制理解的基础，并将产生对其保真度基础的构象转变的见解。2)我们将利用结构信息制造突变，a)改变DNA合成的保真度，b)影响向编辑模式的过渡。总之，这些突变将测试从结构中推断出的特定假设，这些假设形成了核苷酸选择的基础，并有助于活性位点的转换。我们将通过生化和遗传手段来研究这些突变对波尔德功能的影响。3)为了了解Pol31和Pol32亚基对Pold结构和功能的贡献，我们将对Pold全酶进行生化和结构研究。Pol31和Pol32对DNA结合熟练度和poly合成DNA的速度的影响将被确定，并进行稳态前动力学分析，以确定聚合反应的不同步骤对poly的高保真度的贡献。此外，我们将确定与DNA和dNTP三元配合物的Pold全酶的结构。对Pol3和Pold全酶的三元结构进行比较，对于破译Pol31和Pol32对Pold功能的贡献具有重要意义。本文提出的结合结构、生物化学和遗传的方法对于定义波尔德聚合和校对功能的作用机制以及描述其高保真度的结构基础将是重要的。公共卫生相关性：本研究的目的是揭示DNA聚合酶4 （Pol4）高保真度的机制，这对维持基因组稳定性和抑制致癌作用至关重要。降低Pol4保真度的突变会导致小鼠和人类罹患癌症。

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. PUBLIC HEALTH RELEVANCE: The aim of this study is to uncover the mechanisms underlying the high fidelity of DNA polymerase 4 (Pol4), which is crucial for maintaining genomic stability and the suppression of carcinogenesis. Mutations that lower the fidelity of Pol4 cause cancers in mice and humans.