Fidelity of DNA Synthesis

摘要 波尔-？催化人类基因组的复制和其他DNA合成过程 细胞然而，Pol-？在复制中，在内源性和 环境破坏，以及重组仍有待确定。此外，我们缺乏 信息的作用，体细胞突变在波尔？在肿瘤发生和其它疾病中 流程.我们的目标是确定的功能，波尔？在DNA合成和 可能的作用，增加突变的Pol-？在人类癌症的产生过程中。一个主要 方法将是利用突变体的Pol-？其掺入诱变核苷酸 类似物，从而作为识别Pol-？合成的DNA的工具。in vivo.我们 有四个具体目标。在目标1，我们将产生突变体的Pol-？增加 特定核苷酸类似物的掺入。在目标2中，我们将纯化野生型， 突变的Pol-？全酶复合物，并详细表征其催化性能， 包括它们的保真度和类似物掺入的动力学。在目标3中，我们将定义 Pol的角色-？在哺乳动物细胞中引入突变的Pol-？是因为 掺入诱变核苷酸类似物并测量细胞中诱导的突变 进行DNA复制、修复和重组。诱发的突变将 鉴定由Pol-？合成的DNA。在目标4中，我们将评估加强 通过进行系列转移实验， 确定携带突变基因Pol-的哺乳动物细胞具有竞争优势， 以及是否存在削弱这种优势的核苷类似物。叙事 我们的目标是建立DNA聚合酶的作用-？在复制人类 基因组和修复由内源性和环境因素引起的损伤。我们 将决定DNA聚合酶的突变促进遗传不稳定性， 加速模型系统中的肿瘤进展。我们的目标是确定一类新的 化疗药物将延缓肿瘤生长。

Abstract Pol-? catalyzes replication of the genome and other DNA synthetic processes in human cells. However, the precise functions of Pol-? in replication, in repair of endogenous and environmental damage, and in recombination remain to be defined. Further, we lack information on the role of somatic mutations in Pol-? in tumorigenesis and other disease processes. Our goals are to identify the functions of Pol-? in DNA synthesis and the possible role of increased mutation by Pol-? in the generation of human cancer. A major approach will be to exploit mutants of Pol-? that incorporate mutagenic nucleotide analogs and thereby serve as tools to identify DNA synthesized by Pol-? in vivo. We have four specific aims. In Aim 1, we will generate mutants of Pol-? that increase incorporation of a specific nucleotide analog(s). In Aim 2, we will purify wild-type and mutant Pol-? holoenzyme complexes and characterize their catalytic properties in detail, including their fidelity and kinetics of analog incorporation. In Aim 3, we will define the roles of Pol-? in mammalian cells by introducing mutant Pol-?'s that preferentially incorporate mutagenic nucleotide analogs and measuring induced mutation in cells undergoing DNA replication, repair and recombination. The induced mutations will identify the DNA synthesized by Pol-?. In Aim 4, we will assess the role of increased mutagenesis in tumor progression by performing serial transfer experiments to determine if mammalian cells that harbor mutator Pol-? have a competitive advantage, and if there are nucleoside analogs that diminish this advantage. Narrative Our objective is to establish the roles of DNA polymerase-? in replication of the human genome and in repair of damage caused by endogenous and environmental agents. We will determine if mutations in DNA polymerase-? promote genetic instability and accelerate tumor progression in model systems. We aim to identify a new class of chemotherapeutic agents that will retard tumor growth.