Fidelity of DNA Synthesis

描述(由申请人提供)：POL-4催化人类细胞中基因组的复制和其他DNA合成过程。然而，Pol-4在复制、修复内源性和环境损伤以及在重组中的确切功能仍有待确定。此外，我们缺乏关于POL-4的体细胞突变在肿瘤发生和其他疾病过程中的作用的信息。我们的目标是确定Pol-4在DNA合成中的功能，以及Pol-4增加突变在人类癌症发生中的可能作用。一个主要的方法将是利用POL-4的突变体，它结合了诱变的核苷酸类似物，从而作为工具来鉴定体内由POL-4合成的DNA。我们有四个具体目标。在目标1中，我们将产生POL-4的突变体，以增加特定核苷酸类似物的掺入(S)。在目标2中，我们将纯化野生型和突变型Pol-4全酶复合体，并详细表征它们的催化性质，包括它们的保真度和类似物掺入的动力学。在目标3中，我们将通过引入优先整合突变核苷酸类似物的突变体POL-4‘S，并在经历DNA复制、修复和重组的细胞中检测诱导突变，来确定POL-4在哺乳动物细胞中的作用。诱导的突变将鉴定由POL-4合成的DNA。在目标4中，我们将通过进行系列转移实验来评估突变增加在肿瘤进展中的作用，以确定携带突变子Pol-4的哺乳动物细胞是否具有竞争优势，以及是否存在削弱这种优势的核苷类似物。公共卫生相关性：我们的目标是确定DNA聚合酶-4在人类基因组复制和修复内源性和环境因素造成的损害中的作用。我们将在模型系统中确定DNA聚合酶-4的突变是否会促进遗传不稳定和加速肿瘤进展。我们的目标是找到一类新的化疗药物，以延缓肿瘤的生长。

DESCRIPTION (provided by applicant): Pol-4 catalyzes replication of the genome and other DNA synthetic processes in human cells. However, the precise functions of Pol-4 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-4 in tumorigenesis and other disease processes. Our goals are to identify the functions of Pol-4 in DNA synthesis and the possible role of increased mutation by Pol-4 in the generation of human cancer. A major approach will be to exploit mutants of Pol-4 that incorporate mutagenic nucleotide analogs and thereby serve as tools to identify DNA synthesized by Pol-4 in vivo. We have four specific aims. In Aim 1, we will generate mutants of Pol-4 that increase incorporation of a specific nucleotide analog(s). In Aim 2, we will purify wild-type and mutant Pol-4 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-4 in mammalian cells by introducing mutant Pol-4'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-4. 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-4 have a competitive advantage, and if there are nucleoside analogs that diminish this advantage. PUBLIC HEALTH RELEVANCE: Our objective is to establish the roles of DNA polymerase-4 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-4 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.