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重组中的确切功能仍有待确定。此外，我们缺乏关于Pol-4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突变增加在人类癌症产生中的可能作用。一个主要的方法将是利用含有诱变核苷酸类似物的Pol-4突变体，从而作为鉴定体内Pol-4合成的DNA的工具。我们有四个具体目标。在目标1中，我们将生成Pol-4突变体，增加特定核苷酸类似物的掺入。在Aim 2中，我们将纯化野生型和突变型Pol-4全酶复合物，并详细表征它们的催化性能，包括它们的保真度和模拟物掺入动力学。在Aim 3中，我们将通过引入优先包含诱变核苷酸类似物的突变Pol-4，并在DNA复制、修复和重组的细胞中测量诱导突变，来定义Pol-4在哺乳动物细胞中的作用。诱导突变将识别Pol-4合成的DNA。在Aim 4中，我们将通过进行一系列转移实验来评估增加的突变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，以及是否存在核苷类似物削弱这种优势。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内源性和环境因子引起的损伤修复中的作用。我们将确定DNA聚合酶-4的突变是否会促进遗传不稳定并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.