DNA Polymerase in Genome Maintenance and Tumorigenesis

描述（由申请人提供）：尽管DNA损伤的去除和修复对于细胞和生物体的生存至关重要，但所有细胞都可以在存在一些未修复的DNA损伤的情况下完成DNA复制。一组新识别的专门DNA聚合酶有助于完成受损基因组的复制。这些酶将核苷酸插入到通常会阻塞折叠叉的损伤处。该过程有助于细胞在来自内源性和环境来源的遗传毒性损伤中存活，但通常会引入不正确的碱基并具有致突变性。突变的积累可导致细胞的肿瘤转化和生物体中的癌症。在跨损伤合成期间形成突变的关键酶是DNA聚合酶zeta（zeta）。这项研究的长期目标是了解哺乳动物Pol zeta如何有助于生物体的生存和与癌症相关的遗传稳定性。破坏小鼠Rev 3L基因，编码Pol zeta的催化亚基，导致胚胎妊娠中期死亡。很可能Pol zeta对于发育是必不可少的，因为胚胎遵循严格定时的快速分裂程序，该程序依赖于Pol zeta依赖过程对DNA损伤的有效耐受。该提案中的研究将使用允许分析培养细胞和成人细胞中Pol zeta功能的方法。该项目的具体目标：目标1是测试哺乳动物细胞中Pol zeta的缺失是否与对DNA损伤的超敏反应、活力受损和基因组不稳定性有关。我们推测，在发育过程中缺乏Pol zeta导致活力丧失，因为快速分裂的细胞积累了受阻的DNA复制叉和DNA断裂。我们已经分离出一种具有Rev 3L基因破坏的小鼠细胞系，现在可以测试这些预测。将分析Rev 3L（-I-）细胞的生长受损、活力和细胞周期进展、对一系列DNA损伤剂的敏感性、光谱核型分析的总体染色体排列和姐妹染色单体交换频率。这些结果将表明哺乳动物Pol zeta在DNA损伤和修复途径方面的最重要功能。目的2是确定哪些组织最依赖于Rev 3L的正常发育和功能，这将指向该酶最重要的生物学作用。使用条件性基因破坏技术将在小鼠细胞中降低和消除Pol zeta。具有侧接LoxP位点的Rev 3L的关键外显子的构建体将用于在成体组织中或在动物寿命期间的特定时间破坏基因。据预测，最依赖于Pol zeta功能的组织将是最快速增殖的组织，或最高暴露于氧自由基或其他DNA损伤剂的组织。目的3是检验以下假设：具有可在缺乏Pol z的情况下存活的细胞类型的动物将具有改变的自发性和DNA损伤诱导的肿瘤发生率。如我们的初步研究所示，如果Pol zeta（-I-）细胞中存在更多的染色体不稳定性，则预测肿瘤发病率将增加。使用嵌合敲除小鼠和具有组织特异性Pol zeta破坏的小鼠，将开始实验以测量自发和诱导肿瘤的发生率。

DESCRIPTION (provided by applicant): Although the removal and repair of DNA damage is essential for the survival of cells and organisms, all cells can complete DNA replication in the presence of some unrepaired DNA damage. A specialized group of newly recognized DNA polymerases assist in accomplishing replication of damaged genomes. These enzymes insert nucleotides opposite lesions that normally block eplication forks. The process helps cells survive genotoxic insults from both endogenous and environmental sources, but often introduces incorrect bases and is mutagenic. Accumulation of mutations can lead to neoplastic transformation of a cell and cancer in an organism. A key enzyme in forming mutations during translesion synthesis is DNA polymerase zeta (zeta). The long-term objectives of this research are to understand how mammalian Pol zeta contributes to survival of the organism and to genetic stability in relation to cancer. Disruption of the mouse Rev3L gene, encoding the catalytic subunit of Pol zeta, leads to lethality midway through embryonic gestation. It is likely that Pol zeta is essential for development because embryos follow a strictly timed program of rapid division that relies on effective tolerance of DNA lesions by a Pol zeta-dependent process. The research in this proposal will use methods that allow analysis of Pol zeta function in cultured and adult cells. The specific aims of this project: Aim 1 is to test whether the absence of Pol zeta in mammalian cells is associated with hypersensitivity to DNA damage, impaired viability and genome instability. We hypothesize that a lack of Pol zeta during development causes a loss of viability as rapidly dividing cells accumulates blocked DNA replication forks and DNA breaks. We have isolated a mouse cell line with a genetic disruption of Rev3L, making it now possible to test these predictions. Rev3L (-I-) cells will be analyzed for impaired growth, viability, and progression through the cell cycle, sensitivity to a range of DNA damaging agents, gross chromosomal arrangements by spectral karyotyping, and frequency of sister chromatid exchange. The results will indicate the most important functions of mammalian Pol zeta with respect to DNA lesions and repair pathways. Aim 2 is to determine which tissues are most dependent on Rev3L for normal development and function, which will point towards the most important biological roles of the enzyme. Pol zeta will be lowered and eliminated in cells of a mouse using conditional gene disruption technology. A construct having key exons of Rev3L flanked by LoxP sites will be used to disrupt the gene in adult tissues or at specific times during the lifespan of the animal. It is predicted that the tissues most dependent on Pol zeta function will be those most rapidly proliferating, or with the highest exposure to oxygen free- radicals or other DNA damaging agents. Aim 3 is to test the hypothesis that animals with cell types, which can survive in the absence of Pol z will have an altered incidence of spontaneous, and DNA damage-induced tumorigenesis. If there is more chromosomal instability in Pol zeta (-I-) cells as indicated by our preliminary studies, it is predicted that tumor incidence will be increased. Using mosaic knockout mice and mice with tissue-specific disruptions of Pol zeta, experiments will be initiated to measure the incidence of spontaneous and induced tumors.