DNA Polymerase in Genome Maintenance and Tumorigenesis

描述(申请人提供)：尽管DNA损伤的移除和修复对于细胞和生物体的生存是必不可少的，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。一组新发现的DNA聚合酶有助于完成受损基因组的复制。这些酶插入核苷酸，与通常阻止复制叉子的病变相反。这一过程帮助细胞在来自内源和环境来源的遗传毒性侮辱中生存下来，但经常引入错误的碱基并具有突变性。突变的积累可能会导致细胞的肿瘤性转化和生物体中的癌症。在跨损伤合成过程中形成突变的一个关键酶是DNA聚合酶Zeta(Zeta)。这项研究的长期目标是了解哺乳动物Pol Zeta如何有助于有机体的生存和与癌症相关的遗传稳定性。编码Pol Zeta催化亚单位的小鼠Rev3L基因的破坏会导致胚胎怀孕中期的死亡。很可能Pol Zeta对发育是必不可少的，因为胚胎遵循严格定时的快速分裂程序，这依赖于Pol Zeta依赖的过程对DNA损伤的有效耐受。这项提案中的研究将使用能够分析培养细胞和成年细胞中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DNA损伤、生存能力受损和基因组不稳定有关。我们假设，在发育过程中缺乏Pol Zeta会导致活力的丧失，因为快速分裂的细胞会积累受阻的DNA复制叉和DNA断裂。我们已经分离出一种带有Rev3L基因中断的小鼠细胞系，现在可以测试这些预测。将分析Rev3L(-I-)细胞在整个细胞周期中的生长、活力和进展是否受损，对一系列DNA损伤剂的敏感性，通过光谱核型分析总的染色体排列，以及姐妹染色单体交换的频率。这些结果将表明哺乳动物Pol Zeta在DNA损伤和修复途径方面最重要的功能。目标2是确定哪些组织最依赖Rev3L来实现正常的发育和功能，这将指向该酶最重要的生物学作用。利用条件性基因破坏技术，小鼠细胞中的Pol Zeta将被降低并消除。一种带有Rev3L关键外显子的构建物两侧有loxP位点，将用于在成年组织中或在动物生命周期的特定时间干扰该基因。据预测，最依赖Pol Zeta功能的组织将是那些增殖最快的组织，或者是那些暴露于氧自由基或其他DNA损伤剂最高的组织。目的3是测试一种假设，即在没有POLZ的情况下能够存活的细胞类型的动物将会改变自发和DNA损伤诱导的肿瘤形成的发生率。如果我们的初步研究表明，在Pol Zeta(-I-)细胞中存在更多的染色体不稳定性，预计肿瘤的发病率将会增加。利用马赛克基因敲除小鼠和具有组织特异性Pol Zeta干扰的小鼠，将启动实验，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.