Mechanism of Mammalian Translesion DNA synthesis

描述(申请人提供)：细胞DNA受到内源和外源诱变剂的持续破坏。这项提议的长期目标是(I)了解未修复的损伤如何导致突变，最终导致退行性疾病、衰老和癌症，(Ii)从而有助于将其遗传毒性后果降至最低。这些知识也可以用来最大限度地发挥癌症化疗药物的疗效。最近的研究揭示了一个新的哺乳动物DNA聚合酶家族，它们专门合成未修复的DNA损伤的DNA。这些低保真度聚合酶是Pol7、Pol1、Pol9、Pol6和Rev1。它们在突变诱导中发挥着核心作用，被认为对不同类型的DNA损伤都很活跃。由于它们容易错误复制未受损的DNA，因此它们的活动必须受到严格监管。为了研究它们在突变诱导中的作用及其调控机制，将发展一种新的实验方法，该方法由三个主要部分组成：含有化学定义的DNA损伤的DNA、在小鼠细胞中复制的质粒和小鼠细胞，其中特定的基因，如特定DNA聚合酶的基因、调节基因和DNA修复基因，通过基因打靶来失活，从而专门研究目的基因的作用。此外，将突变版本的基因引入基因敲除细胞以检查其功能互补的实验，将允许对跨损伤合成进行机制分析。典型的实验将如下进行：(I)合成含有位点特异性DNA损伤的DNA；(Ii)将该修饰的DNA掺入到质粒中；(Iii)将修饰的质粒导入小鼠宿主细胞；(Iv)回收后代质粒并分析损伤部位的事件；以及(V)评估基因失活对跨损伤合成的影响。这一策略与其他成熟的技术，如利用纯化聚合酶的体外跨损伤合成实验，研究蛋白质-蛋白质相互作用的酵母双杂交实验，以及聚合酶的细胞内定位实验，将研究哺乳动物的诱变机制。

DESCRIPTION (provided by applicant): Cellular DNA is continuously damaged by endogenous and exogenous sources of mutagens. The long-term objectives of this proposal are (i) to understand how unrepaired damage induces mutations that ultimately lead to degenerative diseases, ageing and cancer, and (ii) thereby to contribute to minimize their genotoxic consequences. This knowledge can also be used to maximize the efficacy of cancer chemotherapeutic agents. Recent studies have revealed a new family of mammalian DNA polymerases that are specialized for a DNA synthesis across unrepaired DNA lesions. These low-fidelity polymerases are pol 7, pol :, pol 9, pol 6 and REV1. They play a central role in mutation induction and are thought to be active on different types of DNA lesions. Since they are prone to miscopy undamaged DNA, their activities must be regulated tightly. To study their roles in mutation induction and the mechanism of their regulation, a new experimental approach will be developed, which consists of three major components: DNA containing a chemically defined DNA damage, a plasmid that replicates in mouse cells, and mouse cells, specific genes of which, such as those for specialized DNA polymerases, their regulatory genes and DNA repair genes, are inactivated by gene targeting, thereby the role of the gene of interest is specifically investigated. In addition, experiments, where mutated versions of a gene are introduced into the gene knockout cells to examine their functional complementation, will allow the mechanistic analysis of a translesion synthesis. Typical experiments will be conducted as follows: (i) DNA containing a site-specific DNA lesion is synthesized; (ii) this modified DNA is incorporated into a plasmid; (iii) the modified plasmid is introduced into mouse host cells; (iv) progeny plasmid is recovered and analyzed for the events at the lesion site; and (v) the effect of the gene inactivation on a translesion synthesis is evaluated. With this strategy together with other established techniques such as the in vitro translesion synthesis assay using purified polymerases, the yeast two-hybrid assay for studying protein-protein interaction, and the intracellular localization assay of a polymerase, the mechanism of mammalian mutagenesis will be studied.