Mechanism of Mammalian Translesion DNA synthesis

描述（由申请人提供）：细胞 DNA 不断受到内源和外源诱变剂的损伤。该提案的长期目标是（i）了解未修复的损伤如何诱导突变，最终导致退行性疾病、衰老和癌症，以及（ii）从而有助于最大限度地减少其基因毒性后果。这些知识还可用于最大限度地发挥癌症化疗药物的功效。最近的研究揭示了一个新的哺乳动物 DNA 聚合酶家族，专门用于跨未修复的 DNA 损伤进行 DNA 合成。这些低保真聚合酶是 pol 7、pol :、pol 9、pol 6 和 REV1。它们在突变诱导中发挥着核心作用，并且被认为对不同类型的 DNA 损伤具有活性。由于它们很容易错误复制未受损的 DNA，因此必须严格监管它们的活动。为了研究它们在突变诱导中的作用及其调节机制，将开发一种新的实验方法，该方法由三个主要组成部分组成：含有化学定义的DNA损伤的DNA、在小鼠细胞中复制的质粒以及小鼠细胞中的特定基因，例如专门的DNA聚合酶、其调节基因和DNA修复基因，通过基因靶向失活，从而专门研究感兴趣基因的作用。此外，将基因的突变版本引入基因敲除细胞以检查其功能互补性的实验将允许对跨损伤合成进行机械分析。典型的实验将按如下方式进行： (i) 合成含有位点特异性 DNA 损伤的 DNA； (ii) 将该修饰的 DNA 掺入质粒中； (iii)将修饰的质粒引入小鼠宿主细胞； (iv) 回收子代质粒并分析病变部位的事件； (v)评估基因失活对跨损伤合成的影响。通过该策略与其他已建立的技术（例如使用纯化聚合酶的体外跨损伤合成测定、用于研究蛋白质-蛋白质相互作用的酵母双杂交测定以及聚合酶的细胞内定位测定）一起，将研究哺乳动物诱变的机制。

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.