Mechanism of Mammalian Translesion DNA synthesis

描述（由申请人提供）：细胞DNA不断受到内源性和外源性诱变剂的破坏。本提案的长期目标是：(i)了解未修复的损伤如何诱发最终导致退行性疾病、衰老和癌症的突变，以及（ii）从而有助于尽量减少其遗传毒性后果。这些知识也可以用来最大化癌症化疗药物的疗效。最近的研究揭示了一种新的哺乳动物DNA聚合酶家族，它专门用于在未修复的DNA损伤上进行DNA合成。这些低保真聚合酶是pol 7， pol:, pol 9， pol 6和REV1。它们在突变诱导中起着核心作用，被认为对不同类型的DNA损伤都有活性。由于它们容易错误复制未受损的DNA，它们的活动必须受到严格的调节。为了研究它们在突变诱导中的作用及其调控机制，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，如专门的DNA聚合酶，其调控基因和DNA修复基因，被基因靶向灭活，从而专门研究感兴趣的基因的作用。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的实验将允许对翻译合成进行机制分析。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整合到质粒中；（iii）将修饰后的质粒导入小鼠宿主细胞；（iv）恢复子代质粒并分析病变部位的事件；(5)评价了基因失活对翻译合成的影响。利用这一策略以及其他已建立的技术，如使用纯化聚合酶的体外翻译合成实验，用于研究蛋白质-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. PUBLIC HEALTH RELEVANCE: Accumulation of DNA damage caused by endogenous, environmental and chemotherapeutic agents is suspected to contribute to ageing, degenerative diseases, and cancer. Hence, it is very important to reveal the cellular mechanism by which unrepaired DNA damage exerts genotoxic effects, such as point mutations and chromosome aberrations, and causes cell death.