Mechanism of Mammalian Translesion DNA synthesis

描述（由申请方提供）：内源性和外源性诱变剂持续损伤细胞DNA。该提案的长期目标是：（一）了解未修复的损伤如何诱发突变，最终导致退行性疾病、衰老和癌症，以及（二）从而有助于尽量减少其遗传毒性后果。这些知识也可以用于最大化癌症化疗剂的功效。最近的研究揭示了一个新的哺乳动物DNA聚合酶家族，专门用于未修复的DNA损伤的DNA合成。这些低保真聚合酶是pol 7、pol：、pol 9、pol 6和REV 1。它们在突变诱导中发挥核心作用，并被认为对不同类型的DNA损伤具有活性。由于它们容易错误复制未受损的DNA，因此必须严格控制它们的活动。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，该方法包括三个主要部分：含有化学上确定的DNA损伤的DNA，在小鼠细胞中复制的质粒，和小鼠细胞，其特定基因，如用于特异性DNA聚合酶的基因，它们的调节基因和DNA修复基因，通过基因靶向而失活，从而具体研究了目的基因的作用。此外，将基因的突变形式引入基因敲除细胞以检查其功能互补的实验将允许对translesion合成进行机理分析。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掺入质粒中;（iii）将修饰的质粒引入小鼠宿主细胞中;（iv）回收后代质粒并分析损伤位点的事件;和（v）评估基因失活对跨损伤合成的影响。与此策略连同其他已建立的技术，如在体外translesion合成测定使用纯化的聚合酶，酵母双杂交测定研究蛋白质-蛋白质相互作用，和细胞内定位测定的聚合酶，哺乳动物诱变的机制进行了研究。 公共卫生相关性：由内源性、环境和化学治疗剂引起的DNA损伤的累积被怀疑有助于衰老、退行性疾病和癌症。因此，揭示未修复的DNA损伤发挥遗传毒性效应（如点突变和染色体畸变）并导致细胞死亡的细胞机制是非常重要的。

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.