Cellullar Response to DNA Adducts

描述（由申请人提供）：未修复DNA损伤的DNA合成，称为跨损伤DNA合成（TLS），是化学诱变的主要机制之一;在人类中，该过程与癌症和年龄相关性退行性疾病的发生有关。这项研究的目的是阐明TLS在人类细胞中发生的机制。近年来发现了几种TLS特异性DNA聚合酶（polymerases，pol），如pol h、pol k、pol i、pol z和REV 1，我们将确定这些酶在人类细胞TLS中的作用。两种环外丙脱氧鸟苷（PdG）DNA加合物和1，N6-乙烯脱氧腺苷（edA）将用作靶病变。我们已经建立了这些加合物在人类细胞中的遗传毒性，使用一个独特的穿梭载体系统在我的实验室开发。为了探索细胞中TLS的机制，将首先进行纯化聚合酶的引物延伸研究，以确定其体外效率，保真度和编码特异性。通过比较体内和体外结果，将鉴定负责细胞中TLS事件的候选聚合酶。候选聚合酶基因在人类细胞中的表达将通过RNA干扰技术沉默。然后将此类“工程化”细胞用作遗传毒性分析的宿主。通过比较“野生型”和工程化细胞中的TLS事件，可以评估候选聚合酶在TLS中的作用和功能。工程细胞的提取物将用于体外复制修饰的质粒，用于生化研究。体外TLS研究、体内遗传毒性试验和RNA干扰技术的结合将为TLS聚合酶的基本作用和功能及其对人类细胞化学诱变的贡献提供新的见解。

DESCRIPTION (provided by applicant): DNA synthesis across unrepaired DNA damage, known as translesion DNA synthesis (TLS), is one of the major mechanisms responsible for chemical mutagenesis; in humans this process is associated with the development of cancer and age-related degenerative diseases. The goal of this research is to elucidate the mechanisms of TLS as they occur in human cells. Several TLS-specialized DNA polymerases (pol), such as pol h, pol k, pol i, pol z and REV1, have been discovered recently; we will determine the role of these enzymes in TLS in human cells. Two exocyclic propanodeoxyguanosine (PdG) DNA adducts and 1,N6-ethenodeoxyadenosine (edA) will be employed as target lesions. We have established the genotoxicity of these adducts in human cells, using a unique shuttle vector system developed in my laboratory. To probe the mechanism for TLS in cells, primer extension studies with purified polymerases will be first conducted to determine their efficiency, fidelity, and coding specificity in vitro. By comparing in vivo and in vitro results, candidate polymerase(s) responsible for TLS events in cells will be identified. The expression of the candidate polymerase gene(s) in human cells will be silenced by RNA interference technology. Such "engineered" cells are then used as hosts for genotoxic analyses. By comparing TLS events in "wild type" and engineered cells, the role and function of candidate polymerase(s) in TLS can be assessed. Extracts of engineered cells will be used to replicate modified plasmid in vitro for biochemical studies. The combination of in vitro TLS studies, in vivo assays for genotoxicity, and RNA interference technology will provide novel insights into the fundamental role and function(s) of TLS polymerases and their contribution to chemical mutagenesis in human cells.