Cellullar Response to DNA Adducts

描述(由申请人提供)：未经修复的DNA损伤中的DNA合成，称为跨病变DNA合成(TLS)，是导致化学突变的主要机制之一；在人类中，这一过程与癌症和年龄相关退行性疾病的发展有关。这项研究的目的是阐明TLS在人类细胞中发生的机制。最近发现了几种TLS专化DNA聚合酶，如polh、polk、polI、polz和Rev1，我们将确定这些酶在人类细胞TLS中的作用。两个外环丙基脱氧鸟苷(PDG)DNA加合物和1，N6-乙基脱氧腺苷(EDA)将被用作靶点。我们使用我实验室开发的一种独特的穿梭载体系统，已经确定了这些加合物在人类细胞中的遗传毒性。为了探讨TLS在细胞中的作用机制，将首先用纯化的聚合酶进行引物延伸研究，以确定它们的体外效率、保真度和编码特异性。通过比较体内和体外的结果，将确定负责细胞内TLS事件的候选聚合酶(S)。候选聚合酶基因(S)在人类细胞中的表达将通过核糖核酸干扰技术沉默。这样的“工程”细胞随后被用作遗传毒性分析的宿主。通过比较“野生型”和工程细胞中的TLS事件，可以评估候选聚合酶(S)在TLS中的作用和功能。工程细胞的提取液将用于在体外复制修改后的质粒，用于生化研究。体外TLS研究、体内遗传毒性检测和RNA干扰技术的结合，将为TLS聚合酶的基本作用和功能(S)及其在人类细胞化学诱变中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.