Genetic control of replication through DNA lesions in humans, and carcinogenesis

描述（由申请人提供）：转录合成（TLS）DNA聚合酶（Pos）通过促进DNA损伤的复制，帮助确保复制叉的持续进展。在拟议的研究中，我们将确定一些人类TLS Pol通过环境污染物和致癌物以及细胞氧化损伤诱导的各种DNA损伤在促进复制中的作用。特别是，我们将测试的假设，即复制通过DNA损伤在人类细胞中发生通过两种不同的模式，其中Pos，？，?, k，Rev 1，和？介导的主要是无错TLS和行动在一个高度专业化的方式依赖于DNA损伤，而Pol？以更普遍和更容易出错的方式执行病变旁路。此外，我们将测试的假设，更普遍和容易出错的作用波尔？源自其通过“蛋白质-模板”导向机制插入嘌呤核苷酸（nt），优选A，相对于DNA损伤的能力。为了阐明通过人类DNA损伤进行无错误和致突变复制的遗传基础，我们将进行以下研究。在目标1中，我们将检查各种TLS Pol对人类和小鼠细胞中无错误与致突变病变旁路的贡献。待研究的损伤包括（6- 4）由UV照射诱导的光产物，由自由基攻击DNA中的鸟嘌呤产生的7，8-二氢-8-氧代鸟嘌呤（8-oxoG），由腺嘌呤与由细胞氧化损伤和暴露于化学致癌物引起的脂质过氧化产物相互作用产生的1，N6-亚乙基脱氧腺苷（edA），1，N2-丙-2 ′-脱氧鸟苷（PdG），由脂质过氧化作用产生的丙烯醛的闭环形式，也是普遍存在的环境污染物，以及环境致癌物（+）抗苯并[a]芘二醇二氧化物（BPDE）的N2-dG加合物。在目标2中，将进行生化研究，以检查Pol？与（6-4）TT光产物、8-oxoG、edA、PdG和N2-dG BPDE合成DNA相反。通过稳态动力学分析，我们将确定的催化效率的波尔？插入核苷酸对DNA损伤，并进行随后的延伸反应，和生化研究将进行测试的假设，Pol？通过蛋白质模板导向的机制在DNA损伤的对面插入嘌呤核苷酸。 我们在这里提出的遗传和生物化学研究是高度相关的描绘TLS Pos的作用，在复制过程中促进无错误与诱变损伤旁路，并提供了一个全面的理解的遗传基础的诱变和致癌诱导的环境和细胞DNA损伤剂在人类细胞。我们的建议，一个主要的无错误模式的TLS的波尔斯？，?, k，Rev 1，和？将预测的作用，这些聚合物在癌症抑制，而诱变模式的TLS聚合物？将预测这种Pol在增强基因组不稳定性和致癌作用中的作用。 公共卫生相关性：DNA损伤是由细胞氧化损伤以及暴露于化学和环境致癌物而在人类细胞中产生的。确定各种DNA聚合酶在人类细胞复制过程中促进无错误与诱变损伤旁路的作用对于全面了解环境和细胞DNA损伤剂诱导的诱变和致癌的遗传基础是重要的。

DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (Pols) help ensure the continued progression of the replication fork by promoting replication through DNA lesions. In the proposed studies, we will determine the roles of a number of human TLS Pols in promoting replication through a variety of DNA lesions induced by environmental pollutants and carcinogens and by cellular oxidative damage. In particular, we will test the hypothesis that replication through DNA lesions in human cells occurs via two distinct modes in which Pols, ?, ?, k, Rev1, and ? mediate predominantly error-free TLS and act in a highly specialized manner dependent upon the DNA lesion, whereas Pol? performs lesion bypass in a more generalized and error-prone manner. Further, we will test the hypothesis that the more generalized and error-prone role of Pol? emanates from its ability to insert a purine nucleotide (nt), preferentially an A, opposite DNA lesions via a "protein-template"-directed mechanism. To elucidate the genetic bases of error-free and mutagenic replication through DNA lesions in humans, we will carry out the following studies. In Aim 1, we will examine the contributions of various TLS Pols to error-free vs. mutagenic lesion bypass in human and mouse cells. The lesions to be studied include (6- 4) photoproduct induced by UV irradiation, 7,8-dihydro-8-oxogunaine (8-oxoG) generated from free-radical attack on guanine in DNA, 1,N6-ethenodeoxyadenosine (edA) generated from interaction of adenine with products of lipid peroxidation resulting from cellular oxidative damage and from exposure to chemical carcinogens, 1,N2-propano-2'-deoxyguanosine (PdG), a ring-closed form of acrolein generated from lipid peroxidation and which also is a ubiquitous environmental pollutant, and N2-dG adduct of the environmental carcinogen (+) anti-benzo[a]pyrene diol expoxide (BPDE). In Aim 2, biochemical studies will be done to examine the proficiency of Pol? in synthesizing DNA opposite (6-4) TT photoproduct, 8-oxoG, edA, PdG, and N2-dG BPDE. By steady-state kinetic analyses we will determine the catalytic efficiency of Pol? for inserting nucleotides opposite the DNA lesion and for carrying out the subsequent extension reaction, and biochemical studies will be done to test the hypothesis that Pol? inserts a purine nt opposite DNA lesions via a protein- template-directed mechanism. The genetic and biochemical studies we propose here are highly relevant for delineating the roles of TLS Pols in promoting error-free vs. mutagenic lesion bypass during replication, and for providing a comprehensive understanding of the genetic bases of mutagenesis and carcinogenesis induced by environmental and cellular DNA damaging agents in human cells. Our proposal for a predominantly error-free mode of TLS by Pols ?, ?, k, Rev1, and ? would predict a role for these Pols in cancer suppression, whereas a mutagenic mode of TLS by Pol? would predict a role for this Pol in enhancing genomic instability and carcinogenesis. PUBLIC HEALTH RELEVANCE: DNA lesions are generated in human cells from cellular oxidative damage and from exposure to chemical and environmental carcinogens. The determination of roles of various DNA polymerases in promoting error-free vs. mutagenic lesion bypass during replication in human cells is important for providing a comprehensive understanding of the genetic bases of mutagenesis and carcinogenesis induced by environmental and cellular DNA damaging agents.