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。特别地，我们将测试通过人类细胞中的DNA损伤的复制通过两种不同的模式发生的假设，其中Pos，η，ι，κ，Rev 1和β介导主要无错误的TLS，并以依赖于DNA损伤的高度专门化的方式起作用，而Polθ以更普遍和更容易出错的方式执行损伤旁路。此外，我们将检验这样的假设，即Polθ的更普遍和更容易出错的作用源自其插入嘌呤核苷酸（nt）的能力，优先插入A，通过“蛋白质模板”导向的机制对DNA损伤。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。目的二是研究Polθ在合成（6-4）TT光产物、8-oxoG、PdA、PdG和N2-dG BPDE中的活性。通过稳态动力学分析，我们将确定Polθ在DNA损伤对面插入核苷酸和进行随后的延伸反应的催化效率，并进行生物化学研究以检验Polθ通过蛋白质模板导向机制在DNA损伤对面插入嘌呤nt的假设。 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。我们提出的Pos、η、ι、κ、Rev 1和ε的TLS的主要无错误模式将预测这些Pos在癌症抑制中的作用，而Polθ的TLS的诱变模式将预测这种Pol在增强基因组不稳定性和致癌作用中的作用。

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, η, ι, κ, Rev1, and ζ mediate predominantly error-free TLS and act in a highly specialized manner dependent upon the DNA lesion, whereas Polθ 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 Polθ 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 Polθ in synthesizing DNA opposite (6-4) TT photoproduct, 8-oxoG, ϵdA, PdG, and N2-dG BPDE. By steady-state kinetic analyses we will determine the catalytic efficiency of Polθ 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 Polθ 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, η, ι, κ, Rev1, and ζ would predict a role for these Pols in cancer suppression, whereas a mutagenic mode of TLS by Polθ would predict a role for this Pol in enhancing genomic instability and carcinogenesis.