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

通过人类 DNA 损伤对复制的遗传控制以及致癌作用

• 批准号: 8974412
• 负责人: SATYA PRAKASH
• 金额: $ 34.43万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-27 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8974412
• 关键词: Acrolein; Adenine; Benzo(a)pyrene; Biochemical; Biochemical Reaction; Biological; Bypass; Cells; DNA; DNA Damage; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; Deoxyguanosine; Ensure; Environmental Carcinogens; Environmental Pollutants; Epoxy Compounds; Exposure to; Family; Free Radicals; Frequencies; Genes; Genetic; Genomic Instability; Glycols; Guanine; Human; Kinetics; Lesion; Lipid Peroxidation; Malignant Neoplasms; Mediating; Mus; Mutagenesis; Mutation; Nucleotides; Pathway interactions; Plasmids; Play; Polymerase; Proteins; Purine Nucleotides; Pyrenes; Role; Scheme; Simian virus 40; Small Interfering RNA; System; Testing; adduct; base; carcinogenesis; chemical carcinogen; in vivo; oxidative damage; ultraviolet irradiation

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.

描述（由申请人提供）：跨损伤合成 (TLS) DNA 聚合酶 (Pol) 通过 DNA 损伤促进复制，有助于确保复制叉的持续进展。在拟议的研究中，我们将确定许多人类 TLS Pol 的作用。通过环境污染物和致癌物以及细胞氧化损伤引起的各种 DNA 损伤促进复制。特别是，我们将测试人类细胞中通过 DNA 损伤进行复制通过两种不同模式发生的假设。 Pols、η、ι、κ、Rev1 和 z 主要介导无错误 TLS，并根据 DNA 损伤以高度专业化的方式起作用，而 Polθ 以更通用且容易出错的方式执行损伤旁路。检验以下假设：Polθ 更普遍且更容易出错的作用源自其通过“蛋白质模板”定向插入嘌呤核苷酸 (nt)（优先是 A）相对 DNA 损伤的能力为了阐明人类通过 DNA 损伤实现无错误和诱变复制的遗传基础，我们将在目标 1 中研究各种 TLS Pols 对无错误与诱变病变旁路的贡献。人类和小鼠细胞中待研究的损伤包括由紫外线照射诱导的 (6-4) 光产物，即自由基产生的 7,8-二氢-8-氧代古嘌呤 (8-oxoG)。攻击 DNA 中的鸟嘌呤，1,N6-乙脱氧腺苷 (edA) 是由腺嘌呤与细胞氧化损伤和接触化学致癌物导致的脂质过氧化产物相互作用产生的，1,N2-丙-2'-脱氧鸟苷 (PdG)，由脂质过氧化产生的丙烯醛的闭环形式，也是一种普遍存在的环境污染物，目标 2 中，将进行生化研究以检验 Polθ 合成相反 (6-4) TT 的 DNA 的能力。光产物、8-oxoG、ϵdA、PdG 和 N2-dG BPDE 通过稳态动力学分析，我们将确定 BPDE 的催化效率。 Polθ用于在DNA损伤对面插入核苷酸并进行随后的延伸反应，并且将进行生化研究以检验Polθ通过蛋白质模板引导的机制在DNA损伤对面插入嘌呤nt的假设。我们在此提出的建议与描述 TLS Pols 在复制过程中促进无错误与诱变病变旁路的作用高度相关，并提供对环境和细胞 DNA 诱导的诱变和致癌作用的遗传基础的全面理解我们提出的 Pols、η、ι、κ、Rev1 和 z 的 TLS 主要无差错模式将预测这些 Pols 在癌症抑制中的作用，而 Polθ 的 TLS 诱变模式将预测这些 Pols 在癌症抑制中的作用。预测该 Pol 在增强基因组不稳定性和致癌作用中的作用。