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; 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（由应用程序提供）：透射合成（TLS）DNA聚合酶（POLS）有助于确保通过通过DNA病变促进复制来确保复制叉的持续进展。在拟议的研究中，我们将通过各种由环境污染物和致癌物诱导的多种DNA病变以及通过细胞氧化物损伤来确定许多人类TLS POL在促进复制中的作用。特别是，我们将测试以下假设：通过人类细胞中的DNA病变复制是通过两种不同的模式发生的，在这种模式下，polθ散发出其使用高度专业的方式取决于DNA病变的能力，而Polθ在更普遍的和错误的方式中执行病变旁路。此外，我们将检验以下假设：Polθ的较普遍和易于错误的作用从其插入嘌呤核苷酸（NT）的能力中散发出，优先是通过“蛋白质板”指导的机制的A相反的DNA病变。为了通过人类的DNA病变阐明无错误和诱变复制的遗传碱基，我们将进行以下研究。在AIM 1中，我们将研究各种TLS POLS对人和小鼠细胞中绕过无误与诱变病变的贡献。待研究的病变包括（6-4）由紫外线辐射引起的光产物，7,8-二氢-8-氧气（8-oxog）是由对DNA中的gunine in gunine in dna中的自由基攻击产生的，1，1，N6-乙烯氧化苯甲酸果碱（EDA）来自与脂质氧化剂的相互作用产生的，从而产生的脂质氧化剂，从而产生了脂质的氧化氧化剂的相互作用。致癌物，1，N2-丙烷-2'-脱氧鸟苷（PDG），这是一种由脂质过氧化产生的环蛋白的环形形式，是无处不在的环境污染物，是一种无处不在的环境污染物，以及N2-DG的n2-DG加合物，将环境癌（+）抗Benzo [a] pylene pylene diole dioldiol dioldiol diol dioldemonent（bpde）（bpde）（bp diol diolde）（bpde）（bpde）（bpde）（bpde）。在AIM 2中，将进行生化研究，以检查POLθ在合成相反（6-4）TT PhotoRoduct，8-oxog，ϵDa，PDG和N2-DG BPDE中的熟练程度。通过稳态动力学分析，我们将确定POLθ的催化效率，用于插入与DNA病变相对的核苷酸并进行随后的扩展反应，并将进行生化研究，以测试POLθ通过蛋白质模板脱离的机械插入纯NT的DNA病变的假说。我们在此提出的遗传和生化研究与描述TLS POL在复制过程中复制过程中促进TLS POL的作用高度相关，并为对环境和细胞DNA在人类细胞中诱导的诱变和细胞DNA诱导的遗传学生成的遗传生成的遗传生成具有全面的理解。我们对POL，η，η，〜，κ，Rev1和ζ的TLS主要无错误模式的建议将预测这些POL在癌症抑制中的作用，而Polθ的TLS诱变模式将预测该POL在增强基因组不稳定性和癌变中的作用。