Translesion DNA synthesis in humans

人类跨损伤 DNA 合成

• 批准号: 8049745
• 负责人: LOUISE PRAKASH
• 金额: $ 36.66万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8049745
• 关键词: Attenuated; Benzo(a)pyrene; Binding; Binding Sites; Biochemical; Biological; Bypass; Cancer Etiology; Cell Line; Cells; Complex; DNA Adducts; DNA Damage; DNA Photolyase; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Deoxyguanosine; Elements; Embryo; Ensure; Environmental Carcinogens; Environmental Pollutants; Epoxy Compounds; Exposure to; Family; Fibroblasts; Frequencies; Genes; Genetic; Genetic screening method; Genome Stability; Genomics; Glycols; Human; Incidence; Induced Mutation; Lesion; Lysine; Maintenance; Malignant Neoplasms; Mediating; Modeling; Mus; Mutation; Plasmids; Play; Polymerase; Process; Pyrimidine Dimers; Reaction; Relative (related person); Role; Simian virus 40; Site; Skin Cancer; Syndrome; System; Testing; Transgenes; UV Mutagenesis; UV induced; Ubiquitin; Ubiquitination; Variant; Xeroderma Pigmentosum; Yeasts; base; carcinogenesis; chemical carcinogen; dimer; human DNA; in vivo; oxidative damage; public health relevance; ultraviolet irradiation; ultraviolet lesions; vector

DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (pols) promote replication through DNA lesions. Humans possess four TLS pols that belong to the Y-family, Pols eta, iota, kappa, and Rev1, and another Pol, Pol zeta, that belongs to the B-family. The overall objectives of this proposal are to elucidate the biological roles of these pols in TLS in human cells, to determine whether they function in an error-free or mutagenic manner, and to identify the means by which a TLS Pol gains access to PCNA and thereby to the replication fork stalled at a DNA lesion site. These and related questions will be studied in the following Specific Aims. In Aim 1, the roles of TLS Pols in promoting replication through a diverse set of DNA lesions in human cells will be analyzed using an SV-40 based plasmid system that we have constructed. DNA lesions to be studied include UV photoproducts, and DNA adducts such as 1,N6-ethenodeoxyadenosine, 1,N2-propano-2'-deoxyguanosine, and (+) trans-dG-N2- benzo[a]pyrene diol epoxide, which result from cellular oxidative damage and from exposure to DNA damaging environmental carcinogens. In Aim 2, studies will be done to test the hypothesis that in human cells, Rev1 functions as a structural element for Pols eta, iota, and kappa. In Aim 3, the roles of TLS Pols in promoting replication through UV lesions and whether they do so in an error-free or mutagenic manner will be studied in the chromosomal cII transgene carried in a mouse embryonic fibroblast cell line. The contributions that cyclobutane pyrimidine dimers vs. (6-4) photoproducts make to UV mutations resulting from the action of different TLS Pols will be analyzed. In Aim 4, the model that, in addition to their binding to PCNA via their PIP domain, TLS Pols bind the ubiquitin moiety on PCNA via their ubiquitin binding domain, will be tested by genetic and biochemical studies of mutations in these domains of Pol eta. In Aim 5, biochemical studies will be undertaken to test the hypothesis that upon stalling at a DNA lesion site, the binding of the replicative pol to PCNA is attenuated as a result of PCNA ubiquitination, and that, in turn, promotes the access of the TLS pol to the primer-template junction via its binding to ubiquitinated PCNA (Ub-PCNA). Specifically, these studies will examine how Ub-PCNA promotes synthesis by Pol eta through a cis-syn TT dimer when a processively moving Pol delta has become stalled at the lesion site. By helping ensure the continuity of the replication fork, TLS Pols play an important role in the maintenance of genomic integrity. Furthermore, their proficient abilities for promoting error-free replication through a large variety of DNA adducts that result from cellular oxidative reactions and from exposure to chemical and environmental carcinogens will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. In fact, the inactivation of Pol eta in humans results in highly elevated levels of skin cancers. The proposed studies are highly relevant for cancer etiology as the results will reveal how human cells minimize the mutagenic and carcinogenic consequences of DNA lesions. PUBLIC HEALTH RELEVANCE: DNA lesions are generated in human cells from cellular oxidative reactions and from exposure to a variety of environmental pollutants and carcinogens. By promoting error-free replication through such DNA lesions, translesion synthesis DNA polymerases would play an important role in maintaining genome stability by keeping the rate of mutations and hence the incidence of cancers low. The proposed studies will elucidate the roles of human DNA polymerases in promoting replication through a variety of DNA lesions and they will examine the mechanisms of this process.

描述（由申请人提供）：跨损伤合成（TLS）DNA聚合酶（pols）通过DNA损伤促进复制。人类拥有属于 Y 家族的四种 TLS pol：Pols eta、iota、kappa 和 Rev1，以及另一个属于 B 家族的 Pol，Pol zeta。该提案的总体目标是阐明这些 pol 在人类细胞 TLS 中的生物学作用，确定它们是否以无差错或诱变的方式发挥作用，并确定 TLS Pol 获取 PCNA 和从而使复制叉停滞在DNA损伤位点。这些及相关问题将在以下具体目标中进行研究。在目标 1 中，将使用我们构建的基于 SV-40 的质粒系统分析 TLS Pols 在通过人类细胞中的多种 DNA 损伤促进复制中的作用。要研究的 DNA 损伤包括 UV 光产物和 DNA 加合物，例如 1,N6-乙烯脱氧腺苷、1,N2-丙-2'-脱氧鸟苷和 (+) 反式-dG-N2-苯并[a]芘二醇环氧化物，其中细胞氧化损伤和接触 DNA 破坏性环境致癌物质所致。在目标 2 中，将进行研究来检验以下假设：在人类细胞中，Rev1 作为 Pols eta、iota 和 kappa 的结构元素发挥作用。在目标 3 中，将在小鼠胚胎成纤维细胞系携带的染色体 cII 转基因中研究 TLS Pols 在通过紫外线损伤促进复制中的作用，以及它们是否以无差错或诱变的方式发挥作用。将分析环丁烷嘧啶二聚体与 (6-4) 光产物对不同 TLS Pol 作用引起的 UV 突变的贡献。在目标 4 中，除了通过 PIP 结构域与 PCNA 结合之外，TLS Pols 还通过其泛素结合结构域结合 PCNA 上的泛素部分，该模型将通过对 Pol eta 这些结构域中的突变进行遗传和生化研究来测试。在目标 5 中，将进行生化研究来检验以下假设：在 DNA 损伤位点停滞后，复制性 pol 与 PCNA 的结合会因 PCNA 泛素化而减弱，进而促进复制性 pol 与 PCNA 的结合。通过与泛素化 PCNA (Ub-PCNA) 的结合，将 TLS pol 连接到引物-模板连接处。具体来说，这些研究将研究当持续移动的 Pol δ 在病变部位停滞时，Ub-PCNA 如何通过顺式-顺 TT 二聚体促进 Pol eta 的合成。通过帮助确保复制叉的连续性，TLS Pols 在维护基因组完整性方面发挥着重要作用。此外，它们通过细胞氧化反应以及接触化学和环境致癌物产生的多种DNA加合物促进无差错复制的熟练能力将通过保持低突变率从而减少基因组稳定性，从而对基因组稳定性产生重大影响。人类致癌的发生率。事实上，人类体内 Poleta 失活会导致皮肤癌的发病率大幅升高。拟议的研究与癌症病因学高度相关，因为其结果将揭示人类细胞如何最大限度地减少 DNA 损伤的诱变和致癌后果。公共健康相关性：DNA 损伤是由于细胞氧化反应以及接触各种环境污染物和致癌物而在人体细胞中产生的。通过这种 DNA 损伤促进无差错复制，跨损伤合成 DNA 聚合酶将在保持突变率和癌症发病率较低的情况下，在维持基因组稳定性方面发挥重要作用。拟议的研究将阐明人类 DNA 聚合酶在通过各种 DNA 损伤促进复制中的作用，并将研究这一过程的机制。