Translesion DNA synthesis in humans

人类跨损伤 DNA 合成

• 批准号: 7523137
• 负责人: LOUISE PRAKASH
• 金额: $ 35.3万
• 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/7523137
• 关键词: Attenuated; Benzo(a)pyrene; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological; Bypass; Cancer Etiology; Carcinogens; Cell Line; Cells; Complex; DNA; DNA Adducts; DNA Damage; DNA Photolyase; DNA biosynthesis; DNA chemical synthesis; 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; Glycol; Human; Incidence; Induced Mutation; Lesion; Lysine; Maintenance; Malignant Neoplasms; Mediating; Modeling; Mus; Mutation; PCNA gene; Plasmids; Play; Polymerase; Process; Public Health; Pyrimidine Dimers; Rate; 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; dimer; environmental chemical; in vivo; phosphatidylinositol phosphate; pol genes; 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。人类拥有四个属于Y家族的TLS Pols，Pol Eta，IOTA，Kappa和Rev1，以及另一个属于B家族的Pol，Pol Zeta。这项建议的总体目标是阐明这些POL在人类细胞TLS中的生物学作用，确定它们是以无错误或突变的方式发挥作用，并确定TLS POL获得增殖细胞核抗原从而获得停滞在DNA损伤部位的复制叉的方式。这些问题和相关问题将在以下具体目标下进行研究。在目标1中，将使用我们构建的基于SV-40的质粒系统来分析TLS POLS在通过人类细胞中的一组不同的DNA损伤促进复制方面的作用。待研究的DNA损伤包括紫外线光产物和DNA加合物，如1，N6-乙叉脱氧腺苷、1，N-丙基-2‘-脱氧鸟苷和(+)反式-DG-N-2-苯并[a]二醇环氧化物，它们是细胞氧化损伤和接触DNA破坏环境致癌物的结果。在目标2中，将进行研究以检验这一假设，即在人类细胞中，Rev1作为POLS ETA、IOTA和Kappa的结构元件发挥作用。在目标3中，将在小鼠胚胎成纤维细胞系中携带的染色体CII转基因中研究TLS POLS在通过紫外线损伤促进复制方面的作用以及它们是以无错误还是以诱变的方式做到这一点。将分析环丁烷嘧啶二聚体与(6-4)光产物对不同TLS POL作用所导致的紫外线突变的贡献。在目标4中，除了通过其PIP结构域与增殖细胞核抗原结合外，TLS Pol还通过其泛素结合结构域将泛素部分结合在增殖细胞核抗原上，这一模型将通过对Pol ETA这些结构域的突变的遗传和生化研究来测试。在目标5中，将进行生化研究来验证这样的假设，即当在DNA损伤部位失速时，由于增殖细胞核抗原泛素化的结果，复制的Poll与增殖细胞核抗原的结合被减弱，并反过来通过其与泛素化的增殖细胞核抗原(Ub-PCNA)的结合促进TLS Poll进入引物-模板连接。具体地说，这些研究将检查Ub-PCNA如何通过顺式同步TT二聚体促进Pol ETA的合成，当Pol Delta在病变部位停滞不前时。通过帮助确保复制分叉的连续性，TLS POLS在维持基因组完整性方面发挥了重要作用。此外，它们通过细胞氧化反应和接触化学和环境致癌物产生的各种DNA加合物促进无错误复制的熟练能力，将通过保持低突变率对基因组稳定性产生重大影响，从而降低人类致癌的发生率。事实上，在人类中，POL ETA的失活会导致皮肤癌水平的高度升高。拟议的研究与癌症病因学高度相关，因为研究结果将揭示人类细胞如何将DNA损伤的突变和致癌后果降至最低。与公共卫生相关：DNA损伤是由细胞氧化反应以及暴露在各种环境污染物和致癌物中在人类细胞中产生的。通过促进此类DNA损伤的无错误复制，跨损伤合成DNA聚合酶将通过保持突变率，从而降低癌症发生率，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损伤促进复制方面的作用，并将研究这一过程的机制。