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）：Translesion合成（TLS）DNA聚合酶（POLS）通过DNA病变促进复制。人类拥有四个属于Y家族的TLS pol，Pols Eta，Iota，Kappa和Rev1，以及另一个属于B家族的Pol Zeta。该提案的总体目标是阐明这些POL在人类细胞中TLS中的生物学作用，以确定它们是否以无错误或诱变的方式起作用，并确定TLS POL可以在DNA病变位置停滞的fork fork for pcna访问PCNA的手段。这些和相关的问题将在以下特定目标中进行研究。在AIM 1中，将使用我们已经构建的基于SV-40的质粒系统来分析TLS POLS通过人类细胞中各种DNA病变促进复制的作用。 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.在AIM 2中，将进行研究以检验以下假设：在人类细胞中，Rev1充当POLS ETA，IOTA和KAPPA的结构元素。在AIM 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在通过紫外病变促进复制以及是否以无错误或诱变方式促进复制的作用。将分析环丁烷嘧啶二聚体与（6-4）对因不同TLS POLS作用产生的紫外线突变所产生的光产物的贡献。在AIM 4中，除了通过其PIP结构域与PCNA结合的模型，TLS POLS还通过其泛素结合结构域在PCNA上结合了泛素部分，还将通过POLETA这些结构域突变的遗传和生化研究来测试。在目标5中，将进行生化研究以检验以下假设：陷入DNA病变部位后，由于PCNA泛素化，复制性POR与PCNA的结合被减弱，然后又可以促进TLS POR通过其与ubiquitionpcna（Ubiquitionpcna）（UbiquItna（UbiquItna）的访问，从而促进了TLS POR的访问。具体而言，这些研究将研究UB-PCNA如何通过在病变部位停滞不前的POL Delta停滞时，通过顺式-Syn 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在维持基因组完整性中起着重要作用。此外，它们通过各种各样的DNA加合物促进无错误复制的能力，这些DNA加合物是由细胞氧化反应以及暴露于化学和环境致癌物暴露于化学和环境致癌物的情况下，将对基因组稳定性产生重大影响，从而通过保持较低的突变速率，从而降低人类癌变的发生率。实际上，人类POL ETA的失活导致皮肤癌的高度升高。拟议的研究与癌症病因高度相关，因为结果将揭示人类细胞如何最大程度地减少DNA病变的诱变和致癌后果。公共卫生相关性：从细胞氧化反应以及暴露于各种环境污染物和致癌物的人类细胞中产生的DNA病变。通过通过此类DNA病变促进无误差复制，跨性质合成DNA聚合酶将通过保持突变速率并因此低癌症的发生在维持基因组稳定性方面起重要作用。拟议的研究将阐明人DNA聚合酶通过多种DNA病变促进复制的作用，它们将检查该过程的机制。