Translesion DNA synthesis in humans

描述（由申请人提供）：转录合成（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，Pols eta，iota，kappa和Rev 1，以及另一个属于B家族的Pols，Polzeta。本提案的总体目标是阐明这些pols在人类细胞TLS中的生物学作用，以确定它们是否以无错误或致突变的方式起作用，并确定TLS Pol获得PCNA的途径，从而获得停滞在DNA损伤位点的复制叉。这些问题和相关问题将在以下具体目标中加以研究。在目标1中，TLS Pos在通过人类细胞中的一组不同的DNA损伤促进复制中的作用将使用我们构建的基于SV-40的质粒系统进行分析。待研究的DNA损伤包括UV光产物和DNA加合物，如1，N6-亚乙基脱氧腺苷、1，N2-丙-2 '-脱氧鸟苷和（+）trans-dG-N2-苯并[a]芘二醇环氧化物，其由细胞氧化损伤和暴露于DNA损伤环境致癌物引起。在目标2中，将进行研究以检验以下假设：在人类细胞中，Rev 1作为Pos 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 Pos在通过UV损伤促进复制中的作用以及它们是否以无错误或致突变的方式这样做。将分析环丁烷嘧啶二聚体与（6-4）光产物对由不同TLS Pol的作用引起的UV突变的贡献。在目的4中，除了通过PIP结构域与PCNA结合之外，TLS Pos还通过其泛素结合结构域与PCNA上的泛素部分结合的模型将通过Pol eta的这些结构域中的突变的遗传和生物化学研究进行测试。在目标5中，将进行生物化学研究以检验以下假设：在DNA损伤位点停滞后，复制pol与PCNA的结合由于PCNA泛素化而减弱，并且反过来，通过其与泛素化PCNA（Ub-PCNA）的结合促进TLS pol接近引物-模板连接。具体而言，这些研究将检查Ub-PCNA如何通过顺式-syn TT二聚体促进Pol eta的合成，当一个前向移动的Pol delta在病变部位停滞时。通过帮助确保复制叉的连续性，TLS Pol在维护基因组完整性方面发挥着重要作用。此外，它们通过由细胞氧化反应和暴露于化学和环境致癌物产生的大量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，人类中Pol eta的失活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，因为结果将揭示人类细胞如何最大限度地减少DNA损伤的致突变和致癌后果。公共卫生相关性：DNA损伤是由细胞氧化反应和暴露于各种环境污染物和致癌物在人体细胞中产生的。通过促进通过这种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并因此保持癌症的发生率低而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。

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.