Translesion DNA synthesis in humans

描述（由申请人提供）：翻译合成（TLS） DNA聚合酶（pol）通过DNA损伤促进复制。人类拥有4个属于y家族的TLS节点，分别是pols eta、iota、kappa和Rev1，以及另一个属于b家族的Pol zeta。本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，确定它们是否以无错误或致突变的方式起作用，并确定TLS Pol进入PCNA的途径，从而进入DNA损伤部位的复制叉。这些和相关的问题将在以下具体目标中进行研究。在Aim 1中，将使用我们构建的基于SV-40的质粒系统分析TLS pol在通过多种DNA病变促进人类细胞复制中的作用。待研究的DNA损伤包括紫外线光产物和DNA加合物，如1，n6 -乙基脱氧腺苷、1，n2 -丙基-2'-脱氧鸟苷和（+）反式dg - n2 -苯并[a]芘二醇环氧化物，它们是由细胞氧化损伤和暴露于DNA损伤性环境致癌物引起的。在目标2中，将进行研究以验证在人类细胞中，Rev1作为pol - eta， iota和kappa的结构元件的假设。在Aim 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 pol在通过紫外线损伤促进复制中的作用，以及它们是以无错误还是诱变的方式促进复制。分析了环丁烷嘧啶二聚体与（6-4）光产物对不同TLS pol作用下的紫外突变的贡献。在Aim 4中，除了通过其PIP结构域与PCNA结合外，TLS Pol还通过其泛素结合结构域与PCNA上的泛素片段结合，该模型将通过Pol eta这些结构域突变的遗传和生化研究进行测试。在Aim 5中，将进行生化研究来验证这样的假设，即在DNA损伤部位停滞后，由于PCNA泛素化，复制pol与PCNA的结合减弱，这反过来又促进了TLS pol通过与泛素化PCNA （Ub-PCNA）的结合进入引物-模板连接。具体来说，这些研究将研究当一个不断移动的Pol delta在病变部位停滞时，Ub-PCNA如何通过顺式同步TT二聚体促进Pol eta的合成。通过帮助确保复制分叉的连续性，TLS pol在维持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和暴露于化学和环境致癌物中产生的大量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，从而减少人类致癌的发生率。事实上，人类体内Pol - eta的失活会导致皮肤癌的高度升高。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将DNA损伤的致突变和致癌后果降至最低。公共卫生相关性：DNA损伤是由于细胞氧化反应和暴露于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损伤促进无错误复制，翻译合成DNA聚合酶将通过保持突变率从而降低癌症发病率，在维持基因组稳定性方面发挥重要作用。提出的研究将阐明人类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.