Role of DNA polymerase eta in errorfree bypass of DNA lesions & cancer prevention

描述（由申请人提供）：转录合成（TLS）DNA聚合酶（Pos）通过DNA损伤促进复制。真核TLS Pos在其通过不同类型的DNA损伤进行复制的能力方面显示出高度的专业化。其中，Pol？是独特的，在其熟练的无差错复制能力，通过UV诱导的环丁烷嘧啶二聚体（CPD），和失活的Pol？在人类中引起癌症倾向综合征，着色性干皮病（XPV）的变体。波尔？还通过化学致癌物诱导的DNA损伤和活性氧作用形成的DNA加合物促进熟练和无错误的复制。为了阐明波尔的作用？在病变旁路，我们将使用相结合的生化，遗传和结构的方法来确定如何Pol？通过DNA损伤如UV诱导的顺式-syn TT二聚体、由氧化损伤形成的8-氧代鸟嘌呤和顺铂化疗后形成的顺铂G-G交联介导有效和无错误的复制。在目标1中，我们将确定Pol？的核苷酸掺入的机制，通过比较的二元和三元结构与未受损的DNA。将通过诱变和动力学研究来测试结构，目的是定义这种独特聚合酶的反应途径。在目标2，我们将确定结构的Pol？与含有顺式-顺式T-T二聚体、（6-4）TT光产物、8-氧代鸟嘌呤和顺铂G-G链内交联DNA形成三元复合物。我们假设波尔？与经典的以及其他TLS聚合酶的不同之处在于其活性位点裂缝的“开放性”，使其能够例如容纳T-T二聚体的两个T。这些结构将通过生物化学和遗传学方法进行测试，旨在确定Pol？对DNA损伤的作用在目标3中，使用生物物理和生物化学的方法，我们将测试的假设，即使大多数的错义突变在XP-V患者远离活性位点，他们不利地影响Pol？结构和功能，因为它们的结构稳定性的影响？域.总之，我们在这里提出的研究将作出重要贡献描绘的机制，其中波尔？保护细胞免受阳光诱导的染色体损伤以及内源性氧化反应和化疗诱导的损伤的遗传毒性作用。 波尔的熟练能力？通过暴露于环境和化学致癌物以及细胞氧化反应形成的DNA损伤来介导无错误复制，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。拟议的研究是高度相关的癌症生物学和病因学，因为他们将揭示如何波尔？最大限度地减少人类细胞中DNA损伤的致突变和致癌潜力。

DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions. Eukaryotic TLS Pols display a high degree of specialization in their proficiency for replicating through different types of DNA lesions. Among these Pol? is unique in its proficient ability for error-free replication though UV induced cyclobutane pyrimidine dimers (CPDs), and inactivation of Pol? in humans causes the cancer- prone syndrome, the variant form of xeroderma pigmentosum (XPV). Pol? also promotes proficient and error-free replication through DNA lesions induced by chemical carcinogens and DNA adducts formed from the action of reactive oxygen species. To elucidate the role of Pol? in lesion bypass, we will use a combined biochemical, genetic, and structural approach to determine how Pol? mediates proficient and error-free replication through DNA lesions such as a UV induced cis-syn TT dimer, an 8-oxoguanine formed from oxidative damage, and a cisplatin G-G crosslink formed upon cisplatin chemotherapy. In Aim 1, we will determine Pol?'s mechanism of nucleotide incorporation by a comparison of binary and ternary structures with undamaged DNAs. The structures will be tested by mutagenesis and kinetic studies aimed at defining the reaction pathway of this unique polymerase. In Aim 2, we will determine structures of Pol? in ternary complex with DNAs containing a cis-syn T-T dimer, a (6-4) TT photoproduct, an 8-oxoguanine, and a cisplatin G-G intrastrand crosslink. We hypothesize that Pol? differs from classical as well as other TLS polymerases in the "openness" of its active site cleft, enabling it, for example, to accommodate both Ts of the T-T dimer. The structures will tested by biochemical and genetic methods aimed at defining Pol?'s action on DNA lesions. In Aim 3, using biophysical and biochemical approaches, we will test the hypothesis that even though most of the missense mutations in XP-V patients lie distant from the active site, they adversely affect Pol? structure and function because of their effects on the structural stabilities of Pol? domains. Together, the studies we propose here will make an important contribution toward delineating the mechanisms by which Pol? guards cells against the genotoxic effects of sunlight-induced chromosomal damage as well as damage induced from endogenous oxidation reactions and by chemotherapy. The proficient ability of Pol? for mediating error-free replication through DNA lesions formed from exposure to environmental and chemical carcinogens and from cellular oxidative reactions will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The proposed studies are highly relevant for cancer biology and etiology, as they will reveal how Pol? minimizes the mutagenic and carcinogenic potential of DNA lesions in human cells.