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

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。真核生物的TLS pol在通过不同类型的DNA损伤进行复制方面表现出高度的专业化。在这些人中，波尔？其独特的精通能力，通过紫外线诱导环丁烷嘧啶二聚体（CPDs）无错误复制，并灭活Pol？在人类中引起癌症易感综合征，变色性干皮病（XPV）的变体形式。波尔吗?通过化学致癌物和由活性氧作用形成的DNA加合物诱导的DNA损伤，促进熟练和无差错的复制。为了阐明Pol？在病变旁路手术中，我们将使用生化、遗传和结构相结合的方法来确定Pol？通过DNA损伤，如紫外线诱导的顺式同步TT二聚体，氧化损伤形成的8-氧鸟嘌呤，顺铂化疗形成的顺铂G-G交联，介导高效且无差错的复制。在Aim 1中，我们将确定Pol？通过比较二元和三元结构与未损伤dna的核苷酸结合机制。这些结构将通过诱变和动力学研究进行测试，旨在确定这种独特聚合酶的反应途径。在目标2中，我们将确定Pol？与含有顺式同步T-T二聚体、（6-4）TT光产物、8-氧鸟嘌呤和顺铂G-G链内交联的dna组成的三元配合物。我们假设波尔？与传统的TLS聚合酶以及其他TLS聚合酶的不同之处在于其活性位点间隙的“开放性”，例如，使其能够容纳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，目的是确定Pol？对DNA损伤的作用。在Aim 3中，使用生物物理和生化方法，我们将验证这样的假设，即即使XP-V患者中的大多数错义突变远离活性位点，它们也会对Pol？结构和功能，因为它们对Pol？域。总之，我们在这里提出的研究将对描述Pol？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引起的损伤的遗传毒性作用。精通Pol？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，从而减少人类致癌的发生率。拟议的研究与癌症生物学和病因学高度相关，因为它们将揭示Pol？将人类细胞中DNA损伤的致突变和致癌潜力降至最低。

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.