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

DNA 聚合酶 eta 在 DNA 损伤无错旁路中的作用

• 批准号: 8197899
• 负责人: ANEEL K. AGGARWAL
• 金额: $ 39.76万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8197899
• 关键词: 7,8-dihydro-8-oxoguanine; 8-hydroxyguanosine; Active Sites; Address; Affect; Apoenzymes; Binding; Biochemical; Biochemical Genetics; Bypass; Cancer Biology; Cancer Etiology; Catalytic Domain; Cells; Cisplatin; Cleaved cell; Complex; DNA; DNA Adducts; DNA Damage; DNA Groove Binding; DNA lesion; DNA-Directed DNA Polymerase; Distant; Environmental Carcinogens; Exposure to; Genome Stability; Human; Incidence; Kinetics; Language; Lesion; Maps; Mediating; Methods; Missense Mutation; Molecular; Movement; Mutagenesis; Mutation; Nucleotides; Pathway interactions; Patients; Polymerase; Proteins; Public Health; Pyrimidine Dimers; Rad30 protein; Reaction; Reactive Oxygen Species; Research; Resolution; Role; Solutions; Solvents; Staging; Structure; Syndrome; Testing; Time; Tumor Suppressor Proteins; UV induced; Variant; Xeroderma Pigmentosum; Yeasts; adduct; base; cancer prevention; carcinogenesis; chemical carcinogen; chemotherapy; crosslink; dimer; oxidation; oxidative damage; public health relevance; response; sunlight-induced

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. PUBLIC HEALTH RELEVANCE: DNA polymerase ? (Pol?) promotes proficient and error-free replication through DNA lesions. Hence, inactivation of Pol? in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum. The proposed studies will elucidate the molecular basis of Pol?'s action opposite different types of DNA lesions.

描述（由申请人提供）：跨损伤合成（TLS）DNA聚合酶（Pols）通过DNA损伤促进复制。真核 TLS Pols 在通过不同类型的 DNA 损伤进行复制的能力方面表现出高度的专业化。其中波尔？其独特之处在于其通过紫外线诱导的环丁烷嘧啶二聚体 (CPD) 进行无差错复制的熟练能力以及 Pol? 的失活能力。在人类中引起癌症倾向综合征，即着色性干皮病 (XPV) 的变异形式。波尔？还通过化学致癌剂诱导的 DNA 损伤和活性氧作用形成的 DNA 加合物促进熟练且无差错的复制。阐明波尔的作用？在病变搭桥中，我们将使用结合生化、遗传和结构的方法来确定Pol？通过 DNA 损伤（例如紫外线诱导的 cis-syn TT 二聚体、氧化损伤形成的 8-氧代鸟嘌呤以及顺铂化疗时形成的顺铂 G-G 交联）介导高效且无错误的复制。在目标 1 中，我们将通过比较未受损 DNA 的二元和三元结构来确定 Pol? 的核苷酸掺入机制。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? 的结构。与包含顺式 T-T 二聚体、(6-4) TT 光产物、8-氧代鸟嘌呤和顺铂 G-G 链内交联的 DNA 形成三元复合物。我们假设波尔？与经典以及其他 TLS 聚合酶的不同之处在于其活性位点裂缝的“开放性”，例如使其能够容纳 T-T 二聚体的两个 T。这些结构将通过生化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，我们将使用生物物理和生化方法检验以下假设：尽管 XP-V 患者中的大多数错义突变远离活性位点，但它们会对 Pol? 产生不利影响。结构和功能，因为它们对 Pol 结构稳定性的影响？域。总之，我们在此提出的研究将为描述 Pol? 的机制做出重要贡献。保护细胞免受阳光引起的染色体损伤以及内源性氧化反应和化疗引起的损伤的基因毒性作用。 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，将通过保持较低的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。拟议的研究与癌症生物学和病因学高度相关，因为它们将揭示 Pol？最大限度地减少人类细胞 DNA 损伤的致突变和致癌潜力。 公共卫生相关性：DNA 聚合酶？ (Pol?) 通过 DNA 损伤促进熟练且无差错的复制。因此，Pol? 失活。在人类中会导致癌症倾向综合征，即着色性干皮病的变异形式。拟议的研究将阐明 Pol? 对抗不同类型 DNA 损伤的作用的分子基础。