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

基本信息

批准号：
8580936
负责人：
ANEEL K. AGGARWAL
金额：
$ 39.31万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-01 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8580936
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 Lesion Maps Mediating Methods Missense Mutation Molecular Movement Mutagenesis Mutation Nucleotides Pathway interactions Patients Polymerase Proteins Pyrimidine Dimers Rad30 protein Reaction Reactive Oxygen Species 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

项目摘要

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.

描述（由申请人提供）：Translesion合成（TLS）DNA聚合酶（POLS）通过DNA病变促进复制。真核TLS POLS在通过不同类型的DNA病变复制方面表现出高度的专业化。在这些pol中？通过紫外线诱导的环丁烷嘧啶二聚体（CPD）和POL的失活，其熟练的无错误复制能力是独一无二的吗？在人类中，引起癌症综合征，这是心胚层色素（XPV）的变体形式。 pol？还通过由活性氧的作用形成的化学致癌物和DNA加合物引起的DNA病变促进了熟练和无误的复制。阐明POL的作用？在病变旁路中，我们将使用合并的生化，遗传和结构方法来确定POL？通过DNA病变（例如紫外线诱导的顺式）TT二聚体，由氧化损伤形成的8-氧气氨基以及在顺铂化学疗法后形成的顺铂G-G交叉链接。在AIM 1中，我们将通过比较未损坏的DNA的二元和三元结构来确定核苷酸掺入的机理。这些结构将通过诱变和动力学研究来测试，旨在定义这种独特的聚合酶的反应途径。在AIM 2中，我们将确定POL的结构？在带有DNA的三元络合物中，含有顺式-Syn T-T二聚体，A（6-4）TT光产物，8-氧气甘氨酸和顺铂G-G-G-G-G-G-G内源交叉链接。我们假设那个pol？在其活性部位裂缝的“开放性”中，与经典和其他TLS聚合酶不同，例如，它可以容纳T-T二聚体的两个TS。这些结构将通过旨在定义POL对DNA病变的作用的生化和遗传方法进行测试。在AIM 3中，使用生物物理和生化方法，我们将检验以下假设：即使XP-V患者中的大多数错义突变都远离活性部位，它们也会对POL产生不利影响？结构和功能是因为它们对POL的结构稳定性的影响？域。我们在这里提出的研究将共同为描述pol的机制做出重要贡献？守护细胞抵御阳光引起的染色体损伤以及内源性氧化反应和化学疗法引起的损伤的遗传毒性作用。 POL的熟练能力？为了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病变中介导无错误的复制，将通过保持较低突变速率对基因组稳定性产生重大影响，从而降低人类癌发生的发生率。拟议的研究与癌症生物学和病因非常相关，因为它们将揭示POL的方式？最小化人类细胞中DNA病变的诱变和致癌潜力。