Interaction of BER proteins with DNA adducts in live human cells

BER 蛋白与活人细胞中 DNA 加合物的相互作用

• 批准号: 8701778
• 负责人: RABINDRA ROY
• 金额: $ 7.56万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-06 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8701778
• 关键词: Aging; Animals; Base Excision Repairs; Binding; Biochemical; Biology; Catalysis; Cell Nucleus; Cells; Chemopreventive Agent; Chromatin; Clinical; Complement; DNA; DNA Adducts; DNA Damage; DNA glycosylase; DNA-(apurinic or apyrimidinic site) lyase; DNA-Protein Interaction; Development; Disease; Environmental Carcinogens; Environmental Pollutants; Event; Excision; Fluorescence Resonance Energy Transfer; Free Radicals; Funding; Future; Genes; Genetic Recombination; Genetic Transcription; Goals; Human; In Vitro; Inflammatory; Kinetics; Knockout Mice; Knowledge; Laboratories; Life; Link; Malignant Neoplasms; Measures; Metabolic; Metabolism; Microscopic; Microscopy; Modeling; Molecular; Mutation; Nerve Degeneration; Oxidative Stress; Pathway interactions; Plaque Assay; Play; Preventive; Process; Property; Proteins; Regulation; Research Methodology; Resistance; Resolution; Role; Site; Techniques; Technology; Testing; Therapeutic; Tobacco smoke; Translational Research; Treatment-Related Cancer; adduct; base; cancer cell; carcinogenesis; chemotherapeutic agent; chemotherapy; human APEX1 protein; improved; inhibitor/antagonist; nanometer; nanosecond; neoplastic cell; new technology; novel; outcome forecast; preclinical study; public health relevance; repaired; small molecule; temozolomide; tumor

DESCRIPTION (provided by applicant): Mammalian AP-endonuclease (APE1) initiates the repair of abasic sites (AP-sites), which are directly induced by environmental carcinogens or formed endogenously by free radicals during cellular metabolism, inflammatory diseases, carcinogenesis and aging. AP-sites are also induced by anti-tumoricidal agents or generated by excision of damaged bases by DNA glycosylases via the base excision repair (BER) pathway. Elevated levels of APE1 have been linked to resistance to chemotherapy, poor prognosis, and poor survival. Reducing the activity of the APE1 protein in cancer cells and tumors by small molecule inhibitors sensitizes mammalian tumor cells to a variety of laboratory and clinical chemotherapeutic agents. APE1-null mice died because of their inability to repair toxic endogenous AP-sites, showing the importance of APE1 in repair of toxic adducts. Exploring APE1 in isolation, as well as in cellulo condition, can help interpret and complement animal biology and translational research. APE1 has previously been cloned, expressed, and characterized. However, surprisingly, limited knowledge is available regarding the biophysical and biochemical properties of APE1 as well as its in cellulo mechanisms of AP-site repair, especially the repair of different types of AP-sites (regular, oxidized/reduced) in a comprehensive manner. Therefore, it is important to know the basic properties of APE1 before beginning animal and human studies for translational research. We hypothesize that in cellulo protein-protein and protein-DNA interactions play a crucial regulatory role in the repair of AP-sites of different kinds. We will test our hypothesis in the following two aims using proposed novel confocal microscopic techniques during the 2-year funding cycle: Aim 1: Compare the kinetics of interaction and repair of different AP-site DNA by APE1 in the nucleus of human cells. This aim will be accomplished by assessing the localization of AP-site DNA constructs and their interactions with APE1 in the nucleus, especially in the context of chromatin. The DNA-protein interactions will be measured by co-localization and then by FRET-FLIM technique for direct binding. We will also measure repair kinetics of both types of AP-sites in intact human cells and then compare repair kinetics with interaction kinetics; Aim 2: Determine the effect of pre-treatment of human cells with DNA damaging agents on the interactions of APE1 and other BER proteins with different AP-site DNA in the nucleus of human cells by co-localization and FRET-FLIM (direct interaction) analysis. This portion of the study will elucidate in cellulo repair mechanisms by testing direct interaction of APE1 and other BER proteins with different AP-site DNA in intact human cells at basal level and under alkylating and oxidative stress conditions. APE1 inhibitors are currently in preclinical study for temozolomide-related cancer treatment. Thus, exploring the molecular basis of APE1 catalysis in vitro and in cellulo together may reveal better ways to explore more effective inhibitors for future use in cancer therapeutics and basic BER mechanism studies.

描述（申请人提供）：哺乳动物AP-核酸内切酶（APE1）启动脱碱基位点（AP位点）的修复，这些位点由环境致癌物直接诱导或在细胞代谢、炎症性疾病、癌变和衰老过程中由自由基内源形成。 AP 位点也由抗肿瘤药物诱导或通过 DNA 糖基化酶通过碱基切除修复 (BER) 途径切除受损碱基而产生。 APE1 水平升高与化疗耐药、预后不良和生存率低有关。通过小分子抑制剂降低癌细胞和肿瘤中 APE1 蛋白的活性，使哺乳动物肿瘤细胞对各种实验室和临床化疗药物敏感。 APE1缺失小鼠因无法修复有毒的内源性AP位点而死亡，这表明APE1在修复有毒加合物中的重要性。单独探索 APE1 以及在纤维素条件下，可以帮助解释和补充动物生物学和转化研究。 APE1 先前已被克隆、表达和表征。然而，令人惊讶的是，关于 APE1 的生物物理和生化特性以及其 AP 位点修复的细胞机制，特别是以综合方式修复不同类型的 AP 位点（常规、氧化/还原）的知识有限。 。因此，在开始动物和人体研究以进行转化研究之前，了解 APE1 的基本特性非常重要。我们假设，在纤维素中蛋白质-蛋白质和蛋白质-DNA 相互作用在不同种类的 AP 位点的修复中发挥着至关重要的调节作用。我们将在 2 年的资助周期内使用拟议的新型共焦显微技术来测试以下两个目标的假设： 目标 1：比较人类细胞核中 APE1 相互作用和修复不同 AP 位点 DNA 的动力学。这一目标将通过评估 AP 位点 DNA 构建体的定位及其与细胞核中 APE1 的相互作用（尤其是在染色质环境中）来实现。 DNA-蛋白质相互作用将通过共定位进行测量，然后通过 FRET-FLIM 技术进行直接结合。我们还将测量完整人体细胞中两种类型的 AP 位点的修复动力学，然后将修复动力学与相互作用动力学进行比较；目标 2：通过共定位和 FRET-FLIM（直接相互作用）确定用 DNA 损伤剂预处理人体细胞对 APE1 和其他 BER 蛋白与人体细胞核中不同 AP 位点 DNA 相互作用的影响分析。这部分研究将阐明细胞修复 通过在基础水平以及烷化和氧化应激条件下测试 APE1 和其他 BER 蛋白与完整人类细胞中不同 AP 位点 DNA 的直接相互作用来研究机制。 APE1 抑制剂目前正在进行替莫唑胺相关癌症治疗的临床前研究。因此，在体外和细胞内共同探索 APE1 催化的分子基础可能会揭示更好的方法来探索更有效的抑制剂，以便将来用于癌症治疗和基本 BER 机制研究。