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（由申请人提供）：哺乳动物AP-核酸酶（APE1）启动了无碱性位点（AP-SITE）的修复，这些修复是由环境致癌物直接诱导的，或者在细胞代谢期间由自由基内源形成的，炎症性疾病，炎症性疾病，癌变和衰老。抗肿瘤剂也会诱导AP矿物质，或通过DNA糖基酶通过碱基切除修复（BER）途径切除受损碱而产生的。 APE1水平升高与对化学疗法，预后不良和存活不良的抗性有关。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学治疗剂敏感。 APE1-NULL小鼠死亡是因为它们无法修复有毒的内源性AP位点，表明APE1在修复有毒加合物中的重要性。孤立地探索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，有关APE1的生物物理和生化特性以及其在AP站点修复的纤维素机制中，尤其是以全面的方式修复AP站点修复的纤维素机制（尤其是修复不同类型的AP站点）。因此，重要的是要在开始动物和人类研究进行转化研究之前了解APE1的基本特性。我们假设在纤维素蛋白质蛋白和蛋白-DNA相互作用中，在修复不同种类的AP站点中起着至关重要的调节作用。我们将在2年资金周期中使用拟议的新型共聚焦显微镜技术在以下两个目标中检验我们的假设：AIM 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。通过评估AP位点DNA构建体的定位及其与APE1在细胞核中，尤其是在染色质的情况下，可以实现此目标。 DNA-蛋白质相互作用将通过共定位，然后通过FRET-FLIM技术来测量直接结合。我们还将测量完整人类细胞中两种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）分析，通过DNA损伤剂对APE1和其他BER蛋白与不同AP位置DNA的相互作用确定人类细胞对APE1和其他AP位点DNA的相互作用的影响。这部分研究将阐明纤维素修复 通过测试APE1和其他BER蛋白与在基础水平和烷基化和氧化应激条件下的完整人细胞中不同AP位点DNA的直接相互作用与不同AP位点DNA的直接相互作用的机制。 APE1抑制剂目前正在临床前研究进行替莫唑胺相关的癌症治疗。因此，在体外和纤维素中探索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，以供将来在癌症治疗和基本BER机理研究中使用。