Interaction of BER proteins with DNA adducts in live human cells

描述（由申请人提供）：哺乳动物AP-内切核酸酶（APE 1）启动无碱基位点（AP-位点）的修复，无碱基位点由环境致癌物直接诱导或由细胞代谢、炎性疾病、致癌和衰老过程中的自由基内源性形成。AP位点也由抗肿瘤剂诱导或通过DNA糖基化酶经由碱基切除修复（BER）途径切除受损碱基而产生。APE 1水平升高与化疗耐药性、预后不良和生存不良有关。通过小分子抑制剂降低癌细胞和肿瘤中APE 1蛋白的活性可使哺乳动物肿瘤细胞对多种实验室和临床化疗剂敏感。APE 1基因敲除小鼠由于不能修复毒性内源性AP位点而死亡，这表明APE 1在修复毒性加合物中的重要性。探索APE 1的隔离，以及在细胞条件下，可以帮助解释和补充动物生物学和转化研究。APE 1先前已被克隆、表达和表征。然而，令人惊讶的是，关于APE 1的生物物理和生物化学性质以及其AP位点修复的细胞内机制，特别是以综合方式修复不同类型的AP位点（常规的，氧化/还原的）的知识有限。因此，在开始动物和人类研究之前，了解APE 1的基本性质对于转化研究是重要的。我们推测，在纤维蛋白质-蛋白质和蛋白质-DNA的相互作用中起着至关重要的调节作用，在不同种类的AP-网站的修复。我们将在2年的资助周期内使用拟议的新型共聚焦显微镜技术在以下两个目标中测试我们的假设：目标1：比较APE 1在人类细胞核中与不同AP位点DNA的相互作用和修复动力学。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APE 1的相互作用来实现，特别是在染色质的背景下。将通过共定位测量DNA-蛋白质相互作用，然后通过FRET-FLIM技术进行直接结合。我们还将测量完整人类细胞中两种类型AP位点的修复动力学，然后比较修复动力学与相互作用动力学;目的2：通过共定位和FRET-FLIM（直接相互作用）分析，确定用DNA损伤剂预处理人类细胞对APE 1和其他BER蛋白与人类细胞核中不同AP位点DNA相互作用的影响。这部分的研究将阐明在细胞修复 通过测试APE 1和其他BER蛋白与不同AP位点DNA在基础水平和在烷基化和氧化应激条件下的完整人类细胞中的直接相互作用的机制。APE 1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，探索体外和细胞内APE 1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，以供未来用于癌症治疗和基本BER机制研究。

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.