Interaction of BER proteins with DNA adducts in live human cells

描述(申请人提供)：哺乳动物AP-内切酶(APE1)在细胞代谢、炎症性疾病、癌变和衰老过程中，启动由环境致癌物直接诱导或由自由基内源性形成的碱性位点(AP-位点)的修复。AP位点也可由抗肿瘤药物诱导，或通过碱基切除修复(BER)途径由DNA糖基酶切除受损的碱基而产生。APE1水平升高与化疗耐药、预后差和生存率差有关。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缺失的小鼠死亡是因为它们不能修复有毒的内源性AP位点，这表明APE1在修复有毒加合物方面的重要性。在分离和纤维素条件下探索APE1，可以帮助解释和补充动物生物学和翻译研究。APE1已被克隆、表达和鉴定。然而，令人惊讶的是，关于APE1的生物物理和生化特性以及其在细胞中的AP位点修复机制，特别是对不同类型AP位点(规则、氧化/还原)的全面修复，人们知之甚少。因此，在开始动物和人类研究以进行翻译研究之前，了解APE1的基本性质是很重要的。我们推测，在细胞内，蛋白质-蛋白质和蛋白质-DNA的相互作用在不同类型的AP-位点的修复中起着至关重要的调节作用。在两年的资金周期中，我们将使用提出的新的共聚焦显微镜技术在以下两个目标上验证我们的假设：目标1：比较APE1在人类细胞核中与不同AP位点DNA相互作用和修复的动力学。这一目标将通过评估AP位点DNA结构的定位及其与APE1在细胞核中的相互作用来实现，特别是在染色质的背景下。DNA与蛋白质的相互作用将通过共定位和FRET-FLIM技术进行直接结合来测量。我们还将测量这两种AP位点在完整人类细胞中的修复动力学，并将修复动力学与相互作用动力学进行比较；目的2：通过共定位和FRET-FLIM(直接相互作用)分析，确定DNA损伤剂预处理人类细胞对APE1和其他BER蛋白与不同AP位点DNA在人细胞核中相互作用的影响。研究的这一部分将在纤维细胞修复中阐明。 通过测试APE1和其他BER蛋白与不同AP位点DNA在完整人类细胞中的基础水平以及在烷化和氧化应激条件下的直接相互作用，研究了APE1和其他BER蛋白的相互作用机制。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出更好的方法来探索更有效的抑制剂，以便将来在癌症治疗和基础BER机制研究中使用。

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.