Mechanism of APE2 in genome integrity

项目摘要/摘要 环境毒素或内源性伤害，如活性氧(ROS)，会导致氧化 细胞内的应激，导致DNA或RNA的不同类型的损伤，包括脱嘌呤/脱嘧啶 (AP)位点和单链断裂(SSB)。作为最常见的DNA损伤类型，SSB一直是 与肿瘤发生、衰老和神经退行性疾病有关。分子 对SSB修复途径的了解仍不清楚，这主要是由于缺乏易于处理的实验 系统。我们和其他人最近证明了APE2解决了非洲爪哇单链构象障碍和 萌芽酵母。尽管APE1被发现对包括DNA修复在内的氧化还原调节起关键作用 转录和RNA加工，目前尚不清楚APE2如何在DNA和RNA中发挥重要作用 新陈代谢。确定APE2如何在SSB修复中发挥关键作用以及确切地如何发挥作用具有重要意义 APE2维持基因组的稳定性。我们大量的初步数据表明，APE2‘S 3’-5‘核酸外切酶 活性通过与单链DNA(SsDNA)及其相互作用蛋白的动态相互作用来调节， APE2与RNA和R-loop等含RNA结构相结合。我们将剖析 APE2在核酸代谢中的分子机制通过两个特定的目的：(1)确定 APE2与DNA结构中AP位点和SSB相互作用和处理的机制 用纯化的蛋白和非洲爪哇卵提取液重建系统，以及(2)确定APE2如何 识别和修复RNA或含RNA结构中的AP位点和SSB。生物化学，细胞生物学， 并将利用分子生物学方法来进行这种假说驱动的结构功能 非洲爪哇卵提取液中APE2介导的核酸代谢分析及重组 含有纯化蛋白质的系统。该项目的预期结果将使APE2的范式发生转变- 介导DNA/RNA代谢中的SSB修复以维持基因组稳定。值得注意的是，我们的发现将 为癌症化疗的新策略提供新的见解，例如调节不同的 APE2在基因组完整性中对SSB修复的调节机制。

Project Summary/Abstract Environmental toxins or endogenous insults such as reactive oxygen species (ROS) result in oxidative stress within cells, leading to different types of damage in DNA or RNA, including Apurinic/apyrimidinic (AP) sites and single-strand breaks (SSBs). As the most common type of DNA damage, SSBs have been implicated in association with tumorigenesis, aging, and neurodegenerative disorders. Molecular understanding of SSB repair pathway remains unclear, largely due to the lack of tractable experimental systems. We and others have demonstrated recently that APE2 resolves SSB damage in Xenopus and budding yeast. Although APE1 has been found critical for including DNA repair, redox regulation of transcription, and RNA processing, it remains unclear how APE2 plays important roles in DNA and RNA metabolism. It is significant to determine how APE2 plays an essential role in SSB repair, and how exactly APE2 maintains genome stability. Our substantial preliminary data suggest that APE2’s 3'-5' exonuclease activity is regulated via its dynamic interactions with single-strand DNA (ssDNA) and its interacting proteins, and that APE2 associates with RNA and RNA-containing structures such as R-loop. We will dissect the molecular mechanism of APE2 in metabolism in nucleic acids via two Specific Aims: (1) determine the mechanism of how APE2 interacts with and processes AP sites and SSBs in DNA structures in reconstitution system with purified proteins and in Xenopus egg extracts, and (2) determine how APE2 recognizes and repairs AP site and SSBs in RNA or RNA-containing structures. Biochemical, cell biology, and molecular biology approaches will be utilized to conduct this hypothesis-driven structure-function analysis of APE2-mediated metabolism of nucleic acids in Xenopus egg extracts and reconstitution systems with purified proteins. Anticipated results from this project will make a paradigm shift on APE2- mediated SSB repair in DNA/RNA metabolism to maintain genome stability. Notably, our findings will provide novel insights into new strategies for cancer chemotherapies such as modulating the distinct regulatory mechanisms of APE2 for SSB repair in genome integrity.