哺乳动物DNA2的泛素化修饰参与DNA损伤应答反应的分子机制研究

DNA2 nuclease/helicase plays a role in stabilizing and repairing stalled replication fork during DNA replication and repair in budding yeast. Mammalian DNA2 lacks classic nuclear localization signals found in yeast DNA2 and predominantly localizes in the mitochondria under normal condition. It has been demonstrated that Mammalian DNA2 can alleviate DNA replication stress which happened in cancer cell and at telomere, but how hDNA2 translocates into nucleus and repair the stalled replication forks is not known. We have preliminary data indicating that 1) DNA2 is ubiquitinated then translocates into the nucleus where it forms foci at stalled replication forks. 2) Mutation of a lysine residue in DNA2 affects DNA2 nulcear localization. 3) Protein complex pull-down and mass spectrometry identifies key modification enzymes and interaction partners including ubiquitin ligase E3, nuclear transport protein, DNA repair and replication proteins. To explore the role and molecular mechanisms of DNA2 ubiquitination in DNA damage response, we will determine how DNA2 ubiquitination is regulated in response to DNA replication stresses, how ubiquitination mediates DNA2 nuclear translocation and how DNA2 recruitment to stalled replication fork and its appropriate functions. We also will also determine the effect of DNA2 with defects in ubiquitination/nuclear localization on genome stability and tumorigenesis using knock-in mice. We believe this project will fully elucidate the role of ubiquitination of DNA2 in nuclear translocation and counteracting with replication stress and its underlying mechanisms, which will further highlight the importance of DNA2 PTM in genome stability and cancer prevention and treatment.

酵母DNA2在DNA复制和修复过程起到稳定和修复复制叉的功能。哺乳动物源DNA2主要位于线粒体，没有酵母DNA2所具有的核定位序列，现已证明其在端粒和肿瘤细胞DNA复制过程中起到解除复制压力的作用，但其核转运和参与复制压力解除的机制有待进一步阐述。本项目组前期研究发现DNA2在DNA损伤下被大量泛素化，并被转运至核内形成焦点；突变泛素化位点抑制其入核；并鉴定DNA2的互作蛋白，包括泛素连接酶E3、调控其入核以及参与DNA复制、修复的蛋白。因此，本项目拟从细胞和分子水平上探讨DNA2泛素化修饰、核转运机制以及在核内稳定和修复复制叉的机制，同时在动物水平上探讨泛素化位点突变对维持基因组稳定以及抑制肿瘤发生的影响。本项目将系统揭示DNA2新鉴定的翻译后修饰-泛素化修饰在核转运以及解除复制压力的功能和分子机制，这有助于进一步阐述DNA损伤下翻译后修饰的DNA2对维持基因组稳定和防治肿瘤的重要性。

越来越多的研究显示核酸酶/解旋酶DNA2在DNA双链损伤修复起到关键作用。在哺乳动物中，DNA2主要位于线粒体，并不具有酵母DNA2所编码的核定位序列，其响应基因毒性压力的机制有待进一步阐述。本研究从DNA2的互作蛋白入手，通过免疫沉淀方法寻找到一系列DNA2的互作蛋白，包括泛素化连接酶TRAF6，核孔蛋白以及其他DNA损伤蛋白。我们推测，在双链断裂损伤药物的作用下，线粒体DNA2在TRAF6的作用下进行泛素化修饰，而泛素化的DNA2在核孔蛋白协助下入核与其他DNA损伤蛋白参与损伤修复。体内外实验证明TRAF6能泛素化DNA2，并通过质谱分析鉴定出DNA2的泛素化位点；泛素化促进DNA2的入核以及DNA2蛋白的稳定性。核质分离实验进一步证明DNA2的稳定性由于更多结合到染色质上。本项目进一步在细胞和动物水平明确DNA2敲减对肿瘤发生的作用。本研究发表一篇American Journal of Human Genetics和另一篇SCI文章，另有一篇AJHG文章在审稿中。在项目实施过程中，申请到另一项国家基金面上项目。