The molecular basis and regulation of the DNA damage-induced nuclear export of the human MacroD2 protein

细胞通过激活重要的信号和修复途径对DNA损伤做出快速反应。细胞事件的编排通常涉及翻译后修饰对细胞核和细胞质之间的蛋白质运输的调节。磷酸化和多聚腺苷二磷酸核糖化是调节蛋白质在DNA损伤中功能的主要信号通路。值得注意的是，PAR化是在同源重组和非同源末端连接之间进行双链断裂修复选择的重要调节因子。虽然这种串扰具有临床意义，但其分子基础却鲜为人知。MACROD2是新近发现的完全逆转蛋白质PAR化的末端ADP-核糖基水解酶之一，是磷酸化和PAR化之间相互作用的重要调节因子。MACROD2在PAR化后迅速招募到损伤部位，并从蛋白质中移除末端的ADP-核糖单位，但在DNA损伤反应(DDR)激酶(PIKK)激活的反应下从细胞核输出。利用活细胞成像、生化分析和基于高含量siRNA的筛选相结合的方法，拟议的项目将确定这种新的DNA损伤诱导出口机制的分子基础和调控。此外，我们还将阐明DNA损伤中PAR化和磷酸化网络之间的动态调节联系。特别是，我们将确定MACROD2的出口基序和出口载体，并建立MACROD2出口与DNA损伤反应调控之间的联系。

Cells rapidly respond to DNA damage by activating vital signaling and repair pathways. The orchestration of cellular events often involves the modulation of protein traffic between nucleus and cytoplasm by post-translational modifications. Phosphorylation and poly-ADP-ribosylation (PARylation) constitute major signaling pathways regulating protein function upon DNA damage. Significantly, PARylation is an important regulator of double-strand break repair choice between homologous recombination and non-homologous end-joining. While this crosstalk is of clinical relevance, its molecular basis is poorly understood. MacroD2, one of the newly identified terminal ADP-ribosylhydrolases fully reversing protein PARylation, emerges as an important regulator of the crosstalk between phosphorylation and PARylation. MacroD2 rapidly recruits to damage sites upon PARylation and removes the terminal ADP-ribose unit from proteins, but is exported from the nucleus in response to activation of DNA damage response (DDR) kinases (PIKKs). Using a combination of live-cell imaging, biochemical assays and high-content siRNA-based screening, the proposed project will identify the molecular basis and regulation of this novel DNA damage-induced export mechanism. Furthermore, we will elucidate the dynamic regulatory connections between PARylation and phosphorylation networks upon DNA damage. In particular, we will identify the export motif and the export carrier of MacroD2 and establish the connection between MacroD2 export and the regulation of the DNA damage response.