Zhao Jianxin, Shao Guangcan, Lu Xiaoxuan, Lv Zhuan, Dong Meng-Qiu, Liu Xiaoqian, Li Jing
Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing, China.
National Institute of Biological Sciences, Beijing, China.
J Biol Chem. 2024 Dec;300(12):107956. doi: 10.1016/j.jbc.2024.107956. Epub 2024 Nov 2.
O-linked N-acetylglucosamine (O-GlcNAc) is the most abundant mono-saccharide modification occurring in the cytoplasm, nucleus, and mitochondria. The recent advent of mass spectrometry technology has enabled the identification of abundant O-GlcNAc transferase (OGT) substrates in diverse biological processes, such as cell cycle progression, replication, and DNA damage response. Herein we report the O-GlcNAcylation of Replication Protein A2 (RPA2), a component of the heterotrimeric RPA complex pivotal for DNA metabolism. We found that RPA2 interacts with OGT, and a topoisomerase II inhibitor, etoposide, diminishes the association. Using higher-energy collisional dissociation mass spectrometry, we mapped RPA2 O-GlcNAc sites to be Ser-4/Ser-8, which are well-known PIKK-dependent RPA2 phosphorylation sites involved in checkpoint activation upon replication stress. We further demonstrated that Ser-4/Ser-8 O-GlcNAcylation antagonizes phosphorylation and impairs downstream Chk1 activation. Moreover, RPA2 O-GlcNAcylation sustains H2AX phosphorylation upon etoposide treatment and promotes inappropriate cell cycle progression, indicative of checkpoint defects. Our work not only unveils a new OGT substrate, but also underscores the distinct roles of OGT in replication versus replication stress.
O-连接的N-乙酰葡糖胺（O-GlcNAc）是细胞质、细胞核和线粒体中最丰富的单糖修饰。质谱技术的最新进展使得能够在多种生物学过程中鉴定出丰富的O-连接的N-乙酰葡糖胺转移酶（OGT）底物，如细胞周期进程、复制和DNA损伤反应。在此我们报道了复制蛋白A2（RPA2）的O-GlcNAc糖基化，RPA2是异源三聚体RPA复合物的一个组成部分，对DNA代谢至关重要。我们发现RPA2与OGT相互作用，并且一种拓扑异构酶II抑制剂依托泊苷会减少这种结合。使用高能碰撞解离质谱，我们将RPA2的O-GlcNAc位点定位到Ser-4/Ser-8，这是众所周知的依赖磷脂酰肌醇3-激酶相关激酶（PIKK）的RPA2磷酸化位点，参与复制应激时的检查点激活。我们进一步证明Ser-4/Ser-8的O-GlcNAc糖基化拮抗磷酸化并损害下游Chk1激活。此外，RPA2的O-GlcNAc糖基化在依托泊苷处理后维持H2AX磷酸化并促进不适当的细胞周期进程，表明检查点缺陷。我们的工作不仅揭示了一种新的OGT底物，还强调了OGT在复制与复制应激中的不同作用。
