O-linked N-acetylglucosamine (O-GlcNAc) is the most abundant mono-saccharide modification occurring in the cytoplasm, nucleus and mitochondria. Recent advent of the mass spectrometry technology has enabled 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.

中文翻译：

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RPA2 在 S4/S8 位点的 O-GlcNAcylation 拮抗磷酸化并调节复制应激期间的检查点激活。


O-连接 N-乙酰氨基葡萄糖 （O-GlcNAc） 是存在于细胞质、细胞核和线粒体中最丰富的单糖修饰。质谱技术的最新出现使得能够在多种生物过程（如细胞周期进程、复制和 DNA 损伤反应）中鉴定丰富的 O-GlcNAc 转移酶 （OGT） 底物。在此，我们报道了复制蛋白 A2 （RPA2） 的 O-GlcNAcylation，RPA2 是 DNA 代谢关键的异源三聚体 RPA 复合物的组成部分。我们发现 RPA2 与 OGT 相互作用，拓扑异构酶 II 抑制剂依托泊苷会减少这种关联。使用更高能量的碰撞解离质谱法，我们将 RPA2 O-GlcNAc 位点定位为 Ser-4/Ser-8，这是众所周知的 PIKK 依赖性 RPA2 磷酸化位点，参与复制应激下的检查点激活。我们进一步证明 Ser-4/Ser-8 O-GlcNAcylation 拮抗磷酸化并损害下游 Chk1 激活。此外，RPA2 O-GlcNAcylation 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独特作用。