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Perturbation of O\u2010GlcNAcylation leads to altered cell\u2010cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one\u2010step enzymatic strategy for capture and profiling O\u2010GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O\u2010GlcNAcylation. FEN1 O\u2010GlcNAcylation is dynamically regulated during the cell cycle. O\u2010GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O\u2010GlcNAcylated proteins, and reveals an unknown mechanism of O\u2010GlcNAcylation in regulating cell cycle progression and DNA damage response.<\/jats:p>","DOI":"10.1002\/anie.202110053","type":"journal-article","created":{"date-parts":[[2021,9,30]],"date-time":"2021-09-30T05:08:17Z","timestamp":1632978497000},"page":"26128-26135","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":45,"title":["One\u2010Step Enzymatic Labeling Reveals a Critical Role of O\u2010GlcNAcylation in Cell\u2010Cycle Progression and DNA Damage Response"],"prefix":"10.1002","volume":"60","author":[{"given":"Yinping","family":"Tian","sequence":"first","affiliation":[{"name":"Department of Hepatobiliary and Pancreatic Surgery The First Affiliated Hospital Zhejiang Provincial Key Laboratory of Pancreatic Disease School of Medicine Zhejiang University  Hangzhou China"},{"name":"MOE Key Laboratory 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