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O-GlcNAcylation of RPA2 at S4/S8 antagonizes phosphorylation and regulates checkpoint activation during replication

Jianxin Zhao1, Guangcan Shao2, Xiaoxuan Lu1

  • 1Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing, China.

The Journal of Biological Chemistry
|November 4, 2024
PubMed

Insights

O-linked N-acetylglucosamine (O-GlcNAc) modifies Replication Protein A2 (RPA2), impacting DNA replication and stress responses. This O-GlcNAcylation antagonizes phosphorylation, leading to cell cycle defects.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • O-linked N-acetylglucosamine (O-GlcNAc) is a prevalent post-translational modification in the nucleus and cytoplasm.
  • O-GlcNAc transferase (OGT) regulates numerous cellular processes by glycosylating proteins.
  • Replication Protein A2 (RPA2) is crucial for DNA replication and repair.

Purpose of the Study:

  • To investigate the O-GlcNAcylation of RPA2.
  • To determine the functional consequences of RPA2 O-GlcNAcylation on DNA replication and stress response pathways.

Main Methods:

  • Mass spectrometry to identify O-GlcNAc sites on RPA2.
  • Co-immunoprecipitation to assess protein interactions.
  • Western blotting to detect protein phosphorylation and activation states.

Main Results:

  • RPA2 was identified as a novel substrate for O-GlcNAc transferase (OGT).
  • O-GlcNAcylation occurred at Ser-4/Ser-8, sites also targeted by phosphorylation.
  • RPA2 O-GlcNAcylation antagonized phosphorylation, impaired Chk1 activation, and sustained H2AX phosphorylation, promoting cell cycle progression under replication stress.

Conclusions:

  • RPA2 is a new substrate for OGT, linking O-GlcNAcylation to DNA replication and stress response.
  • O-GlcNAcylation of RPA2 plays a distinct role in regulating replication versus replication stress.
  • This modification impacts checkpoint activation and cell cycle control, highlighting OGT's complex regulatory functions.
Keywords:
O-GlcNAcRPA2checkpointphosphorylationreplication stress

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