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GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity

Yang Han1, Hongling Zhao1, Gang Li2,3

  • 1Department of Radiation Biology, Beijing Key Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, Beijing, China.

British Journal of Cancer
|April 4, 2024
PubMed
Abstract

Insights

Crotonylation of DNA-PKcs at K525, mediated by GCN5, is crucial for DNA repair and DNA-PK complex assembly. This finding offers a potential strategy to enhance cancer radiotherapy outcomes.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Biochemistry

Background:

  • DNA double-strand breaks (DSBs) are critical in cancer radiotherapy and cell survival.
  • The DNA-dependent protein kinase (DNA-PK) complex, crucial for DSB repair, is regulated by post-translational modifications (PTMs).
  • The specific PTMs of DNA-PKcs and their functional impact remain incompletely understood.

Purpose of the Study:

  • To investigate the role of crotonylation, a PTM, on DNA-PKcs.
  • To identify the enzymes responsible for DNA-PKcs crotonylation and decrotonylation.
  • To elucidate how crotonylation of DNA-PKcs affects DNA-PK complex assembly, DNA binding, and DSB repair.

Main Methods:

  • Mass spectrometry to identify crotonylation sites on DNA-PKcs.
  • Co-immunoprecipitation, western blotting, and in vitro assays to confirm crotonylation and identify modifying enzymes.
  • Laser microirradiation, DNA binding, DNA-PK assembly, and immunofluorescence assays to assess functional impacts.
  • In vivo studies using mouse xenografts to evaluate radiotherapy sensitization.

Main Results:

  • K525 was identified as a key crotonylation site on DNA-PKcs, upregulated by irradiation.
  • GCN5 was identified as the crotonylase and HDAC3 as the decrotonylase for K525.
  • K525 crotonylation enhances DNA-PKcs DNA binding, DNA-PK complex assembly, and autophosphorylation.
  • GCN5-mediated crotonylation is essential for DSB repair via the NHEJ pathway.
  • GCN5 suppression sensitized tumors to radiotherapy in vivo.

Conclusions:

  • K525 crotonylation of DNA-PKcs is vital for DNA-PK complex assembly and DSB repair through the NHEJ pathway.
  • Targeting GCN5-mediated K525 crotonylation of DNA-PKcs presents a potential therapeutic strategy to improve cancer radiotherapy efficacy.

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