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Ku70 suppresses alternative end joining in G1-arrested progenitor B cells

Zhuoyi Liang1,2,3, Vipul Kumar1,2,3, Marie Le Bouteiller4

  • 1HHMI, Boston Children's Hospital, Boston, MA 02115.

Proceedings of the National Academy of Sciences of the United States of America
|May 19, 2021
PubMed

Insights

Ku protein suppresses alternative end-joining (A-EJ) of DNA double-strand breaks (DSBs) in Lig4-deficient cells during G1 phase. This Ku-dependent blockage of A-EJ inhibits DSB repair, impacting V(D)J recombination and potentially other cellular processes.

Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) and is crucial in G1 phase when homologous recombination is inactive.
  • Core C-NHEJ factors Ku70/80 (Ku) and XRCC4/ligase IV (Lig4) are essential for DSB sensing and joining, respectively.
  • While XRCC4/Lig4 are vital for V(D)J recombination in G1, cells lacking them can utilize alternative end-joining (A-EJ) pathways for chromosomal DSBs.

Purpose of the Study:

  • To investigate the role of Ku in regulating alternative end-joining (A-EJ) pathways in the absence of XRCC4/ligase IV (Lig4).
  • To understand how Ku influences the repair of various DNA double-strand breaks (DSBs) initiated by different nucleases in Lig4-deficient cells.
  • To elucidate the mechanisms underlying the differential effects of Ku and Lig4 deficiencies on V(D)J recombination and other cellular processes.

Main Methods:

  • Utilized Lig4-deficient G1-arrested progenitor B cell lines.
  • Introduced DSBs using RAG1/2, Cas9:gRNA, and Zinc finger nucleases.
  • Assessed DSB rejoining and translocation levels in cells with varying deficiencies in Ku70 and Lig4.
  • Analyzed the impact of Ku deficiency on A-EJ in Lig4-deficient cells.

Main Results:

  • Ku suppresses A-EJ of DSBs generated by RAG1/2, Cas9:gRNA, and Zinc finger nucleases in Lig4-deficient G1-arrested progenitor B cells.
  • Deletion of Ku70 in Lig4-deficient cells significantly increases DSB rejoining and translocation levels.
  • RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient cells but occurs via a translocation-like A-EJ mechanism in Ku70-deficient and Ku70/Lig4 double-deficient cells.
  • Lig4-deficient G1-arrested progenitor B cells exhibit functionally suppressed end-joining due to Ku-dependent A-EJ blockage.

Conclusions:

  • Ku actively inhibits A-EJ in Lig4-deficient G1-arrested progenitor B cells, leading to suppressed DSB repair.
  • Ku-mediated inhibition of A-EJ during G1 phase contributes to the differential outcomes observed in Ku versus Lig4 deficiencies in V(D)J recombination, neuronal apoptosis, and embryonic development.
  • The findings highlight a critical regulatory role of Ku in controlling alternative DNA repair pathways during specific cell cycle phases.

Keywords:
Cas9DSB repairG1 phaseV(D)J recombinationend joining

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