DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination

Proc Natl Acad Sci U S A. 2020 Sep 15;117(37):22953-22961. doi: 10.1073/pnas.2007455117. Epub 2020 Aug 31.


The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical nonhomologous end-joining (cNHEJ) factor. Naïve B cells undergo class switch recombination (CSR) to generate antibodies with different isotypes by joining two DNA double-strand breaks at different switching regions via the cNHEJ pathway. DNA-PK and the cNHEJ pathway play important roles in the DNA repair phase of CSR. To initiate cNHEJ, KU binds to DNA ends and recruits and activates DNA-PK. Activated DNA-PK phosphorylates DNA-PKcs at the S2056 and T2609 clusters. Loss of T2609 cluster phosphorylation increases radiation sensitivity but whether T2609 phosphorylation has a role in physiological DNA repair remains elusive. Using the DNA-PKcs5A mouse model carrying alanine substitutions at the T2609 cluster, here we show that loss of T2609 phosphorylation of DNA-PKcs does not affect the CSR efficiency. Yet, the CSR junctions recovered from DNA-PKcs5A/5A B cells reveal increased chromosomal translocations, extensive use of distal switch regions (consistent with end resection), and preferential usage of microhomology-all signs of the alternative end-joining pathway. Thus, these results uncover a role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ repair pathway choice during CSR.

Keywords: DNA-PKcs; T2609 autophosphorylation; alternative end joining; class switch recombination; nonhomologous end joining.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B-Lymphocytes / immunology
  • DNA Repair / physiology
  • DNA-Activated Protein Kinase / genetics*
  • DNA-Activated Protein Kinase / metabolism*
  • DNA-Binding Proteins / metabolism
  • Female
  • Gene Rearrangement
  • Humans
  • Immunoglobulin Class Switching / genetics*
  • Immunoglobulin Class Switching / physiology
  • Immunoglobulin Switch Region / genetics
  • Immunoglobulins / genetics
  • Ku Autoantigen / metabolism
  • Male
  • Mice
  • Mice, 129 Strain
  • Phosphorylation
  • Recombination, Genetic / genetics
  • Translocation, Genetic


  • DNA-Binding Proteins
  • Immunoglobulins
  • DNA-Activated Protein Kinase
  • Ku Autoantigen