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

The DNA-dependent protein kinase (DNA-PK), which is composed of the KU heterodimer and the large catalytic subunit (DNA-PKcs), is a classical non-homologous 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 micro-homology – 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. To understand the role of T2609 phosphorylation in CSR and DSB repair specifically, we evaluated the role of T2609 phosphorylation in physiological DSB repair during CSR using germline mouse models. As in the case of V(D)J recombination, T2609A mutation also does not affect CSR efficiency. But CSR junctions from DNA-PKcs5A/5A B cells revealed a preferential loss of Sμ-Sε junctions, increased MH usage, increased use of downstream switch regions (consistent with excessive end resection) and increased inter-chromosomal translocations ((+) strand preys)– all consistent with cNHEJ deficiency and compensatory Alt-EJ-mediated CSR. Together these findings support the role of DNA-PKcs T2609 phosphorylation in promoting cNHEJ-mediated CSR. In the absence of T2609 phosphorylation, the increased dependence on the Alt-EJ pathway, together with the extensive length of the Sg1 region (12.5kb in 129SV strain), the dense RGYW/AGCT sequence motifs, and the sequence-independent 3D interactions (synapses) between Sµ-Sg1, lead to robust Alt-EJ-mediated CSR to IgG1 in DNA-PKcs5A/5A B cells. Thus, our analyses identify a novel role of DNA-PKcs T2609 phosphorylation in repair pathway choice during CSR.