Phosphorylation of DNA-PKcs at the S2056 cluster ensures efficient and productive lymphocyte development in XLF- deficient mice

The nonhomologous end-joining (NHEJ) pathway is a major DNA double -strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activat-ing the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA- PKcs deletion only moderately impairs end-ligation, the expression of kinase- dead DNA- PKcs completely abrogates NHEJ. Active DNA- PK phosphoryl-ates DNA- PKcs at two clusters-PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid- based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. Xlf-/- mice have substan-tial peripheral lymphocytes that are completely abolished by the loss of DNA- PKcs, the related ATM kinases, other chromatin-associated DNA damage response fac-tors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2- C-terminal regions, sug-gesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA- PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf-/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from DNA-PKcsPQR/PQRXlf -/- mice are less efficient and the residual junc-tions display decreased fidelity and increased deletion. These findings establish a role for DNA- PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA- PKcs in end-ligation.

Automated summary

The nonhomologous end-joining (NHEJ) pathway is crucial for DNA double-strand break repair and lymphocyte development. The Ku70 and Ku80 heterodimer initiates NHEJ by recruiting DNA-dependent protein kinase (DNA-PKcs). Phosphorylation of DNA-PKcs at S2056 is important for end-ligation, but its physiological significance is still unclear. Xlf deficiency leads to defective lymphocyte development, which can be restored by DNA-PKcs phosphorylation. These findings highlight the role of DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ and its synergy with XLF.