GSE129895
Phosphorylation at the S2056 1 cluster of DNA-PKcs is dispensable for lymphocyte development and class switch recombination in mice.
The classical non-homologous end-joining (cNHEJ) pathway is a major DNA double-strand break repair pathway in mammalian cells and is required for lymphocyte development and maturation. The DNA-dependent protein kinase (DNA-PK) is a cNHEJ factor that encompasses the Ku70-Ku80 (KU) heterodimer and the large catalytic subunit (DNA-PKcs). In mouse models, loss of DNA-PKcs (DNA-PKcs-/-) abrogates end-processing (e.g., hairpin-opening), but not end-ligation, while expression of the kinase-dead DNA-PKcs protein (DNA-PKcsKD/KD) abrogates end-ligation, suggesting a kinases-dependent structural function of DNA-PKcs during cNHEJ. Lymphocyte development is abolished in DNA-PKcs-/- and DNA-PKcsKD/KD mice due to the requirement for both hairpin-opening and end-ligation during V(D)J recombination. DNA-PKcs itself is the best-characterized substrate of DNA-PK. The S2056-cluster is the best characterized auto-phosphorylation site on human DNA-PKcs. Here we show that radiation can induce phosphorylation of murine DNA-PKcs at the corresponding S2053 and generated knockin mouse models with alanine- (DNA-PKcsPQR) or phospho-mimetic aspartate (DNA-PKcsSD) substitutions at the S2053 cluster.  Despite moderate radiation sensitivity in the DNA-PKcsPQR/PQR fibroblasts and lymphocytes, both DNA-PKcsPQR/PQR and DNA-PKcsSD/SD mice retain normal kinase activity, and undergo efficient V(D)J recombination and class switch recombination, indicating that phosphorylation at the S2053-cluster of mouse DNA-PKcs (corresponding to S2056 of human DNA-PKcs), although important for radiation resistance, is dispensable for the end-ligation and hairpin-opening function of DNA-PK essential for lymphocyte development. Overall design: HTGTS analyzes thousands of CSR junctions between the 5' of Sµ and its joining partner genome wide, including both internal deletion (Sµ-Sµ) and potentially productive switching (Sµ-S?1). We also used HTGTS to uncover a prominent shift to MH, extensive erosion within switch region, and relative increase of inversion in DNA-PKcs-/- B cells with only moderate CSR defects. So we performed HTGTS analyses of CSR junctions from DNA-PKcsPQR/PQR B cells along with control DNA-PKcs-/- B cells.  While the results confirmed S region erosion and increased MH usage in DNA-PKcs-/- B cells, no alteration in MH usage and Sµ junction distribution is noted in DNA-PKcsPQR/PQR B cells.