Kinase-dependent structural role of DNA-PKcs during immunoglobulin class switch recombination

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD, but not DNA-PKcs-/- B cells. Meanwhile, the residual joints from DNA-PKcsKD/KDcells and the efficient Sμ-Sγ1 junctions from DNA-PKcs-/- B cells both display similar preferences for small (2–6 nt) microhomologies (MH). In DNA-PKcs-/- cells, Sμ-Sγ1 joints are more resistant to inversions and extensive resection than Sμ-Se and Sμ-Sμ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR. To ascertain how different DNA-PKcs mutations (null vs KD) affect CSR in an isotype dependent manner, we employed the High Throughput Genome Translocation Sequencing (HTGTS) (1) method to analyze CSR junctions in DNA-PKcsKD/KD and DNA-PKcs-/- B cells with preassembly IgH and L chains (HL). We measured switching efficiency, efficiency and size of small MH in DNA-PKcs-/- and DNA-PKcsKD/KD B cells in contrast to cNHEJ-deficient Xrcc4-/- B cells, as well as insertions and deletions than both Sμ-Sμ and Sμ-Sε junctions. Finally our analyses also identified long MH mediated inter-chromosomal translocations in DNA-PKcsKD/KD B cells and a reduced number of G mutations in 5’Sμ in repair deficient B cells. Since all our experimental mice carry the preassembled IgH on the 129 ackground, we replaced the IgH switch region (from JH4 to the last Cα exon, chr12 114, 494, 415–114, 666, 816) of the C57/BL6-based mm9 with the corresponding region in the AJ851868.3 (NCBI gene accession no. AJ851868.3) 129 IgH sequence (1415966–1592715) to generate the mm9sr (switch region replacement) genome. 1. Hu J, et al. (2016) Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11:853–871.