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

Abstract

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 (<i>DNA-PKcs</i>^{<i>KD</i>/<i>KD</i>} ) 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 <i>DNA-PKcs</i>^{<i>KD</i>/<i>KD</i>} , but not <i>DNA-PKcs</i>^<i>-/-</i> , B cells. Meanwhile, the residual joints from <i>DNA-PKcs</i>^<i>KD/KD</i> cells and the efficient Sµ-Sγ1 junctions from <i>DNA-PKcs</i>^<i>-/-</i> B cells both display similar preferences for small (2-6 nt) microhomologies (MH). In <i>DNA-PKcs</i>^<i>-/-</i> cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Sε 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.