Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining

Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway that is required for assembly of antigen receptor variable region gene segments by V(D)J recombination. Recombination activating gene endonuclease initiates V(D)J recombination by generating DSBs between two V(D)J coding gene segments and flanking recombination signal sequences (RS), with the two coding ends and two RS ends joined by C-NHEJ to form coding joins and signal joins, respectively. During C-NHEJ, recombination activating gene factor generates two coding ends as covalently sealed hairpins and RS ends as blunt 5'-phosphorylated DSBs. Opening and processing of coding end hairpins before joining by C-NHEJ requires the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). However, C-NHEJ of RS ends, which do not require processing, occurs relatively normally in the absence of DNA-PKcs. The XRCC4-like factor (XLF) is a C-NHEJ component that is not required for C-NHEJ of chromosomal signal joins or coding joins because of functional redundancy with ataxia telangiectasia mutated kinase, a protein that also has some functional overlap with DNA-PKcs in this process. Here, we show that XLF has dramatic functional redundancy with DNA-PKcs in the V(D)J SJ joining process, which is nearly abrogated in their combined absence. Moreover, we show that XLF functionally overlaps with DNA-PKcs in normal mouse development, promotion of genomic stability in mouse fibroblasts, and in IgH class switch recombination in mature B cells. Our findings suggest that DNA-PKcs has fundamental roles in C-NHEJ processes beyond end processing that have been masked by functional overlaps with XLF.