To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining (NHEJ) is the DNA repair process that fixes double-strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination (CSR). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC4, DNA ligase 4, DNA-PKcs, Artemis, and XLF. Paralog of XRCC4 and XLF (PAXX) is the recently described accessory NHEJ factor that structurally resembles XRCC4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom-generated and commercially available NHEJ-deficient human haploid HAP1 cells, PAXX^Δ, XRCC4^Δ , and XLF^Δ . In our studies, HAP1 PAXX^Δ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild-type controls. By contrast, XRCC4^Δ and XLF^Δ HAP1 cells possessed significant DNA repair defects measured as sensitivity to double-strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR, we generated and characterized Paxx^-/- and Aid^-/- murine lymphoid CH12F3 cells. CSR to IgA was nearly at wild-type levels in the Paxx^-/- cells and completely ablated in the absence of activation-induced cytidine deaminase (AID). In addition, Paxx^-/- CH12F3 cells were hypersensitive to zeocin when compared to wild-type controls. We concluded that Paxx-deficient mammalian cells maintain robust NHEJ and CSR.

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