Robust DNA repair in PAXX‐deficient mammalian cells

Abstract

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, XRCC 4, DNA ligase 4, DNA ‐ PK cs, Artemis, and XLF . Paralog of XRCC 4 and XLF ( PAXX ) is the recently described accessory NHEJ factor that structurally resembles XRCC 4 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 HAP 1 cells, PAXX Δ , XRCC 4 Δ , and XLF Δ . In our studies, HAP 1 PAXX Δ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild‐type controls. By contrast, XRCC 4 Δ and XLF Δ HAP 1 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 CH 12F3 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 −/− CH 12F3 cells were hypersensitive to zeocin when compared to wild‐type controls. We concluded that Paxx ‐deficient mammalian cells maintain robust NHEJ and CSR .

Normal development of mice lacking PAXX, the paralogue of XRCC4 and XLF

“…Furthermore, it did not affect growth, fertility, development of lymphoid organs, or genomic stability of mice, when compared to heterozygous or WT littermates. In addition, PAXX was dispensable for CSR to IgG1 in primary B splenocytes, which is in line with data obtained using knockout B-cell CH12F3 cell lines, where inactivation of Paxx did not affect CSR to IgA [14,27]. Finally, we analyzed brain sections of WT and Paxx À/À mice and found no difference in neurodevelopment using two independent methods, Nissl staining and TUNEL assay (not shown).…”

Generation of a Mouse Model Lacking the Non-Homologous End-Joining Factor Mri/Cyren

“…Fluorescence-activated cell sorting (FACS) analysis was performed as previously described [11,32]. Briefly, spleens and thymi were isolated from 2-month-old mice, and splenocytes and thymocytes were counted using Countess™ Automated Cell Counter (Invitrogen, Carlsbad, CA, United States); the cell suspension was spun down and diluted with PBS to obtain a final cell concentration of 2.5 × 10 7 /mL.…”

“…The DSBs sensitivity assay was performed as previously described [10,32,33]. Human nearly-haploid HAP1 cells were generated by the Horizon Discovery Group (Waterbeach, Cambridge, UK, #HZGHC005061c001 and #HZGHC005061c004) and are commercially available.…”

“…Deficiency for XRCC4, LIG4, XLF, or DNA-PKcs results in hypersensitivity to DSBs in human HAP1 cells [10,32,33]. To determine the effect of Mri on DSB sensitivity, we obtained two independent clones of MRI-deficient HAP1 cells as well as WT and XRCC4-deficient controls.…”

Corrigendum to: Robust DNA repair in PAXX‐deficient mammalian cells

“…In Figure B of the paper by Dewan et al . , the western blot lanes were incorrectly labelled. From left to right, the lanes should have been labelled as WT, PAXX ∆ , DNA‐PKcs ∆ and PAXX ∆ DNA‐PKcs ∆ .…”