Robust DNA repair in PAXX-deficient mammalian cells

Alisa Dewan; Mengtan Xing; Marie Benner Lundbæk; Raquel Gago-Fuentes; Carole Beck; Per Arne Aas; Nina-Beate Liabakk; Siri Sæterstad; Khac Thanh Phong Chau; Bodil Merete Kavli; Valentyn Oksenych

doi:10.1002/2211-5463.12380

Robust DNA repair in PAXX-deficient mammalian cells

FEBS Open Bio. 2018 Feb 7;8(3):442-448. doi: 10.1002/2211-5463.12380. eCollection 2018 Mar.

Authors

Affiliations

¹ Department of Clinical and Molecular Medicine (IKOM) Norwegian University of Science and Technology Trondheim Norway.
² Present address: Centre for Immune Regulation and Department of Immunology University of Oslo and Oslo University Hospital-Rikshospitalet Oslo Norway.
³ Present address: KG Jebsen Coeliac Disease Research Centre University of Oslo Oslo Norway.
⁴ St. Olavs Hospital Trondheim University Hospital, Clinic of Medicine Norway.

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, 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.

Keywords: CH12F3; HAP1; IgA; class switch recombination; etoposide; zeocin.