DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair

Abstract

XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.

Fig. 1

Crystal structure of PAXX. (A) Domain architecture of human PAXX and other XRCC4-superfamily members. Sequence identities between human PAXX and XRCC4, XLF and SAS6 are 10.9, 11.2 and 10.1% respectively. (B) Structure of a PAXX dimer with the two polypeptide chains shown in cyan and pink. The N- and C-termini of the structure are indicated as N-ter and C-ter respectively. (C) Residues mediating the PAXX dimerization interface, with β-sheet sandwich-like packing between protomers in adjacent asymmetric units. The packing of each strand from different protomers is shown in surface and stick representations. Black dotted lines are hydrogen bonds between side-chain pairs (S95 and S99, T97 and T97, S99 and S95) and between the side-chain of D108 of each protomer with the main chain of A104 of the adjacent protomer in the crystals. (D) ES-MS profile showing that PAXX^1-204 is a dimer. Three charge states are observed for the dimer. The main charge state 13+ is labeled in the mass spectrum. (E) Comparison of XRCC4-superfamily members. The head domains of PAXX (cyan), XRCC4 (silver), XLF (magenta) and SAS6 (lime) are superimposed.

Fig. 2

The PAXX C-terminus interacts with Ku. (A) GFP-pulldowns showing that GFP-PAXX^WT but not GFP-PAXX^1-145 transiently overexpressed in 293FT cells interacts with Ku. (B) Co-IP from HeLa nuclear extracts showing that endogenous PAXX and Ku interact. (C) Sequence alignment of the C-termini of PAXX orthologs. Conserved residues are indicated with reverse shading and similar residues are highlighted in grey. (D) Peptide pulldowns from HeLa nuclear extracts using control (H2AX) or PAXX^177-204 peptides analysed by silver staining. “M” represents protein markers, and numbers represent molecular weights in kDa. (E) GFP-pulldown showing that PAXX residues V199 and F201 are required for Ku binding in the context of full-length PAXX.

Fig. 3

PAXX is required for DSB repair in human cells. (A) GFP-tagged PAXX accumulates at sites of laser micro-irradiation in U2OS cells. White arrowheads indicate the path of the laser used to induce DSBs. (B) Clonogenic survival assay showing that PAXX depletion in U2OS cells causes radiosensitivity and that this is rescued by exogenous expression of PAXX^WT but not PAXX^V199A/F201A. In this experiment and those below, error bars represent the standard error of the mean (SEM) from 3 independent experiments. (C) Clonogenic survival assay showing that PAXX^−/− cells are radiosensitive and that PAXX loss is epistatic with XRCC4 depletion. (D) PAXX^−/− cells display persistent γ-H2AX foci after IR. Cells with >5 foci were scored as positive, and were co-stained with Cyclin A to eliminate S and G2 cells from analysis. At least 100 cells were scored per condition. “cl.” indicates clone number. (E) PAXX is required for cellular DSB repair as measured by neutral comet assay. R/D ratios represent mean tail length of cells treated with 40 μg/ml phleomycin for 2 hours and allowed to recover (R) for 2 hours over mean tail length of cells damaged (D) for 2 hours without recovery.

Fig. 4

PAXX promotes NHEJ in vitro and stabilizes NHEJ proteins on damaged chromatin. (A) EMSA of Ku and PAXX derivatives with 50-bp 6-FAM(6-carboxyfluorescein)-labelled DNA. 200 nM PAXX and 20 nM Ku were added where indicated. (B) Stimulation of DNA-end ligation by PAXX. 50 ng of pcDNA3.1(-) digested by EcoRV was incubated with XRCC4/LIG4 (25 nM), Ku (25 nM), PAXX (250 nM) and PAXX^V199A/F201A (250 nM) as indicated (left). Ligation efficiency (right) was calculated as a percentage of ligated plasmid from four independent assays. (C) Chromatin fractionation of PAXX^+/+ and PAXX^−/− cells treated with phleomycin as indicated. Note that DNA-damage-dependent chromatin recruitment of proteins such as RPA, that function in DNA repair pathways other than NHEJ, were unaffected by PAXX loss. (D) Model of PAXX in NHEJ. Two Ku-bound DNA-ends are bound by a PAXX dimer via its C-termini, which stabilizes the NHEJ machinery to promote DNA-end ligation.