PAXX/Ku interaction is rate limiting for repair of double-strand DNA breaks requiring end processing

Abstract

In mammalian cells, DNA double strand breaks (DSBs) are primarily repaired via classical non-homologous end joining (c-NHEJ)-one of the most essential DNA repair pathways. As NHEJ does not utilize a template, this type of repair is the default mechanism for eliminating DSBs occurring in non-cycling cells. NHEJ is a crucial process in mammals, and defects of this repair pathway often result in immunological impairment owing to failure of somatic recombination in lymphocytes and improper neuronal biogenesis. The NHEJ machinery assembles in a stepwise process at DSBs and proceeds via several key repair phases including break recognition, mediated by Ku proteins (Ku70/80 heterodimer binding to DNA), DNA ends processing, and finally DNA ligation. DNA end-bound Ku recruits the large kinase protein DNA-PKcs, and downstream repair-facilitating components such as PAXX, XLF and XRCC4/Ligase IV complex that together facilitate the repair reaction. Processing of DNA breaks can require both nucleotide removal and incorporation, involving a plethora of enzymes such as nucleases and polymerases. It is currently not known which step, if any, limits the completion of the repair process. Here, we describe a single conserved amino acid substitution in PAXX protein Ku70/80 contact interface that dramatically stabilizes the repair complex. This mutation leads to co-dependent mislocalization of PAXX and Ku to the nucleoli. Surprisingly, this novel PAXX gain-of-function mutation accelerates NHEJ repair but only of DSBs that require end processing such as radiation-induced DSBs. Thus, in mammalian NHEJ, the repair complex stability is rate-limiting for the overall repair reaction of DSBs.

Keywords: DNA damage; DNA damage response; DNA repair; DSB; NHEJ; PAXX; double strand break; molecular biology; non-homologous end joining; nucleolus.

Figure 1

PAXX K193R mutation leads to constitutive DNA-independent interaction with Ku70.A, PAXX and Ku70 proteins linear cartoons with key interacting and functional regions and all PAXX lysine (K) residues indicated. Arrows point to major intermolecular interactions identified in this study. B, immunoprecipitation of endogenous Ku70 protein with FLAG-PAXX WT/K193R variants transiently transfected into U2OS PAXX knockout cells. Indicated above the blot image, increasing amounts of ethidium bromide (EtBr). Bar graphs above the WB panel indicate the normalised mean signal intensity for Ku70 (n = 4; SD; ∗p = 0,011). Position of molecular weight marker is depicted on the right. C, electrophoretic mobility shift assay (EMSA) of purified Ku dimer incubated with either DNA alone, Ku only or decreasing amounts of WT/K193R recombinant PAXX protein (protein concentration is depicted above lanes in μM). Arrows depict DNA/Ku/PAXX complexes containing either a single Ku70/Ku80 molecule or two Ku70/Ku80 molecules bound to the probe. Single asterisk depicts PAXX bound to a complex with a single Ku70/Ku80 molecules. Double asterisk depicts PAXX bound to a complex with a two Ku70/Ku80 molecules. In lanes three and seven no Ku protein was added.

Figure 2

PAXX K193R mutation results in nucleolar PAXX/Ku complex formation.A, immunofluorescence staining showing representative images of various PAXX mutants (FLAG-tagged; plasmid-derived) and Ku80 (endogenous) proteins in U2OS PAXX knockout background. DAPI – depicts nuclear stain. B, quantification of immunofluorescence experiments from panel A. “Nuclear” pattern is defined absence of staining in the nucleoli. “Nuclear and nucleolar” pattern is defined by the presence of staining both in the nucleus and the nucleoli. n – the number of scored nuclei; scale bar = 50 μm.

Figure 3

PAXX K193R mutation accelerates repair of “blocked” DSBs.A–C, DSB repair foci count at indicated time points after exposure of U2OS PAXX KO cells expressing either PAXX WT or PAXX K193R protein to zeocin, X-rays (IR) or etoposide. (Ctrl) denotes the number of observed DSB foci prior to exposure to DNA-damaging agent (for A and B n = 3, median, ∗∗∗∗p < 0,001; for C n = 2, median). Numbers above 4 h and 8 h data points indicate median number of unrepaired foci. D, in vitro DNA ligation assays. The graph shows quantification of ligation reactions from three independent experiments (n = 3), where 100% was the level of ligation observed in the absence of PAXX. The gel picture in the lower panel shows a representative migration of the ligation products and the non-ligated template DNA. E, leftpanel shows the quantification of mean indel size (n = 3, SD; n.s. denotes statistically not significant) whereas right panel depicts proportion of deletions versus insertions observed after repair of CRISPR-induced chromosomal DSBs (n = 3).

Figure 4

Cryo-EM structure of Ku70/80 with the PAXX peptide mutant K193R.A, cryo-EM map of Ku70/80 with the PAXX peptide mutant K193R to 2.8 Å resolution. Ku70 is in yellow, Ku80 in green, DNA in gray, and the PAXX peptide in blue with an arrow indicating the location of the mutated residue. B, enlarged inset of the R193 residue shown in blue, interacting with the backbone oxygens of Ku80 shown in green, and E250 of Ku70 C) Enlarged inset of the PAXX WT, K193 residue shown in blue with surrounding Ku80 residues labeled and shown in green.