SFPQ•NONO and XLF function separately and together to promote DNA double-strand break repair via canonical nonhomologous end joining

Abstract

A complex of two related mammalian proteins, SFPQ and NONO, promotes DNA double-strand break repair via the canonical nonhomologous end joining (c-NHEJ) pathway. However, its mechanism of action is not fully understood. Here we describe an improved SFPQ•NONO-dependent in vitro end joining assay. We use this system to demonstrate that the SFPQ•NONO complex substitutes in vitro for the core c-NHEJ factor, XLF. Results are consistent with a model where SFPQ•NONO promotes sequence-independent pairing of DNA substrates, albeit in a way that differs in detail from XLF. Although SFPQ•NONO and XLF function redundantly in vitro, shRNA-mediated knockdown experiments indicate that NONO and XLF are both required for efficient end joining and radioresistance in cell-based assays. In addition, knockdown of NONO sensitizes cells to the interstrand crosslinking agent, cisplatin, whereas knockdown of XLF does not, and indeed suppresses the effect of NONO deficiency. These findings suggest that each protein has one or more unique activities, in addition to the DNA pairing revealed in vitro, that contribute to DNA repair in the more complex cellular milieu. The SFPQ•NONO complex contains an RNA binding domain, and prior work has demonstrated diverse roles in RNA metabolism. It is thus plausible that the additional repair function of NONO, revealed in cell-based assays, could involve RNA interaction.

Figure 1.

SFPQ•NONO and XLF independently stimulate end joining in vitro. (A) Alignment of human DBHS proteins. DBHS homology region (orange) mediates RNA binding, dimerization, and higher-order multimerization. N-terminal IDR of SFPQ (teal) mediates DNA binding. Other IDRs (grey) are shown. (B) SFPQ•NONO Superdex 200 gel filtration profile, showing A₂₈₀ as a function of elution volume. Positions of SFPQ•NONO elution peak and molecular weight standards (run independently) are indicated. Refer to Supplementary Data for selectivity curve. (C) SDS-PAGE analysis of indicated region of gel filtration profile. Positions of SFPQ and NONO are indicated at right, and molecular weight markers at left (kDa). (D) SDS-PAGE analysis of NHEJ factor preparations. Red arrowheads indicate relevant polypeptides. The 70 kDa species in the DNA ligase IV•XRCC4 preparation is believed to be an unrelated contaminant. (E) Results of end joining reactions. Assays contained 100 nM of 1503 bp BglII-linearized DNA substrate, 100 nM DNA ligase IV•XRCC4 (L4•X4), 100 nM Ku, and other factors as indicated (nM). Size markers are indicated at left (bp). Substrate (S), product (P), and upper dye marker (M, 17 000 bp) are indicated at right. (F) Selected samples from Panel E, displayed as electropherogram traces. Peaks are labeled as in panel E. All panels are presented at the same scale. (G) Fraction of substrate converted to product. Mixtures are as follows S•N/X, SFPQ•NONO/XLF; X/P, XLF/PAXX; S•N/P, SFPQ•NONO/PAXX. Dotted line indicates level of activity in basal reactions without stimulatory factors.

Figure 2.

Dependence of end joining on DNA end concentration. (A) End-joining reactions were performed as in Figure 1 using equal masses of each of the indicated substrates. DNA end concentrations are indicated (nM). Reactions contained 100 nM of the indicated factors. Labeling as in Figure 1. (B) Quantification of Panel A, plotted as fraction substrate converted to product. (C) Quantification of Panel A, showing molar concentration of product (joints) versus initial concentration of substrate (ends) on a log–log scale.

Figure 3.

Dependence of end joining on DNA end structure. Substrates were of uniform 1503 bp length and had cohesive 5΄ overhang (BglII), blunt (SmaI), or cohesive 3΄ overhang (SacI) ends. (A) Results of end joining assay. Reactions contained 100 nM of indicated factors. Labeling as in Figure 1. (B) Quantification of results from panel A.

Figure 4.

Polarity of end joining. (A) Heteroduplex pairing model. Ends are designated as ‘a’ or ‘b’ with arrow denoting a to b polarity. Substrates have an off-center BamHI site to facilitate analysis of product distribution. In this model, pairing is constrained to a side-by-side, parallel arrangement, favoring a-a and b-b junctions. (B) End joining in different relative orientations creates diagnostic BamHI fragments as indicated. Distribution of reaction products discriminates between a heteroduplex pairing model and a sequence-independent, protein-mediated pairing model. (C) Results of end joining assay. Reactions contained 100 nM of indicated factors. Indicated samples were subjected to limit digestion with BamHI. Positions of size markers are indicated at left (bp), and diagnostic BamHI fragments arising from unligated substrate and different types of joints at right. (D) Quantification of results from panel C.

Figure 5.

Clonogenic survival assays. (A) Lentiviral shRNA-mediated knockdown of NONO and XLF. Proteins from total cell lysates were resolved by SDS-PAGE and immunoblotting was performed separately with anti-NONO, anti-XLF, and anti β-actin as a loading control. Panels are from same experiment. (B) Survival following γ-ray exposure at indicated doses. HeLa cells were transduced with indicated lentiviral shRNA vectors, exposed after 48 h to ¹³⁷Cs γ-rays, and incubated to allow colony formation. Surviving fraction was normalized to reflect plating efficiency of non-irradiated control cells. Assays were performed in triplicate. Error bars (smaller than symbols in some groups) denote standard deviation. Dotted lines indicate dose reduction factor (NONO versus control shRNA) at 10% survival. (C) Survival following cisplatin exposure at indicated concentrations for 1 h. Results were pooled from two independent experiments, each performed in triplicate, for a total of six replicates for each condition. Surviving fraction was normalized to plating efficiency of control cells in each experiment. Error bars denote standard deviation.

Figure 6.

DSB repair assays. (A) Plasmid rejoining assay design. Substrate is a linearized plasmid with EcoRV and AfeI ends. Boxes denote 6 bp of identical sequence at each end. Precise c-NHEJ-mediated joining results in a 6 bp direct repeat. Microhomology-mediated end joining (MHMEJ) results in a 6 bp deletion, leaving one copy of the repeat and creating a novel BstXI site. Positions of PCR primers are indicated. Drawing is not to scale. (B) Results of plasmid rejoining assay. HeLa cells were transduced with indicated lentiviral shRNA vectors and after 48 h transfected with EcoRV/AfeI-linearized reporter plasmid. After a further 48 h, episomal DNA was recovered by Hirt extraction, junctional sequences were amplified by PCR, and products were resolved using an Agilent Bioanalyzer. Indicated samples were subjected to limit digestion with BstXI. Positions of BstXI-resistant PCR product (180 bp, resolves as doublet, see text), larger BstXI cleavage product (120 bp), and lower size marker (M, 50 bp) are indicated. Lower size marker obscures the smaller BstXI cleavage product. (C) Results of repair foci assay. Assays were performed using HeLa cells as described in Materials and Methods. Cells were irradiated and allowed to recover as indicated, processed, and 50 cells per treatment group were scored for 53BP1 foci. Results are presented as histograms, with mean number of foci per cell and standard deviation indicated. Statistical analysis was performed by ANOVA.

Figure 7.

Model. Substrate DNAs are shown at top, with ends designated as ‘a’ and ‘b’ and arrow showing a to b polarity. Complexes (I) and (II) depict side-by-side and end-to-end substrate pairing mediated by DNA ligase IV/XRCC4/XLF filament. Pairing is protein-mediated and DNA sequence-independent. Joining occurs with random polarity (equal probability of ab-ab, ba-ba, ab-ba, ba-ab joints). Complexes (III) and (IV) depict an alternative side-by side pairing geometry, which has not been observed directly but is consistent with known biochemical characteristics of SFPQ•NONO (see text). Loop structure allows joining by protomeric DNA ligase IV•XRCC4 complex (XLF not required). Joining occurs with random polarity. Complexes (V) and (VI) depict side-by-side pairing stabilized by sequence-dependent DNA strand invasion. Because substrates are constrained to a parallel geometry, inverted (ab-ba, ba-ab) joints are favored. This model is excluded by data in Figure 3. Complexes also contain Ku protein, although the stoichiometry and position relative to other factors is uncertain. For simplicity, Ku is not shown.