A single XLF dimer bridges DNA ends during nonhomologous end joining

Abstract

Nonhomologous end joining (NHEJ) is the primary pathway of DNA double-strand-break repair in vertebrate cells, yet how NHEJ factors assemble a synaptic complex that bridges DNA ends remains unclear. To address the role of XRCC4-like factor (XLF) in synaptic-complex assembly, we used single-molecule fluorescence imaging in Xenopus laevis egg extract, a system that efficiently joins DNA ends. We found that a single XLF dimer binds DNA substrates just before the formation of a ligation-competent synaptic complex between DNA ends. The interaction of both globular head domains of the XLF dimer with XRCC4 is required for efficient formation of this synaptic complex. Our results indicate that, in contrast to a model in which filaments of XLF and XRCC4 bridge DNA ends, binding of a single XLF dimer facilitates the assembly of a stoichiometrically well-defined synaptic complex.

Figure 1:. XLF-XRCC4 interaction is required for NHEJ in Xenopus egg extract.

(A) Bio-layer interferometery (BLI) measurements of interaction between point mutants of XRCC4 and C-terminally truncated XLF (XLF¹⁻²²⁶). Traces represent the average and the shading around each trace represents the standard deviation of three replicates. (B) Alignment of human (Hs) and Xenopus laevis (Xl) XLF and XRCC4 around residues required for the XLF-XRCC4 interaction: XlXLF L68 (HsXLF L65, blue), XlXLF L117 (HsXLF L115, red), XlXRCC4 K104 (HsXRCC4 K99, cyan)^,, and XlXRCC4 F111 (HsXRCC4 F106, chartreuse). (C-D) Bulk NHEJ assay in egg extract immunodepleted of XLF (C) or XRCC4 (D) and supplemented with purified recombinant protein. The molar ratio of recombinant protein:DNA ends for XLF and XRCC4:LIG4 in the rescue experiments shown in C and D is 500:1 and 50:1, respectively. Δmock, immunodepletion with nonspecific rabbit IgG; ΔX4, XRCC4 depletion, ΔXLF, XLF depletion; X4:L4, recombinant XRCC4:LIG4 complex. DNA species: scc, supercoiled closed-circular; lin, linear; oc, open-circular; dim, dimer; mult, multimer. Uncropped images of C and D are in Supplementary Data Set 1.

Figure 2:. XLF-XRCC4 interaction is required for SR-complex formation

(A) Schematic of intramolecular single-molecule FRET reporter for monitoring short-range synaptic complex (SR-complex) formation. DNA is tethered internally via a biotin-streptavidin interaction and labeled 7 bp from each end with Cy3 (Donor) and Cy5 (Acceptor) fluorophores. No FRET is detected when DNA ends are untethered or in the long-range synaptic complex (LR-complex). Close alignment of DNA ends within the SR-complex is indicated by energy transfer between Cy3 and Cy5. The data shown in B and C were collected using a 0.5 s exposure time, alternating between two frames of Cy3 excitation and one frame of Cy5 excitation. The data shown in D and E were collected using a 1 s exposure with a 1 s delay between exposures, alternating between four frames of Cy3 excitation and one frame of Cy5 excitation. (B-C) Kinetics of SR-complex formation in extract depleted of XLF or XRCC4 and supplemented with purified recombinant protein, as in Fig. 1C-D. The mean fraction of FRET-positive (SR-complex or ligated) substrates is plotted as a function of time after extract addition. Error bars represent the minimum and maximum values obtained from multiple experimental replicates. See Supplementary Table 2 for sample sizes. (D) Sample smFRET trajectories showing SR-complex formation and dissociation in XRCC4-immunodepleted extract supplemented with either XRCC4^WT:LIG4^K278R or XRCC4^K104E:LIG4^K278R. Catalytically inactive LIG4^K278R was used here and in panel E to prevent ligation of DNA ends. (E) Cumulative distribution functions of SR-complex lifetimes. The difference between the two conditions was not statistically significant (p = 0.45, log-rank test). See Supplementary Table 3 for sample sizes.

Figure 3:. Three-color imaging of XLF binding and SR-complex formation

(A) 3-color single-molecule imaging of FRET between Cy3 and Cy5-labeled DNA ends, along with binding of Halo-tagged XLF protein labeled with Alexa Fluor 488 (AF488-XLF). Top panel: Intensity in the Cy3 (cyan) and Cy5 (red) channels with excitation of Cy3. Second panel: Calculated Cy3-Cy5 FRET efficiency. Third panel: AF488 intensity, expressed as a multiple of the intensity of single-AF488 reference spots. Bottom panel: Cy5 intensity with direct excitation. See Supplementary Fig. 2F for additional traces. (B) Histogram of AF488 intensity, expressed as a multiple of the average intensity of single-AF488 reference spots. The black curve includes all substrates, while the red curve includes only the subset of frames within 10 s of the transition to high FRET. (C) Average of AF488-XLF and Cy5-XLF stoichiometry for traces aligned at the time of FRET onset. AF488 intensity, normalized to the intensity of single-AF488 reference spots, was divided by the protein labeling fraction to give XLF protein stoichiometry. Cy5 intensity, normalized to single-Cy5 intensity (see Supplementary Fig. 3A), was divided by protein labeled fraction after subtracting 1 to account for the single Cy5 label on the DNA. Black curve shows data from a mock AF488 imaging experiment without AF488-XLF protein. (D) Average stoichiometry of XLF nonspecifically labeled on cysteines with Cy5-maleimide, aligned at the time of FRET onset. Cy5-mal-XLF stoichiometry was calculated as for Cy5-XLF in panel (C). Black curve shows data from a control experiment in the absence of Cy5-mal-XLF. The decay of the black curve below zero results from photobleaching of the Cy5 label on DNA.

Figure 4:. End joining in the presence of a synthetic tandem dimer of XLF

(A) Schematic of XLF tandem dimer (tdXLF) construct. Two XLF coding sequences are concatenated by a flexible 15-amino acid-long linker. Asterisks denote point mutations. (B) Dose-dependence of end joining as a function of tdXLF concentration for WT/WT and WT/L68D,L117D constructs. Black wedges represent 2-fold serial dilution series from 910 nM to 0.44 nM. Control reactions not supplemented with tdXLF are labeled “-”. lin, linear DNA substrate; oc, open-circular product; mult, dimeric and multimeric products. An uncropped image of B is in Supplementary Data Set 1. (C) Quantification of product formation in the gel in panel (B). Fraction joined was quantified as background-subtracted intensity of product bands divided by total background-subtracted intensity of substrate and product bands. (D) Histogram of FRET efficiency from tdXLF rescue of smFRET intramolecular circularization experiments, accumulated over 20 min.

Figure 5:. Characterization of human XLF^L115A and Xenopus XLF^L117A

(A) XLF-XRCC4 DNA pull-down assay. Beads coated with a biotinylated 1000 bp DNA fragment were incubated with a soluble 500 bp DNA fragment and the indicated proteins. Deproteinized supernatant and bead-bound fractions were separated on a 1x TBE agarose gel. 1000 and 500 bp DNA fragments alone were loaded in the first two lanes. (B)) Bulk NHEJ assay in egg extract immunodepleted of XLF (ΔXLF) and supplemented with purified recombinant protein. lin, linear substrate; oc, open-circular products; scc, supercoiled closed-circular products; mult, dimeric and higher-order multimeric products. Uncropped images of A and B are in Supplementary Data Set 1. (C)) Bio-layer interferometry of XRCC4 binding by wild-type and L115A/L117A mutant human and Xenopus XLF. Response curves represent the average of two experimental replicates for each condition. XLF variants were tested at a concentration of 250 nM, with the exception of hXLF^{1−224,L115D}, which was tested at 2 μM. Fitted K_on and K_off values are listed in Supplementary Table 1. (D) Model of the role of XLF in short-range synaptic complex assembly. Binding of both head domains of XLF to XRCC4:LIG4 is required for SR-complex formation, which may involve (i) direct engagement of both DNA ends by the XLF:XRCC4:LIG4 ternary complex, or (ii) a conformational change in the DNA-PK holoenzyme that is facilitated allosterically by binding of the XLF:XRCC4:LIG4 ternary complex. In the latter model, conformational flexibility in LIG4 (red double arrows) permits binding and unbinding of the LIG4 catalytic domain to DNA ends.