A Mechanism to Minimize Errors during Non-homologous End Joining

Abstract

Enzymatic processing of DNA underlies all DNA repair, yet inappropriate DNA processing must be avoided. In vertebrates, double-strand breaks are repaired predominantly by non-homologous end joining (NHEJ), which directly ligates DNA ends. NHEJ has the potential to be highly mutagenic because it uses DNA polymerases, nucleases, and other enzymes that modify incompatible DNA ends to allow their ligation. Using frog egg extracts that recapitulate NHEJ, we show that end processing requires the formation of a "short-range synaptic complex" in which DNA ends are closely aligned in a ligation-competent state. Furthermore, single-molecule imaging directly demonstrates that processing occurs within the short-range complex. This confinement of end processing to a ligation-competent complex ensures that DNA ends undergo ligation as soon as they become compatible, thereby minimizing mutagenesis. Our results illustrate how the coordination of enzymatic catalysis with higher-order structural organization of substrate maximizes the fidelity of DNA repair.

Keywords: NHEJ; end processing; non-homologous end joining; single-molecule; smFRET.

Figure 1.. Joining of diverse end structures in Xenopus egg extracts.

(A) Model of two-stage DNA end synapsis during NHEJ (Graham et al., 2016). (B) Linear DNA molecules with the indicated ends were added to egg extracts and incubated for 90 min. DNA was extracted, and joints were amplified by PCR and analyzed by paired-end Illumina sequencing. Indel length is relative to direct ligation of the top strand depicted for each end pair. Light blue sequence corresponds to nucleotides inserted during joining. Bar graphs were generated by identifying junction reads with the indicated indel length and normalizing to the total number of reads. See Methods for controls regarding fidelity of amplification, library preparation, and sequencing. Analysis of aligned sequencing reads is reported in Data S1.

Figure 2.. Involvement of pol λ and Tdp1 in end processing.

(A to C) Radiolabeled linear DNA molecules with the depicted ends were added to the indicated egg extracts, and reaction samples were stopped at the indicated time points. DNA was extracted, digested, and analyzed by denaturing PAGE and autoradiography. Lower panels, un-joined DNA ends; upper panels, joined DNA ends. Red asterisk indicates radiolabeled strand. Immunodepletion of pol λ alone blocked joining of the ends depicted in (A) (see Figure S1H, lanes 22 to 24). Immunodepleted extracts were supplemented with the following final concentrations of recombinant protein as indicated: pol λ and pol μ, 10 nM; Tdp1, 40 nM. S, input substrate; I, processing intermediate; P, ligation product. Upper panels depict “head-to-tail” joints (see Figure S1B). “Head-to-head” joints show similar products and are shown in Figures S1C, S1E, and S1F.

Figure 3.. Disruption of short-range synapsis inhibits end processing by pol λ and Tdp1.

(A and B) Denaturing PAGE analysis of processing and joining of depicted DNA ends in indicated extracts as in Figure 2. X4, XRCC4; L4^WT and L4^ci, wild-type and catalytically-inactive Lig4, respectively. Recombinant Lig4-XRCC4 and XLF were added to final concentrations of 50 nM and 100 nM, respectively. PKcs-i in DMSO was added to a final concentration of 50 μM. S, input substrate; I, processing intermediate; P, ligation product.

Figure 4.. pol λ acts within the short-range synaptic complex.

(A) Experimental scheme for single-molecule assay of DNA-end synapsis and polymerase activity. D, Cy3 donor fluorophore; A, Cy5 acceptor fluorophore; Q, BHQ10 quencher. Fluorophore positions are noted in Table S1. (B) Example trajectory. The Cy3 donor fluorophore was excited and donor emission (green) and Cy5 acceptor emission (magenta) were recorded (upper panel) and used to calculate FRET efficiency (blue, lower panel). (C) Frequency of quenching events as a function of FRET efficiency immediately prior to quenching under the indicated conditions. Blue, extracts supplemented with dUTP^Q, adenine DNA template base (see (A)) substrate (n = 145 events from 9 independent experiments); red, extracts supplemented with dTTP, adenine DNA template base substrate (n = 16 events from 6 independent experiments); yellow, extracts supplemented with dUTP^Q, thymine DNA template base (n = 8 events from 6 independent experiments). See Methods for calculation of quenching frequency and Figure S4A for kinetic analysis and histograms represented as probability density. (D) Histogram of the FRET efficiency of all frames for all molecules prior to censoring. Colors as in C. (E) Quenching survival kinetics (Kaplan-Meier estimate) under the indicated conditions (colors as in C). The x-axis indicates dwell time in the high-FRET (solid line) or low-FRET (dashed lines) state prior to quenching. Shaded areas, 95% confidence intervals.

Figure 5.. Short-range synapsis accelerates Tdp1 activity.

(A) Experimental scheme for single-molecule assay of Tdp1 activity and DNA-end synapsis. The DNA substrate was a derivative of the 3'-adduct substrate depicted in Figure 2B (see Figure 1, #10 for chemical structure), with Cy3B conjugated to one of the 3'-adducts. D, Cy3B donor fluorophore; A, ATTO647N acceptor fluorophore. (B) Example trajectory. The Cy3B donor fluorophore was excited and donor emission (green) and ATTO647N acceptor emission (magenta) were recorded (upper panel) and used to calculate FRET efficiency (blue, lower panel). (C) Frequency of donor loss events as a function of FRET efficiency immediately prior to donor loss under the indicated conditions. Blue, mock-depleted extracts (n = 222 events from 2 independent experiments); red, Tdp1-depleted extracts (n = 65 events from 2 independent experiments); yellow, Tdp1-depleted extracts supplemented with 40 nM recombinant Tdp1 (n = 339 events from 2 independent experiments). See Methods for calculation of donor loss frequency and Figure S5D-S5F for kinetic analysis and histograms represented as probability density. (D) Histograms of FRET efficiency of all frames for all molecules prior to censoring. Colors as in C. (E) Donor survival kinetics (Kaplan-Meier estimate) under the indicated conditions. The x-axis indicates dwell time in the high-FRET (solid line) or low-FRET (dashed lines) state prior to donor loss. Blue, donor survival kinetics in mock-depleted extract ( n = 76 events, dashed line; 107 events, solid line); red, donor survival kinetics in Ku-depleted extract (n = 687 events); shaded areas, 95% confidence intervals.

Figure 6.. Ku protects DNA ends from premature and off-pathway processing.

(A) Western blot analysis of end-processing enzyme recruitment to a DSB (blunt ends introduced by XmnI digestion) in mock-treated or DNA-PKcs-inhibited extracts (see Figure S6A for a representative experiment and explanation of quantification). Fold-enrichment is relative to undigested DNA (n = 6, mean ± s.d., *p < 0.001, **p < 0.001, two-tailed t-test). (B and C) Denaturing PAGE analysis of end processing and joining of the depicted DNA ends in the indicated extracts as in Figure 2. S, input substrate; I, processing intermediate; P, ligation product.

Figure 7.

Model of DNA end processing during NHEJ.