Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET

Abstract

The Mre11/Rad50/Nbs1 (MRN) complex initiates and coordinates DNA repair and signaling events at double-strand breaks. The interaction between MRN and DNA ends is critical for the recruitment of DNA-processing enzymes, end tethering, and activation of the ATM protein kinase. Here we visualized MRN binding to duplex DNA molecules using single-molecule FRET, and found that MRN unwinds 15-20 base pairs at the end of the duplex, holding the branched structure open for minutes at a time in an ATP-dependent reaction. A Rad50 catalytic domain mutant that is specifically deficient in this ATP-dependent opening is impaired in DNA end resection in vitro and in resection-dependent repair of breaks in human cells, demonstrating the importance of MRN-generated single strands in the repair of DNA breaks.

Keywords: DNA structure; DNA–protein interaction.

Fig. 1.

MRN opens DNA duplexes. (A) Schematic of the DNA molecules used in this study. The duplex DNA contains Cy3 and Cy5 located 5 bp apart in the duplex, which has a 4-nt 3′ overhang. The 15- and 22-nt Y substrates contain noncomplementary ends that replace 15 and 22 bp, as shown, and the hairpin DNA contains a 4-nt hairpin and a 51-bp stem. (B) FRET histograms of immobilized constructs in the absence of MRN. (C) Difference plot showing the FRET histogram from the duplex subtracted from those of the DNAs with noncomplementary ends (Y DNAs) (D–F) FRET histograms and fits of the duplex construct in the presence of increasing concentrations of MRN. F₁ and F₂ peaks were fit with a Gaussian function (green and blue lines as indicated), and N molecules were analyzed. (G) Difference plot showing a histogram for the DNA duplex in the absence of MRN subtracted from data in the presence of various concentrations of MRN.

Fig. 2.

The MRN duplex opening is ATP-dependent and requires a DNA end. (A) Difference plots as in Fig. 1 showing the effects of ATP on MRN-induced low-FRET states with Cy3/Cy5 duplex DNA. The dotted curve represents the difference plot from Fig. 1G, the effect of 10 nM MRN on FRET values in the presence of ATP. (B and C) Difference plots as in A but with MR and Mre11 protein, respectively. (D) Difference plot as in A but with hairpin substrate (Fig. 1A). (E and F) Representative FRET traces for individual molecules of immobilized DNA with DNA only (E) or with MRN and ATP (F). (G and H) Average FRET values for Cy3/Cy5 duplex DNA molecules incubated alone (G) or with MRN and ATP (H), with the SD of the FRET value over the collection period (σ_FRET) plotted on the y-axis.

Fig. 3.

(A) Diagram of duplex DNA substrate and binding to immobilized MRN. (B and C) FRET histograms for duplex DNA bound to immobilized WT MRN in the absence (B) or presence (C) of ATP. (D) Difference plot showing the effect of ATP on FRET level for DNA bound to WT MRN. (E) K_d values for MRN–DNA binding, measured by immobilized DNA and mOrange MRN binding (Upper) or by immobilized mOrange MRN protein and duplex binding (Lower). (F and G) FRET histograms for duplex DNA bound to MR(S1202R)N in the absence (F) and presence (G) of ATP. (H) Difference plot showing the effect of ATP on FRET level for DNA bound to MR(S1202R)N.

Fig. 4.

RAD50-S1202R is deficient in promoting resection. (A) Resection assays on linearized plasmid DNA performed in vitro with purified Exo1, Ku, WT MRN, and MR(S1202R)N complexes as indicated. Reaction products were separated by agarose gel electrophoresis and analyzed by SYBR Green staining (Top), Southern blot analysis with an RNA probe specific for the 3′ strand at one end (Middle), and quantitative PCR (Bottom), as described previously (11). (B) Diagram of the SA-GFP reporter integrated into U2OS cells. The SSA product between two tandem GFP fragments restores a GFP⁺ cassette and causes a 2.7-kb deletion. (C) Immunoblot analysis of clonal stable cell lines harboring RAD50-WT and RAD50-S1202R expression cassettes resistant to an siRNA directed against endogenous RAD50 (siRAD50#1/si50). Shown are RAD50 and actin immunoblot signals from the parental cell line (U2OS SA-GFP) treated with a nontargeting siRNA (siCTRL/siC) and the parental cell line and RAD50-WT and RAD50-S1202R cell lines treated with siRAD50#1. (D) Cell lines used in B were treated with siRNA before transient expression of I-SceI and subsequent analysis of GFP⁺ cells by FACS. Shown is the frequency of SSA for siRAD50#1-treated samples relative to parallel siCTRL-treated samples for each cell line. *P < 0.0001 vs. parental siRAD50#1-treated; n = 12. Error bars represent SD. (E) Diagram of the DR-GFPhyg reporter for homology-directed repair (HDR) (27), which was integrated into cells derived from a Rad50-deficient human patient (RAD50^R/X) (28) between the tandem GFP fragments restores a GFP⁺ cassette. (F) Immunoblot signals of RAD50^R/X cells transfected with RAD50 WT, RAD50-S1202R (S1202R), or EV. (G) RAD50^R/X cells with DR-GFPhyg were transfected with expression vectors for I-SceI along with RAD50 expression vectors or EV as indicated, and GFP⁺ cells were subsequently analyzed by FACS. The frequency of HDR (GFP⁺ cells) normalized to parallel EV transfections is shown. *P < 0.01 vs. EV and S1202R; n = 4. Error bars represent SD.