Gel shift assays with recombinant SOSS1 T117E and RPA. (A) Gel shift assays were performed with SOSS1 complex and RPA on a ³²[P]-labelled substrate containing a 44-nt ssDNA 3′ overhang and a 35-bp duplex region as shown. Each reaction contained 1.125, 2.25, 4.5, 9, 18, 36, or 72 nM SOSS1 complex or RPA. The reactions were separated in a native 8% acrylamide gel which was analysed by phosphorimager. (B) DNA binding assays were performed as in (A) with substrates containing a 44-nt 5′ overhang, a 44-nt 3′ overhang, or a 50-bp duplex DNA, and quantification of the results from a representative experiment is shown. Reactions contained 4.5, 9, 18, 36, or 72 nM SOSS1 or RPA. (C) Binding assays were performed with SOSS1 as in (A) with various lengths of radiolabelled ssDNA (28, 31, or 34 nt) or substrates containing 28, 31, or 34 nt 5' overhangs adjacent to 31 nt duplex DNA and the quantified results from a representative set of experiments are shown. (D) Binding assays were performed with SOSS1 and RPA as in (A) with a substrate containing an internal ssDNA gap as shown. Each reaction contained 2.25, 4.5, 9, 18, 36, or 72 nM of SOSS1 or RPA. Quantification of these results is shown in (E).

Single-molecule studies of RPA and SOSS1 binding to ssDNA. (A) A schematic representation of reaction steps for single-molecule FRET measurements. m and n are in the unit of nucleotides. (B) FRET efficiency histograms for (dT)₁₆₊₂₄ DNA only, and RPA binding to (dT)₁₆₊₂₄ at different protein concentrations. Three distinct peaks centred at FRET=0.53, 0.42, and 0.29 were observed at varying RPA concentrations (blue arrows), representing 0, 1, and 2 RPA binding to (dT)₁₆₊₂₄, respectively. (C) Time evolution of the FRET histogram for (dT)₁₆₊₂₄ after incubating with 2 nM RPA and flushing out the excess unbound RPA. (D) A single-molecule FRET-time trace for (dT)₁₆₊₂₄ obtained after incubating with 2 nM RPA and flushing out the excess unbound RPA, showing an RPA dissociation event (the black arrow). After the event, there was still one RPA remaining bound, giving an FRET of 0.42. (E) FRET efficiency histograms for (dT)₁₆₊₂₄ DNA only, and SOSS1 binding to (dT)₁₆₊₂₄ at different protein concentration. Two distinct peaks centred at FRET=0.53 and 0.37 were observed at varying SOSS1 concentrations (blue arrows), representing 0 and 1 SOSS binding to (dT)₁₆₊₂₄, respectively. (F) Time evolution of the FRET histogram for (dT)₁₆₊₂₄ after incubating with 60 nM SOSS1 and flushing out the excess unbound SOSS1. (G) A representative single-molecule FRET-time trace for (dT)₁₆₊₂₄ obtained after incubating with 60 nM SSOS1 and flushing out the excess unbound SOSS1. At t=30 s, Cy5 was photobleached. (H) Average cross-correlations of Cy3 and Cy5 intensity-time traces for (dT)₁₆₊₂₄ DNA only, one or two RPA bound, and one SOSS1 bound. Each cross-correlation was averaged over >100 molecules. (I) Average cross-correlations of Cy3 and Cy5 intensity-time traces for other two DNA substrates, (dT)₁₆₊₄₆ and (dT)₃₂₊₃₉. The FRET-time traces for cross-correlation analysis were obtained after flushing out unbound RPA or SOSS1, in order to assure the time-resolved anti-correlation comes only from the bound proteins. Single exponential fits are also shown (solid lines).

SOSS1 promotes resection by hExo1. (A) Resection reactions were performed with 0.375 nM Exo1 wild-type or nuclease-deficient (D78A/D173A), 40 nM SOSS1(T117E) complex or 40 nM RPA, and 31.25 nM wild-type MRN. Reactions were separated in a 1% non-denaturing agarose gel and further analysed by SYBR Green staining (top panel) for total DNA and by non-denaturing Southern hybridization (middle panel) using an RNA probe complementary to the 3′ end of DNA adjacent to one of the break sites. The small products observed in the southern are likely generated by simultaneous digestion of the DNA from both ends of the molecule (). (B) Resection reactions were performed with 0.375 nM Exo1 and 14, 42, or 126 nM SOSS1 and the amount of single-stranded DNA produced was quantified through quantitative PCR (qPCR) using two sets of primers to measure ssDNA levels at sites located 29 or 1025, nt from the DNA end as described previously (). The average per cent of ssDNA from three experiments is shown; error bars indicate standard deviation.

The SOSS1 complex can block RPA inhibition of Exo1. (A) Resection assays were performed as in except that each reaction contained 0.375 or 1.5 nM hExo1. (B) Resection assays as in (A) with either 0.375 nM (top) or 0.75 nM (bottom) Exo1, quantified as in by qPCR.

The SOSS1 complex promotes both exo- and endonucleolytic cleavage of DNA by Exo1. (A) Resection assays were performed using a 1.7-Kb DNA substrate internally labelled with [³²P], containing 0.16 nM wild-type Exo1 and Exo1(D78A/D173A), 15 nM MRN, 36 nM SOSS1(T117E) complex, and 36 nM RPA. Reaction products were separated by thin layer chromatography, analysed by phosphorimager, and the amount of labelled NMP released by the exonuclease activity of hExo1 was quantified by the ImageQuant software. (B) Resection assays as in (A) with 0.16 nM wild-type Exo1 and 36 nM SOSS1(T117E) complex; the average of three experiments is shown, with standard deviation. (C) Resection assays were performed using a Cy3/Cy5 labelled 717 bp DNA substrate, incubated with wild-type Exo1 (5 nM), SOSS1(T117E) complex (72 nM) and RPA (72 nM). Reaction products were analysed on a 20% denaturing sequencing gel and scanned for Cy5 emission.

The SOSS1 complex recruits Exo1 to DNA ends. (A) hExo1 D78A/D173A binding to DNA 5′ ends was tested through DNA crosslink and gel shift assays using a 717-bp double-stranded DNA substrate internally labelled with [³²P](asterisks) and containing three azide groups (N₃) at the 5′ end. The substrate was incubated with 10 or 20 nM hExo1(D78A/D173A) protein in the presence of 208 nM SOSS1(T117E) complex or 208 nM RPA as indicated. After incubation, each reaction was UV crosslinked (254 nm) then loaded and separated in a 1% native agarose gel. The gel was dried and visualized by exposure to phosphorimager. Arrows indicate position of crosslinked protein-DNA species formed in the presence of both Exo1 and SOSS1. (B) DNA binding of hExo1(D78A/D173A) was measured using a 717-bp PCR product containing biotin and three azide groups as shown. The substrate was pre-incubated with magnetic streptavidin beads for immobilization. The immobilized substrate was incubated with 80 nM Exo1(D78A/D173A) and 21.6, 86.4, or 345.6 nM SOSS1(T117E) complex as indicated, the beads were washed, and bound protein was separated on a 6% SDS–PAGE and visualized by western blot using an antibody against Exo1.

MRN promotes Exo1-mediated resection of DNA ends in the presence of Ku70/80. (A) Resection assays were performed as in , except that the reactions contained higher levels of Exo1 (1.5 nM), 40 nM SOSS1(T117E) complex, 40 nM RPA, 32 nM MRN, and 100 nM Ku. (B) Reaction assays were performed as in (A) except with 16 nM Dna2, 10 nM BLM, 100 nM Ku, 80 nM RPA, 80 nM SOSS1, and 32 nM MRN.