Single-molecule observation of ATP-independent SSB displacement by RecO in Deinococcus radiodurans
- Department of Chemistry, Seoul National University, Republic of Korea
- Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), United States
- Daegu National Science Museum, Republic of Korea
- Department of Physics, Pohang University of Science and Technology, Republic of Korea
Figures
Figure 1 with 2 supplements
Displacement of drSSB from individual diffusing ssDNA by drRecO.
(A) Schematic illustration of the single-molecule FRET measurement for freely diffusing drSSB- and/or the drRecO–ssDNA complex by ALEX-FRET. Cy3B (donor) and Cy5 (acceptor) were attached to the ends …
Figure 1—figure supplement 1
Schematic illustration of ALEX microscope setup and data interpretation.
(A) Schematic description of microscope set-up and data acquisition for ALEX-FRET measurement. (AOM, acoustic optical modulator; MR, mirror; DM, dichroic mirror; PH, pinhole; APD, avalanche …
Figure 1—figure supplement 2
Dissociation kinetics of drSSB–ssDNA.
We performed a surface-tethering experiment to measure the dissociation rate of drSSB by employing a Total internal reflection fluorescence (TIRF) microscope. drSSB was incubated with the …
Figure 2 with 1 supplement
Real-time observation of drSSB displacement from immobilized ssDNA by drRecO.
(A) Schematic descriptions of TIRF microscopy and the experimental scheme for the real-time measurement of drRecO-mediated drSSB displacement from ssDNA. dT70 ssDNA was tethered to the glass surface …
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Figure 2—source data 1
Data summary table for the results shown in Figure 2E.
- https://cdn.elifesciences.org/articles/50945/elife-50945-fig2-data1-v2.docx
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Figure 2—source data 2
Data summary table for the results shown in Figure 2G.
- https://cdn.elifesciences.org/articles/50945/elife-50945-fig2-data2-v2.docx
Figure 2—figure supplement 1
The fluorescence intensity time traces and cumulated FRET histograms of drSSB and drRecO binding to dT70 and drRecO binding to drSSB-coated dT70.
(A, B) The time traces of the donor and acceptor intensity and the cumulated FRET histograms for drSSB binding to dT70, presented in Figure 2B. 138 time traces were accumulated. The FRET peak …
Figure 3 with 1 supplement
The exchange rates by drRecO in the preformed and competitive conditions.
(A) Schematic illustration of the preformed condition. (B–D) The concentration-dependent exchange rates by drRecO in the preformed condition. drSSB was washed out when drRecO was injected. We added …
Figure 3—figure supplement 1
Observation of drSSB and drRecO binding to short ssDNAs (dT20, dT30 and dT40) by ALEX-FRET.
We performed additional experiments to understand why the reverse reaction was not favored. The number of free nucleotides may be an important factor. To check this possibility, we used ALEX-FRET to …
Figure 4 with 2 supplements
Colocalization of drSSB and ssDNA after drSSB displacement by drRecO.
(A) Three models of SSB displacement from ssDNA by RecO. In Model 1, SSB is completely dissociated from ssDNA by RecO. In Model 2, RecO displaces SSB, but SSB remains on the complex by binding to …
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Figure 4—source data 1
Data summary table for the results shown in Figure 4D.
- https://cdn.elifesciences.org/articles/50945/elife-50945-fig4-data1-v2.docx
Figure 4—figure supplement 1
Colocalization measurement of fluorescently labeled drSSB and ssDNA.
Schematic representation of the prism-type TIRF system and the inside of a flow channel on a microscope slide. Fluorescently labeled drSSB and ssDNA were used. dT70 was labeled with Cy3B on its 3′ …
Figure 4—figure supplement 2
Measurement of photo-bleaching and blinking on TIRF imaging.
(A) Schematic representation of the measurement of blinking (a temporary dark state of fluorophore) and bleaching (permanent ‘switching off’) of the acceptor in the smFRET experiment using TIRF …
Figure 5
Observation of the intermediate state during drSSB displacement from ssDNA by drRecO.
(A) Schematic illustration of the binding of drRecO to the preassembled drSSB–dT70 complex. The same measurement is presented in Figure 2D. (B, C) FRET time traces of the binding of drRecO to the …
Figure 6 with 2 supplements
Real-time observation of drRecO binding to various lengths of ssDNA.
(A) Schematic illustration and representative FRET time trajectories for drRecO binding to dT40 (left), dT50 (middle), and dT60 (right). The orange dashed lines denote the time of drRecO injection. …
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Figure 6—source data 1
Data summary table for the results shown in Figure 6B.
- https://cdn.elifesciences.org/articles/50945/elife-50945-fig6-data1-v2.docx
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Figure 6—source data 2
Data summary table for the results shown in Figure 6C.
- https://cdn.elifesciences.org/articles/50945/elife-50945-fig6-data2-v2.docx
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Figure 6—source data 3
Data summary table for the results shown in Figure 6D.
- https://cdn.elifesciences.org/articles/50945/elife-50945-fig6-data3-v2.docx
Figure 6—figure supplement 1
FRET distributions of 40-nt ssDNA and the intermediate state of dT40-drRecO complex.
(A) Representative time trace of drRecO binding to dT40, showing the fluctuating dynamic of the FRET state for dT40 (gray box) and the intermediate state of dT40–drRecO (magenta box). (B) Cumulative …
Figure 6—figure supplement 2
drRecO binding to various lengths of ssDNA by TIRF measurement.
(A) The FRET histograms for drRecO binding to dT40, dT50 and dT60, obtained from the FRET time traces shown in Figure 6A. The numbers of time traces used for the histograms for dT40, dT50, and dT60 …
Figure 7
The binding modes of drRecO mutants to ssDNA.
(A) 1D FRET histograms for dT70 only, wt-drRecO–dT70, K35E/R39E drRecO (N-terminal mutation)–dT70, and R195E/R196E drRecO (C-terminal mutation)–dT70, obtained by ALEX-FRET. One of the ssDNA-binding …
Figure 8 with 1 supplement
drRecO-mediated drSSB displacement depending on the length of ssDNAs.
(A, B) Representative time trajectories for (A) drSSB binding to dT40 and (B) drRecO-mediated drSSB displacement from dT40. The drSSB–dT40 complex was formed by adding 125 nM drSSB to …
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Figure 8—source data 1
Data summary table for the results shown in Figure 8C.
- https://cdn.elifesciences.org/articles/50945/elife-50945-fig8-data1-v2.docx
Figure 8—figure supplement 1
The observation of drSSB displacement by drRecO on various lengths of ssDNA.
(A) The cumulative FRET histograms and (B) the representative time trajectories for drSSB binding dynamics and drRecO-mediated drSSB displacement on 50-nt and 60-nt ssDNAs. The left panels in (A) …
Figure 9 with 1 supplement
Real-time observation of drSSB displacement by drRecO mutants with lower ssDNA-binding affinity.
(A, B) Representative FRET time trajectories for the binding of K35E/R39E drRecO and R195E/R196E drRecO mutants to the preformed drSSB–dT70 complex. The injection time of the mutants is marked by …
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Figure 9—source data 1
Data summary table for the results shown in Figure 9C.
- https://cdn.elifesciences.org/articles/50945/elife-50945-fig9-data1-v2.docx
Figure 9—figure supplement 1
R121A drRecO mutation hinders both drRecO–ssDNA binding and formation of the intermediate state.
(A) Position of the 121-arginine residue that locates in the middle of the estimated ssDNA-binding site in drRecO. (B) FRET distribution of R121A drRecO–dT70 complex. R121A mutation showed a large …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3) | Invitrogen | Competent cells | |
| Genetic reagent (D. radiodurans) | RecO | DOI:10.2210/pdb1U5K/pdb | ||
| Commercial assay kit | PrimeSTAR DNA polymerase | Takara, Japan | DNA polymerization for PCR | |
| Software, algorithm | MATLAB | Mathworks Matlab Version R2018 | The source code is distributed by T.J Ha group (http://bio.physics.Illinois.edu/) | |
| Sequence-based reagent | DNA oligos | DNA oligos were ordered from IDT |
