(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 …
(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 …
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 …
(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 …
Data summary table for the results shown in Figure 2E.
Data summary table for the results shown in Figure 2G.
(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 …
(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 …
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 …
(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 …
Data summary table for the results shown in Figure 4D.
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′ …
(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 …
(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 …
(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. …
Data summary table for the results shown in Figure 6B.
Data summary table for the results shown in Figure 6C.
Data summary table for the results shown in Figure 6D.
(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 …
(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 …
(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 …
(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 …
Data summary table for the results shown in Figure 8C.
(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) …
(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 …
Data summary table for the results shown in Figure 9C.
(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 …
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 |