Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function
- University of California, Davis, United States
- Graduate Group in Biophysics, United States
Figures
Figure 1 with 1 supplement
Visualization of salt-induced intramolecular condensation of single molecules of SSBAF488-ssDNA complexes.
(A) Bacteriophage λ dsDNA (48.5 kbp) was biotinylated, denatured, coated with SSBAF488, and then (B) attached to a streptavidin-coated glass coverslip of a microfluidic chamber where it was extended …
Figure 1—figure supplement 1
Intensity and length measurements during salt-induced intramolecular condensation.
Representative plots showing the change in intensity (leftmost panels) or length (rightmost panels) of SSBAF488-coated ssDNA molecules for the condition where [NaOAc] was increased from (A, B) 0 to …
Figure 2 with 1 supplement
The length change upon salt-induced condensation of SSBAF488-coated ssDNA is nearly reversible in the absence of free SSB protein.
(A) A montage of frames from a video recording of a single molecule of SSBAF488-coated ssDNA contracting in length as the salt concentration is increased from 0 to 100 mM NaOAc, and then …
Figure 2—figure supplement 1
SSBAF488 partially dissociates from ssDNA during salt transitions in the absence of free protein.
(A) Schematic showing the injection system used to transiently increase the salt concentration. (B–E) Representative plots showing the change in intensity (leftmost panels) or length (rightmost …
Figure 3 with 6 supplements
The extent of SSB-ssDNA condensation is greater than anticipated based on known ssDNA-wrapping transitions.
(A) Poly(dT) was titrated into 100 nM SSBf (tetramer) and the average fluorescence enhancement of SSBf from three titrations was plotted as a function of ratio of poly(dT) to SSB tetramer. The data …
Figure 3—figure supplement 1
Salt back-titrations to determine the concentration at which SSBf dissociates from ssDNA.
Plot of the fluorescence enhancement of pre-formed SSBf-poly(dT) complexes as a function of NaOAc concentration, normalized relative to the fluorescence of SSBf in the absence of ssDNA. NaOAc was …
Figure 3—figure supplement 2
Length distributions of single molecules of SSBf-coated ssDNA as a function of [NaOAc].
Distribution of lengths of SSBf-coated ssDNA at increasing concentrations of NaOAc (N = 216). The data were fit to a Gaussian distribution, and the mean and standard deviation from the fits were …
Figure 3—figure supplement 3
Intensity of SSBf-ssDNA molecules as a function of [NaOAc].
Scatter-plot showing the intensity of individual SSBf-ssDNA complexes imaged at each concentration of NaOAc. The intensity of each molecule was normalized to the average intensity of the molecules …
Figure 3—figure supplement 4
Length distributions of single molecules of SSBf--coated ssDNA as a function of [Mg(OAc)2].
Distribution of lengths of SSBf-coated ssDNA at increasing concentrations of Mg(OAc)2 (N = 156). The data were fit to a Gaussian distribution and the mean and standard deviation from the fits were …
Figure 3—figure supplement 5
Length distributions of single molecules of SSBf-coated ssDNA as a function of [NaGlu].
Distribution of lengths of SSBf-coated ssDNA at increasing concentrations of NaGlu (N = 205). The data were fit to a Gaussian distribution, and the mean and standard deviation from the fits were …
Figure 3—figure supplement 6
Length distributions of single molecules of SSBf-coated ssDNA as a function of [NaGlu] in the presence of 1 mM Mg(OAc)2.
Distribution of lengths of SSBf-coated ssDNA at increasing concentrations of NaGlu in the presence of 1 mM Mg(OAc)2 (N = 214). The data were fit to a Gaussian distribution, and the mean and standard …
Figure 4 with 1 supplement
The binding of SSB eliminates hysteresis from the force-extension behavior of ssDNA measured by single-molecule magnetic tweezer force spectroscopy
(A) A DNA substrate was made by ligating biotin- and DIG-containing ‘handles’ (i.e. ∼2-kb products from PCR containing biotin-dGTP or DIG-dUTP) to the flanking ends of a 13.5-kb DNA substrate. The …
Figure 4—figure supplement 1
Force extension curve of SSB-coated ssDNA at low force.
Data from Figure 4B,C,D re-scaled and plotted from 0 to 1 pN on the y-axis. Black squares are ssDNA alone in 0 mM NaOAc. Circles represented measurements made with SSB-coated ssDNA at the …
Figure 5 with 2 supplements
RecO and RecOR alter SSB-ssDNA wrapping to induce nucleoprotein fiber condensation
(A) The work (i.e. ΔE) stored in the ssDNA or SSB-coated ssDNA molecules was determined from the area under the curves from the data in Figure 4C,D, as shown in Figures 5—figure supplements 1, 2, …
Figure 5—figure supplement 1
Diagram of the area under the force-extension curves used to calculate the changes in energy in the absence and presence of SSB.
(A) The work (i.e. energy) stored in the ssDNA was determined from the area under the curves from Figures 4C,D (250 mM NaOAc is used here as the example) and plotted relative to ssDNA alone in the …
Figure 5—figure supplement 2
Difference in the change in energy contributed from SSB binding at increasing salt concentrations.
The difference in the work (i.e. ΔΔE) stored in the SSB-coated ssDNA molecule, determined by subtracting the ΔE measured for relaxing ssDNA (open red circles, dashed line) and pulling ssDNA (closed …
Author response image 1
Videos
Video 1
Salt-induced intramolecular condensation of SSBAF488-ssDNA.
Video recording of a single molecule of SSBAF488-coated ssDNA, imaged using TIRF microscopy, upon increasing [NaOAc] from 0 to 100 mM. The video frames were rendered into a topological intensity …
Video 2
Condensation of SSBAF488 in the absence of free protein during a transient increase from 0 to 100 mM NaOAc.
Video recording of a single molecule of SSBAF488-coated ssDNA contracting in length as the salt concentration is increased from 0 to 100 mM NaOAc, and then subsequently reduced back to zero mM, …
Video 3
Condensation of SSBAF488 in the absence of free protein during a transient increase from 0 to 400 mM NaOAc.
Video recording of a single molecule of SSBAF488-coated ssDNA contracting in length as the salt concentration is increased from 0 to 400 mM NaOAc, and then subsequently reduced back to zero mM, …
