ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements
- Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Spain
- University of Bristol, United Kingdom
Figures
Figure 1
Possible scenarios for CTD-induced decondensation of ParB-DNA networks.
(A) Model for ParB network formation and condensation via ParB-DNA and ParB-ParB interactions. ParB monomers comprise a central DNA-binding domain (CDBD) with specific and possibly non-specific …
Figure 2 with 2 supplements
Combined lateral MT and TIRF microscopy to study ParB-DNA interactions.
(A) Cartoon of the MT-TIRF setup used to visualise ParB-DNA interactions at the single-molecule level with controlled external force. A magnet pulls laterally on the distal end of a DNA molecule …
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Figure 2—source data 1
Fluorescence intensity of 15 DNA molecules in the presence of 500 nM ParBAF as a function of time.
- https://doi.org/10.7554/eLife.43812.006
Figure 2—figure supplement 1
Purification and activity assays of ParBAF and CTDAF.
(A) ParBAF and CTDAF labelling efficiency. (B) ParBAF had non-specific (in TBE) and parS specific (in TBM) DNA-binding activity equivalent to that of wild-type ParB, as measured in an EMSA, in EDTA …
Figure 2—figure supplement 2
Control experiments showing increasing intensity due to ParBAF binding and constant intensity due to protein exchange.
(A) Averaged decrease of intensity due to introduction of protein-free buffer (n = 26). (B) Representative traces of intensity decay of formaldehyde crosslinked ParBAF in the presence and absence of …
Figure 3 with 2 supplements
ParB binding and unbinding kinetics.
(A) Scheme of the multilaminar flow system employed to fast-exchange of buffers. The fluid cell contains two inlets and a single outlet. Switching the velocities of both channels shifts the boundary …
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Figure 3—source data 1
Normalised integrated fluorescence intensity for a representative DNA molecule in a laminar-flow experiment with 250 nM ParBAF as a function of time.
- https://doi.org/10.7554/eLife.43812.014
Figure 3—figure supplement 1
Correct performance of the syringes demonstrated by tracking DNA tethers and boundary shift fluorescence measurements.
(A) Vertical Magnetic Tweezers. (B) Lateral Magnetic Tweezers. Peaks in Y and Z (vertical) and X and Y (lateral) correlate with changes in flow, showing a correct performance by computer controlled …
Figure 3—figure supplement 2
Measuring and for different ParBAF concentrations.
(A) Representative integrated fluorescence intensity for a DNA molecule in a laminar-flow experiment, as a function of time, coated with 125, 250 or 500 nM ParBAF. Protein injection correlates with …
Figure 4 with 2 supplements
ParB binding kinetics in the presence and absence of Mg2+and the effect of the free CTD.
(A) Unbinding rate and observed binding rate values for 250 nM ParBAF. We observed a reduction in (slower unbinding) in the case of EDTA but no significant difference was observed in valu…
Figure 4—figure supplement 1
The CTD is capable of binding DNA.
(A) This DNA molecule is visible due to binding by 10 µM CTDAF in buffer supplemented with EDTA, but not with Mg2+. ParBAF complexes are also visible in buffer supplemented with EDTA. (B) …
Figure 4—figure supplement 2
Measuring the effect of the CTD on the dissociation rate in EDTA and Mg2+ buffer conditions.
Representative traces of normalised fluorescence intensity as a function of time for data shown in Figure 4B. Protein binding and unbinding were cyclically measured as described before (Figure 2B)…
Figure 5
Competition of the CTD in ParB binding and unbinding kinetics, and effects in condensation.
(A) Visualisation of condensation of a single DNA molecule induced by 250 nM ParBAF binding. The volume of the bead causes the DNA to be slightly tilted with respect to the surface such that …
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Figure 5—source data 1
Integrated fluorescence intensity for 3 molecules throughout the cycles of ParBAF and ParBAF +CTD (constant intensity) or ParBAF and ParBAF +unlabelled ParB (intensity changes).
- https://doi.org/10.7554/eLife.43812.023
Author response image 1
CTDAF binds to full-length ParB bound to DNA.
(A) Fluorescence signal of CTDAF crosslinked with formaldehyde in Mg2+ conditions in the absence of ParB (left) and in the presence of ParB (right). (B) Quantification of fluorescence signal in both …
Author response image 2
Preparation of functional fluorescently labelled ParB.
(A) SDS-PAGE of finally purified ParBS68C and wild type ParB, selectively and non-selectively, respectively, conjugated to Alexa 488. (B-D) Representative TBM- and TBE-EMSAs to assess the activity …
Author response image 3
The CTD of ParB is required for the correct folding of the N-terminal and/or central DNA-binding domains.
(A) SDS-PAGE of wild-type ParB and ParBΔCTD E227. 2-6 µg of each was loaded as indicated. (B-C) Representative TBM- and TBE- EMSAs for ParBΔCTD E227 monitoring binding of parS-containing or …
Author response image 4
Author response image 5
Author response image 6
Author response image 7
Videos
Video 1
Movie showing that ParBAF binding increases the intensity along a DNA molecule.
Flow rate of 250 µl/min. Movie is 5X accelerated.
Video 2
Movie showing that intensity remains constant when imaging a ParBAF coated DNA molecule (250 nM).
Movie is 5X accelerated.
Video 3
Movie showing that the intensity of DNA coated with 250 nM ParBAF decays flowing buffer (250 µl/min).
Movie is 5X accelerated.
Video 4
Movie showing that the intensity of DNA coated with 250 nM ParBAF is recovered in FRAP experiment.
Movie is 5X accelerated.
Video 5
Movie showing the laminar flow switching assay.
250 nM ParBAF binding increases intensity along a DNA molecule and unbinding decreases it (total flow rate 200 µl/min). Movie is 5X accelerated.
Video 6
Movie showing that intensity remains constant when imaging 10 µM CTDAF, similar to ParBAF.
Movie is 5X accelerated.
Video 7
Movie showing that 250 nM ParBAF is able to condense DNA under permissive forces.
Movie is 5X accelerated.
Video 8
Movie showing that 250 nM ParBAF does not decondense upon force increase, contrary to ParB in previously published magnetic tweezers assays.
Movie is 5X accelerated.
Video 9
Movie showing that 250 nM ParBAF in the presence of an excess of CTD (2.5 µM) is not able to condense DNA.
Movie is 5X accelerated.
Video 10
Movie showing that 250 nM ParBAF in the presence of an excess of CTD (2.5 µM) is not able to condense DNA even in the absence of magnetic force.
Movie is 5X accelerated.
Video 11
Movie showing that a bright dot sometimes appears at the anchoring point of a DNA molecule in the presence of 250 nM ParBAF.
Movie is 5X accelerated.
Tables
Table 1
Student’s t-test p-values for values.
https://doi.org/10.7554/eLife.43812.019| WT - Mg2+ | WT + CTD Mg2+ | WT - EDTA | WT + CTD - EDTA | |
|---|---|---|---|---|
| WT – Mg2+ | 1 | 0.1235 | 5E-4 | 0.0019 |
| WT + CTD Mg2+ | 1 | 0 | 0 | |
| WT - EDTA | 1 | 6E-5 | ||
| WT + CTD - EDTA | 1 |
Table 2
Student’s t-test p-values for values.
https://doi.org/10.7554/eLife.43812.020| WT - Mg2+ | WT + CTD Mg2+ | WT - EDTA | WT + CTD - EDTA | |
|---|---|---|---|---|
| WT - Mg2+ | 1 | 0.079 | 0.9542 | 0.1139 |
| WT + CTD Mg2+ | 1 | 0.0243 | 0.0103 | |
| WT - EDTA | 1 | 0.1129 | ||
| WT + CTD - EDTA | 1 |
Additional files
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Source code 1
Custom-written ImageJ script to assemble movies including a time stamp based on individual frames.
- https://doi.org/10.7554/eLife.43812.029
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Transparent reporting form
- https://doi.org/10.7554/eLife.43812.030
