Distinct architectural requirements for the parS centromeric sequence of the pSM19035 plasmid partition machinery

  1. Andrea Volante
  2. Juan Carlos Alonso
  3. Kiyoshi Mizuuchi  Is a corresponding author
  1. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States
  2. Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones, Científicas, Spain
6 figures, 2 tables and 1 additional file

Figures

Figure 1 with 3 supplements
ParApSM ATPase activation by ParBpSM, parSpSM, and nsDNA.

(A) Efficient activation of ParApSM ATPase by ParBpSM exhibits critical dependency on parSpSM heptad-sequence-repeat number. The ATPase reactions contained ParApSM (2 μM), pBR322 DNA (60 μM in bp, unless noted otherwise), ParBpSM (at the concentration indicated), and parSpSM duplex substrates (4.4 μM of the 7 bp consensus sequence repeats) or equal amount of a scrambled sequence duplex (scram). (B) Comparison of parSpSM containing four contiguous repeats with different heptad orientation arrangements. (C) parSpSM containing different heptad arrangements of three contiguous repeats and two contiguous triple heptad repeats with a gap fails to fully activate the ParApSM ATPase. (D) 7R-parSpSM concentration dependence of ParApSM ATPase activity. Reaction mixtures contained ParApSM (2 μM), ParBpSM (2 μM), pBR322 DNA (60 μM in bp), and increasing concentration of 7R-parSpSM duplex (duplex fragment concentration shown, the ratio of parSpSM heptad repeat sequence to ParBpSM dimers are indicated on top). (E) The numbers and arrangement of the heptad repeats of the parSpSM fragments used in this study (also see Figure 1—figure supplement 2). Data points represent means and standard errors of mean (SEM) of N repeated experiments (N* represents repeats for majority of data points, see Figure 1—source data 1 for details). Curves were fitted after subtraction of the background in the absence of ParApSM to an equation v-v0 = (vmax[B]n)/(KAn + [B]n). The maximum turnover rates (vmax) cited in the text represent mean ± 95% confidence intervals (symmetrized to the larger estimated errors from the mean for simplicity).

Figure 1—figure supplement 1
ParApSM-ATPase stimulation by ParBpSM at different ParApSM concentrations, ParBpSM1-27, and ParBpSM-7R-parSpSM in the absence of nsDNA.

(A) The ATPase reactions contained ParApSM at concentrations indicated, nonspecific plasmid DNA (30 μM in bp), ParBpSM (at the concentration indicated), and 7R-parSpSM duplex (34.6 μM in bp or 4.4 μM of the ParBpSM-binding consensus sequence). (B) The ParBpSM1-27 (black circles) and its variant K10A (red triangles) weakly stimulated ParApSM ATPase (2 μM) in the presence of nsDNA. No stimulation was observed with ParBP11-30, which specifically interacts with P1 plasmid ParAP1 (purple square) or when nsDNA was omitted from the reaction (green circle). (C) Reactions with two concentrations of 7R-parSpSM dsDNA fragment, 0.75 μM (filled circles) and 0.15 μM (open circles) in parSpSM fragment concentration (or ~5 μΜ and ~1 μΜ of the ParBpSM-binding consensus sequence) in the absence of nsDNA were compared in the presence of a range of ParBpSM concentrations indicated. Data points represent means and standard errors of mean (SEM) of N repeated experiments (N* represents repeats for majority of data points; see Figure 1—figure supplement 1—source data 1).

Figure 1—figure supplement 2
DNA duplex substrates.

Analysis of annealed dsDNA fragments (500 ng per well) used in this study by 6% PAGE stained with EtBr.

Figure 1—figure supplement 3
Binding affinity of ParBpSM to 4R- and 3R-parSpSM.

Binding affinity of ParBpSM to 4R- (panel A) and 3R-parSpSM (panel B) was compared to a scrambled sequence DNA fragment (55 bp, nsDNA, panel C) by EMSA. Indicated concentrations of ParBpSM were preincubated with 0.05 nM 32P-labeled DNA substrate in 50 mM Tris–HCl pH 7.4, 100 mM KCl, 2 mM MgCl2, 1 mM DTT at room temperature for 15 min. The samples were analyzed by 4–12% PAGE and autoradiography. The smear between the specific complex and free DNA bands in the gel due to dissociation of the complex during electrophoresis was not included in the analysis but assumed to be proportional to the measured quantity of the specific complex. Significant quantity of nonspecific complex, which stayed at the top of the gel, was detected only at the highest concentrations of ParBpSM with nsDNA. Data points represent means and standard errors of mean (SEM) of N repeated experiments (N* represents repeats for majority of data points, see Figure 1—figure supplement 3—source data 1 for details.)

Figure 2 with 3 supplements
Kinetics of ParApSM disassembly from the nsDNA-carpet.

(A) Schematic of two-inlet flow cell used for visualizing the association and dissociation of fluorescent proteins on nsDNA-carpet. Binding and washing solutions each containing proteins, double-stranded DNA fragments or buffer alone as specified were infused at different flow rates (5 μl/min or 0.5 μl/min) from two inlets on the left into a Y-shaped flow cell. At the middle of the flow channel just downstream of the flow convergence point where the observations are made, content of the faster infusion syringe flows over the observation point. By switching the flow rates of the two syringes, protein binding to the nsDNA-carpet and dissociation during the washing cycle can be recorded. (B) ParBpSM in the presence of parSpSM with at least four contiguous ParBpSM-binding sequence repeats stimulates the ParApSM-GFP dissociation from the nsDNA-carpet. ParApSM-GFP (1 μM) preincubated with 1 mM ATP was infused into the nsDNA-carpeted flow cell at 5 μl/min, while the washing solution containing the specified components was infused at 0.5 μl/min. When the ParApSM-GFP density on the nsDNA-carpet reached 5–10% of the saturation density (t = 0) (~4000 ParApSM dimers/μm2), the flow rates were switched to start the wash with solution containing: buffer alone, 55 bp scramDNA fragment (65 nM in bp), or with ParBpSM (1 μM) without or with different parSpSM fragment (0.5 μM parSpSM heptad repeat sequence). The Y-axis shows the ParApSM-GFP intensity normalized to that at t = 0. Each time point represents the mean with error bar corresponding to the standard errors of mean (SEM) of N repeated experiments.

Figure 2—figure supplement 1
ATPase activity of ParApSM-GFP and the hydrolysis-deficient ParApSMD60E-GFP.

The ATPase reaction contained 2 μM ParApSM-GFP (black) or ParApSMD60E-GFP (cyan), pBR322 DNA (30 μM bp), ParBpSM-Alexa647 (1% labeled), and 7R-parSpSM DNA (4.4 μM 7 bp consensus sequence repeats). Measurements are averages of at least three independent measurements, and the error bars represent the standard errors of mean. N* indicates some lower concentrations of ParBpSM were not included in some of the experimental repeats.

Figure 2—figure supplement 2
ParApSM, ParApSMD60E, and ParBpSM interactions with the DNA-carpet measured each separately.

(A) ParApSM-GFP, ParApSMD60E-GFP (top) and ParBpSM-Alexa647 (bottom) binding to the nsDNA-carpet separately. ParApSM-GFP (1 μM, black), ParApSMD60E-GFP (1 μM, cyan), or ParBpSM-Alexa647 (1% labeled,1 μM, red) were preincubated with 1 mM ATP and separately infused into the nsDNA-carpeted flow cell at 5 μl/min for 15 min. ParApSM-GFP (apo) (green line, top) was without ATP. (B) Dissociation kinetics of proteins separately bound to the nsDNA carpet. ParApSM-GFP (black) and ParApSMD60E-GFP (cyan) (top) or ParBpSM-Alexa647 (bottom, red) densities on the nsDNA-carpet during wash with buffer containing ATP. Each time point represents means and standard errors of mean (vertical spread) of N experiments.

Figure 2—figure supplement 3
ParBpSM-activated dissociation of ParApSM-GFP or ParApSMD60E-GFP bound to nsDNA-carpet to saturation density.

ParApSM-GFP (A) or ParApSMD60E-GFP (B) (1 μM) was preincubated with 1 mM ATP and infused into the nsDNA-carpeted flow cell at 5 μl/min up to saturation binding. At t = 0, the solution flowing over the observation area was switched to the ATP-containing wash buffer with the indicated cofactors: ParBpSM, 1 μM with or without; 3R-, 4R-, or 7R-parSpSM (0.5 μM consensus sequence), or 55 bp scrambled sequence duplex (5.5 μM in bp) as indicated. ParBpSM and parSpSM fragments were preincubated prior to use. The fluorescence intensity of ParA-GFP proteins were normalized to that at t = 0. Each time point represents means and standard errors of mean (vertical spread) of N experiments.

ParBpSM-parSpSM concentration affects kinetics of ATP hydrolysis-dependent ParApSM disassembly from the nsDNA-carpet.

(A) ParApSM-GFP (1 μM) preincubated with 1 mM ATP was infused into the nsDNA-carpeted flow cell at 5 μl/min and when the ParApSM-GFP density on the nsDNA-carpet reached ~10% of the saturation density, the flow over the observation area was switched (t = 0) to wash solution containing ParBpSM ([a–d] 1000 nM, [e–h] 250 nM, or [i–l] 62.5 nM) and the stoichiometric concentration of parSpSM fragments indicated in the ‘parSpSM in wash’ column on the left side of each row of panels. Fluorescence signal was converted to protein density and plotted (dimers/μm2, ParApSM-GFP: black; and ParBpSM-Alexa647: red). Each time point represents mean and standard errors of mean (SEM) (vertical spread) of N repeated experiments. Dashed vertical lines indicate the peak ParBpSM density on the nsDNA-carpet. (B) The time course of the ParBpSM:ParApSM molar ratio (B/A) for the four panels in the columns in (A) with matching ParBpSM concentration with different parSpSM (7R: black; 4R: purple, 3R: red; none: gray) in the wash solution. (C) ATP hydrolysis is required for accelerated ParApSM release from the nsDNA-carpet. The experiments shown in (A) (middle column) were repeated using ParApSMD60E-GFP (1 μM) bound to the nsDNA-carpet and ParBpSM (250 nM) plus parSpSM fragment, (m) 7R, (n) 4R, (o) 3R, or (p) without parSpSM (125 nM heptad concentration) in the wash solution. Fluorescence signal was converted to protein density (dimers/μm2, ParApSMD60E-GFP: cyan; ParBpSM-Alexa647: red) and plotted. Each time point represents mean and SEM (vertical spread) of N experiments. (D) The time course of the ParBpSM:ParApSMD60E molar ratio (B/AD60E) for the four panels of (C) (7R: black; 4R: purple; 3R: red; none: gray). Each time point represents the mean with error bar corresponding to the SEM of N repeated experiments.

Stable ParApSM-ParBpSM complex is formed prior to ATP hydrolysis in the presence of functionally active parSpSM.

(A) ParApSM-GFP (1 μM) and ParBpSM-Alexa647 (1% labeled, 1 μM) were preincubated with ATP (1 mM) plus (a) 7R-, (b) 4R-, (c) 3R-parSpSM, or (d) without parSpSM. The preincubated sample was infused into the nsDNA-carpeted flow cell at 5 μl/min for 15 min and then the solution flowing over the observation area was switched to a buffer containing ATP (t = 0). Fluorescence signals of ParApSM (black) and ParBpSM (red) were converted to protein density on the nsDNA-carpet (dimers per μm2) and plotted. Each time point represents mean and SEM (vertical spread) of N experiments. (B) Time course of the carpet-bound ParBpSM:ParApSM molar ratio for the four panels of (A) (7R: black; 4R: purple; 3R: red; none: gray). (C) Continued supply of ParBpSM and parSpSM in the washing solution is required to sustain high rate of ParApSM disassembly. The experiment in the presence of 7R-parSpSM in panel (Aa) was repeated with addition of ParBpSM and/or 7R-parSpSM in the wash solution. Fluorescence signals of ParApSM were normalized to the value at t = 0. Each time point represents mean and standard errors of mean (SEM) (vertical spread) of N experiments.

ParApSM-ATP stabilizes functionally active parSpSM-bound ParBpSM on the nsDNA-carpet.

Complexes containing ParApSM-GFP, and ParBpSM-Alexa647 bound to the DNA-carpet together with different parSpSM DNA fragments, (a) 7R, (b) 4R, (c) 3R, or (d) without parSpSM, were washed with either a buffer containing 1 mM ATP (black) or the same buffer containing ParApSM-GFP (0.5 μM, red), in addition to ATP. The ParBpSM-Alexa647 dissociation curves normalized to the protein density at t = 0 were plotted. Each time point represents mean and standard errors of mean (SEM) (vertical spread) of N experiments.

A model of nsDNA-bound ParApSM-ATPase activation by parSpSM-bound ParBpSM.

(A) ParApSM-ATP dimers bound to nsDNA in their basal state can interact with ParA-activation domains protruding from ParBpSM dimers bound to parSpSM sequence repeats. However, multiple ParApSM dimers in a mini-filament cannot simultaneously interact with ParBpSM dimers bound to a set of repeated sequence copies within a parSpSM site and the two proteins dissociate quickly. We propose the interacting pair of proteins at this stage are not fully engaged and these ParApSM dimers are not in the ATPase-activated state. Here, we assume two ParA-interacting domains belonging to one ParBpSM dimer binds one ParApSM dimer. (B) We propose torsional (or other conformational) thermal Brownian dynamics of the mini-filaments allow the ParBpSM dimer at the fourth position to establishes interaction with another ParApSM dimer, locking in the non-equilibrium conformation of the individual mini-filaments prior to the formation of this second bridge. The distortion promotes conformational transition of the ParApSM dimers to the ATPase active state with fully engaged ParBpSM. (C) The conformational transition also destabilizes ParBpSM-parS interaction, releasing the parSpSM DNA from the activated nsDNA-ParApSM-ParBpSM complex prior to ATP hydrolysis and disassembly of the complex. This allows reloading of fresh ParBpSM to the released parSpSM, which recycles to disassemble the remaining ParApSM on the nsDNA. Meanwhile, fully engaged ParBpSM dimers left on the ParApSM dimers cause ATP hydrolysis and disassemble ParApSM dimers from nsDNA.

Tables

Table 1
The accelerated ParApSM dissociation phase of the curves in Figure 3Aa,c,i and b,f,j after the peak ParBpSM/ParApSM ratio points were fitted to single exponential curves to estimate the koff values.

The means with error bars corresponding to the standard errors of mean (SEM) of N repeated experiments in Figure 3 are shown.

[ParBpSM] (nM)7R-parSpSM4R-parSpSM
ParApSM koff (min–1)ParApSM koff (min–1)
10001.808 ± 0.0451.909 ± 0.057
2501.495 ± 0.0241.462 ± 0.040
62.51.184 ± 0.0411.101 ± 0.033
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)BL21 DE3 AIInvitrogenC607003Protein expression strain
Recombinant DNA reagentpET11aEMD Millipore9436Protein expression vector
Recombinant DNA reagentpT712ωWelfle et al., 2005ParBpSM overexpression plasmid
Recombinant DNA reagentpCB746 (pT712 vector)Pratto et al., 2008ParApSM overexpression plasmid
Recombinant DNA reagentpCB755 (pT712 vector)Pratto et al., 2008ParApSMD60A overexpression plasmid
Recombinant DNA reagentpET11a- ParApSMD60EThis workParApSMD60E overexpression plasmid
Recombinant DNA reagentpCB1033 (pT712 vector)This workParBpSM-GCE overexpression plasmid
Recombinant DNA reagentpET11a- ParApSM-EGFPThis workParApSM-EGFP overexpression plasmid
Recombinant DNA reagentpET11a- ParApSMD60A-EGFPThis workParApSMD60A-EGFP overexpression plasmid
Recombinant DNA reagentpET11a- ParApSMD60E-EGFPThis workParApSMD60E-EGFP overexpression plasmid
Sequence-based reagentScrambled 55mer DNA oligo + strandThis workGGGATCAAACACTTGATAGACAAGTCTTTGACCTAATTGTGAAAATTATGAAGGG
Sequence-based reagentScrambled 55mer DNA oligo - strandThis workCCCTTCATAATTTTCACAATTAGGTCAAAGACTTGTCTATCAAGTGTTTGATCCC
Sequence-based reagent7R parS DNA oligo + strandThis workGGGAATCACAAATCACAAGTGATTAATCACAAATCACTTGTGATTTGTGATTGGG
Sequence-based reagent7R parS DNA oligo - strandThis workCCCAATCACAAATCACAAGTGATTTGTGATTAATCACTTGTGATTTGTGATTCCC
Sequence-based reagent6R parS DNA oligo + strandThis workGGGAATCACAAGTGATTAATCACAAATCACTTGTGATTTGTGATTGGG
Sequence-based reagent6R parS DNA oligo - strandThis workCCCAATCACAAATCACAAGTGATTTGTGATTAATCACTTGTGATTCCC
Sequence-based reagent5R parS DNA oligo + strandThis workGGGAATCACAAATCACAAATCACTTGTGATTTGTGATTGGG
Sequence-based reagent5R parS DNA oligo - strandThis workCCCAATCACAAATCACAAGTGATTTGTGATTTGTGATTCCC
Sequence-based reagent4R(1) parS DNA oligo + strandThis workGGGAATCACAAATCACTTGTGATTTGTGATTGGG
Sequence-based reagent4R(1) parS DNA oligo - strandThis workCCCAATCACAAATCACAAGTGATTTGTGATTCCC
Sequence-based reagent4R(2) parS DNA oligo + strandThis workGGGAATCACTTATCACAAGTGATTAATCACTGGG
Sequence-based reagent4R(2) parS DNA oligo - strandThis workCCCAGTGATTAATCACTTGTGATAAGTGATTCCC
Sequence-based reagent4R(3) parS DNA oligo + strandThis workGGGAATCACTTATCACAAATCACAAATCACTGGG
Sequence-based reagent4R(3) parS DNA oligo - strandThis workCCCAGTGATTTGTGATTTGTGATAAGTGATTCCC
Sequence-based reagent3R-2nc-3R parS DNA oligo + strandThis workGGGAATCACAAATCACAAATCACATCATAGTTCATAGTTGTGATTTGTGATTTGTGATTGGG
Sequence-based reagent3R-2nc-3R parS DNA oligo - strandThis workCCCAATCACAAATCACAAATCACAACTATGAACTATGATGTGATTTGTGATTTGTGATTCCC
Sequence-based reagent3R-1nc-3R parS DNA oligo + strandThis workGGGAATCACAAATCACAAATCACATCATAGTTGTGATTTGTGATTTGTGATTGGG
Sequence-based reagent3R-1nc-3R parS DNA oligo - strandThis workCCCAATCACAAATCACAAATCACAACTATGATGTGATTTGTGATTTGTGATTCCC
Sequence-based reagent3R-1nc parS DNA oligo + strandThis workGGGAATCACAAATCACTTGTGATTTCATAGTGGG
Sequence-based reagent3R-1nc parS DNA oligo - strandThis workCCCACTATGAAATCACAAGTGATTTGTGATTCCC
Sequence-based reagent3R(1) parS DNA oligo + strandThis workGGGAATCACAAATCACTTGTGATTGGG
Sequence-based reagent3R(1) parS DNA oligo - strandThis workCCCAATCACAAGTGATTTGTGATTCCC
Sequence-based reagent3R(2) parS DNA oligo + strandThis workGGGAATCACTTATCACAAATCACAGGG
Sequence-based reagent3R(2) parS DNA oligo - strandThis workCCCTGTGATTTGTGATAAGTGATTCCC
Sequence-based reagent2R parS DNA oligo + strandThis workGGGAATCACTTGTGATTGGG
Sequence-based reagent2R parS DNA oligo - strandThis workCCCAATCACAAGTGATTCCC
Sequence-based reagent1R parS DNA oligo + strandThis workGGGAATCACTGGG
Sequence-based reagent1R parS DNA oligo - strandThis workCCCAGTGATTCCC
Peptide, recombinant proteinParBpSM1-27This workMIVGNLGAQKAKRNDTPISAKKDIMGD
Peptide, recombinant proteinParBpSM1-27 K10AThis workMIVGNLGAQAAKRNDTPISAKKDIMGD
Peptide, recombinant proteinParBP11-30This workMSKKNRPTIGRTLNPSILSGFDSSSASGDR
Chemical compound, drug[γ−32P]ATPPerkinElmerNEG002A250UC
Chemical compound, drugBiotin-17-dCTPInvitrogen65601
Chemical compound, drugTerminal transferaseNew England BiolabsM0315
Chemical compound, drugAlexa Fluor 488 C5 maleimideThermo FisherA10254
Chemical compound, drugAlexa Fluor 594 C5 maleimideThermo FisherA10256
Chemical compound, drugAlexa Fluor 647 C2 maleimideThermo FisherA20347
Chemical compound, drugAntifoam Y-40 emulsionSigmaA5758
Chemical compound, drugEDTA-free Sigmafast protease inhibitor cocktail tabletSigmaS8830
Chemical compound, drugDOPCAvanti Polar Lipids850375P
Chemical compound, drugDOPE-BiotinAvanti Polar Lipids870273C
Chemical compound, drugBiotin-14-dCTPThermo Fisher19518018
Software, algorithmPrism 9GraphPadPrism 9Used for curve fitting, and fitting parameters and their error estimation
Software, algorithmMetaMorph 7Molecular DevicesMetaMorph 7Used for TIRF microscope data acquisition
Software, algorithmImageJ/FijiNational Institutes of HealthImageJUsed for TIRF microscope image analysis
Other (instrument)Prism type TIRF microscopeIn-house; Vecchiarelli et al., 2013, Ivanov and Mizuuchi, 2010Used for ParAF-ParBF complex assembly–disassembly experiments

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  1. Andrea Volante
  2. Juan Carlos Alonso
  3. Kiyoshi Mizuuchi
(2022)
Distinct architectural requirements for the parS centromeric sequence of the pSM19035 plasmid partition machinery
eLife 11:e79480.
https://doi.org/10.7554/eLife.79480