DNA reshaping by MukB. Right-handed knotting, left-handed supercoiling

Abstract

MukB is a bacterial SMC (structural maintenance of chromosome) protein required for faithful chromosome segregation in Escherichia coli. We report here that purified MukB introduces right-handed knots into DNA in the presence of type-2 topoisomerase, indicating that the protein promotes intramolecular DNA condensation. The pattern of generated knots suggests that MukB, similar to eukaryotic condensins, stabilizes large right-handed DNA loops. In contrast to eukaryotic condensins, however, the net supercoiling stabilized by MukB was negative. Furthermore, DNA reshaping by MukB did not require ATP. These data establish that bacterial condensins alter the shape of double-stranded DNA in vitro and lend support to the notions that the right-handed knotting is the most conserved biochemical property of condensins. Finally, we found that MukB can be eluted from a heparin column in two distinct forms, one of which is inert in DNA binding or reshaping. Furthermore, we find that the activity of MukB is reversibly attenuated during chromatographic separation. Thus, MukB has a unique set of topological properties, compared with other SMC proteins, and is likely to exist in two different conformations.

FIGURE 1. MukB is eluted from heparin column as two peaks

Following nickel-chelate chromatography, MukB was further purified using two rounds of heparin chromatography. A, protein profiles following elution from the heparin columns. Protein concentrations were determined by a Bradford assay. The high salt fractions from the Heparin I column (indicated by the black bar) were pooled, and 1 mg of the pooled protein was resolved on the Heparin II column. The fraction numbers eluted from Heparin II are indicated below the plot. The gray bar indicates Heparin I fractions used to study reversible inactivation of MukB. B, SDS-PAGE analysis of purified MukB (Heparin I pool). The indicated amounts of MukB were mixed with SDS-PAGE gel loading buffer, incubated either for 20 min at 23 °C (23C) or for 2 min at 100 °C (100C), resolved by electrophoresis through a 14% (top) or 8% (bottom) gel, and visualized either by staining with Coomassie Blue (top) or immunoblotting with anti-MukB antibody (bottom). Positions of MukB, ACP, MukB oligomers (×), proteolytic products of MukB (P), and unidentified proteins p30 and p24 are indicated on the right of the gels. C, silver-stained SDS-polyacrylamide gel that analyzes Heparin II fractions. Positions of the bands described in Fig. 1B are shown on the right. Note that ACP and p30 comigrate only with the low salt peak of MukB. D and E, DNA binding profile of eluted fractions. In D, 3-μl aliquots were tested for binding to supercoiled pBR322. In E, 1-μl aliquots were assayed with linear pBR322. L0.2 and L1.0, reactions were done using 0.2 or 1.0 μg of purified MukB, respectively NC, nicked circular DNA; SC, supercoiled DNA. F, DNA supercoiling assay using 1-μl aliquots. FT, flow-through. G, DNA knotting reactions using 1-μl aliquots. H, protein concentration dependence of DNA knotting found for various size aliquots of fractions 21–24 and 26. For comparison, the dependences for the loaded Heparin I pool (Load) and combined Heparin II fractions (Pool) are shown. For D and H, the fractions were dialyzed against 20 mM HEPES, 40 mM NaCl, 1 mM EDTA, 8% glycerol, 1 mM DTT prior to setting up activity assays to remove excessive salt.

FIGURE 2. MukB is a slow ATPase

The Heparin I pool of MukB was additionally purified by gel filtration. A, the ATPase rate of MukB was measured as described under “Materials and Methods” in the presence or absence of phage φX174 DNA (ssDNA) or pBR322 DNA (dsDNA), and the data are expressed as the number of ATP molecules hydrolyzed by MukB dimer/min. B, NaCl dependence of ATPase rate found for 2 mM MgCl₂ in the presence of single-stranded DNA. Also shown are the data for the high salt (HS) and the low salt (LS) fractions of MukB obtained after subsequent separation using heparin chromatography. The average of two experiments is shown. Error bars, S.D.

FIGURE 3. Gel shift analysis of MukB binding to DNA

Increasing amounts of MukB were incubated with 10 ng of pBR322 plasmid (4.36 kb), linearized pBR322 plasmid, PCR-generated 443-bp linear DNA, or φX174 DNA (5386 nucleotides); the reactions were quenched, the mixtures were resolved by gel electrophoresis, and the DNA was visualized by staining with SYBR Gold (Molecular Probes). The amount of MukB used in reactions is shown above the lanes as the protein/DNA molar ratio. The position of supercoiled (SC) and nicked circular (NC) DNA is marked. The stars indicate distinct gel-shifted bands. Except for in B, the high salt Heparin II pool was used in reactions. A, MukB binding to supercoiled, linear, and short linear DNA requires similar amounts of protein. The reactions were done in the presence of 1 mM MgATP. B, DNA binding by the low salt MukB. C, MukB binding to pBR322 DNA in the presence or absence of 1 mM MgATP or when MgCl₂ in the standard Reaction Buffer was replaced with 1 mM EDTA. D, MukB binding to the phage φX174 DNA.

FIGURE 4. Right-handed knotting and left-handed supercoiling by MukB

A, DNA knotting by MukB. 10 ng of singly nicked pBR322 DNA was treated with phage T2 topoisomerase in the presence of increasing amounts of MukB. Following deproteinization, DNA samples were resolved by gel electrophoresis. Marked on the left are positions for different topological forms of DNA, including linear DNA (L), nicked circular DNA (NC), catenanes and concatamers (cats), and three-, four-, five-, and six-noded knots. The amount of topoisomerase and MukB used in each reaction is shown above the gel as a molar ratio of the enzyme to DNA. M, knot marker obtained as described in Ref. . B, DNA supercoiling by MukB. 10 ng of relaxed pBR322 plasmid DNA was treated with wheat germ topoisomerase I in the presence of MukB. The amount of each protein used in the reaction is indicated above the gels as a molar ratio of the protein to DNA. After deproteinization, the DNA was analyzed by gel electrophoresis. Positions of relaxed (Rlx) and supercoiled (SC) DNA topoisomers are indicated. C, electron micrographs of DNA knots generated in the presence of MukB. The majority of generated knots were right-handed trefoils, as shown in the figure. D, MukB generates negative supercoiling. 10 ng of relaxed pBR322 DNA was treated with 35 fmol of wheat germ topoisomerase I in the presence or absence (mock) of 2.9 pmol of MukB. The deproteinized DNA was then analyzed by two-dimensional gel electrophoresis. DNA topoisomers form a distinctive arch on a two-dimensional gel. Completely relaxed topoisomer has the lowest mobility in the first dimension. Negatively supercoiled topoisomers ((−) sc) are located on the left-hand side of the arch; positively supercoiled topoisomers ((+) sc) are located on the right. E, time course of DNA supercoiling by MukB. 10 ng of pBR322 DNA was treated for the indicated times with 35 fmol of wheat germ topoisomerase I in the presence of 2.9 pmol of MukB and in the presence or absence of 1 mM MgATP or 1 mM MgAMPPNP. F, DNA supercoiling in the absence of magnesium. Reactions were done as described for B, except that MgCl₂ and MgATP were replaced with 1 mM EDTA for one set of reactions.

FIGURE 5. DNA reshaping activities copurify with MukB during gel filtration

180 μg of the Heparin I pool of MukB were purified by gel filtration through the 1.9-ml Sephacryl S300 column using 20 mM HEPES, 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 5% glycerol as the running buffer. 105-μl fractions were collected, and the aliquots were then analyzed for protein composition and DNA reshaping. A, SDS-PAGE analysis of the eluted fractions. B, copurification of DNA binding with MukB. DNA binding was done as described in the legend to Fig. 3 using 1-μl aliquots of eluted fractions. L, DNA binding using 500 ng of purified MukB. C, protein concentration and ATPase activity (2-μl aliquots) profiles in eluted fractions. D, copurification of topoisomerase-1-dependent DNA supercoiling with MukB. Reactions were done as described in Fig. 4B using 2-μl aliquots of the fractions. S, mock reaction; L, supercoiling reaction using 500 ng of MukB.

FIGURE 6. MukB undergoes reversible changes during chromatographic separation

MukB was passed through the Sephacryl S300 column, the peak fractions (fractions 7–9 in Fig. 5) were pooled and subjected to heparin chromatography. The high salt peak eluted from the heparin column was dialyzed as in Fig. 1D and tested for DNA supercoiling (A) or the binding to linear pBR322 DNA (B) alongside the pooled fractions obtained after gel filtration and the starting pool of MukB.

FIGURE 7. A working model of DNA organization by MukB

Right-handed DNA loops are stabilized by the binding to individual molecules of MukB. The size of DNA loops depends on the density of MukB on DNA and decreases at high concentrations of the protein. MukB is further postulated to assemble into a macromolecular structure. As a result, DNA loops are arranged in space to favor only specific crossings between randomly colliding DNA segments. Topoisomerase-catalyzed DNA transport between such segments generates right-handed trefoils (left-hand side of the figure). Distortions within the nucleoprotein assembly alter the knotting pattern. The two structures in the figure differ only in how the loops are connected yet give rise to different knots; the structure on the right produces a four-noded knot rather than trefoil. Note that the extent of knotting in a topoisomerase-2-coupled assay depends on the frequency of intersegment collisions between the DNA loops. The knotting frequency would be reduced if the loops are sufficiently small and are facing away from each other. The suboptimal relative orientation of DNA loops would be achieved, for example, if condensins twist around the symmetry axis, giving rise to a double-helical filament.