Direct Observation of Single MuB Polymers Eric C Greene, Kiyoshi Mizuuchi  Molecular Cell  Volume 9, Issue 5, Pages 1079-1089 (May 2002) DOI: 10.1016/S1097-2765(02)00514-2

Figure 1 A System for Observing Single Transposition Target Complexes (A) Molecules of biotinylated lambda DNA were tethered to a neutravidin-coated flow cell. An argon laser was reflected off the surface of the flow cell allowing illumination to occur with total internal reflection geometry. Laser penetration depth was ∼120 nm, and the total depth of the flow cell was ∼25 μm. DNA molecules were extended with buffer flow to confine them within the evanescent wave. With this configuration, only molecules of EGFP-MuB within the evanescent wave are detected. (B) Target complexes were assembled by rinsing the DNA with 500 nM EGFP-MuB and 2 mM ATP (assembly buffer). Disassembly was initiated by rinsing the tethered complexes with buffer lacking both EGFP-MuB and nucleotide (disassembly buffer). Molecular Cell 2002 9, 1079-1089DOI: (10.1016/S1097-2765(02)00514-2)

Figure 2 Individual Polymers of MuB at Conserved Regions on the DNA during Disassembly (A) Immobilized target complexes were assembled and disassembly was initiated. A typical DNA molecule is shown, and the time point of each image is indicated at the top of the figure. The positions of three prominent fluorescent regions centered at 0.5, 3.5, and 7.0 μm are indicated with brackets on the right side of the figure. The grayscale and contrast were adjusted to emphasize the spatial distribution of MuB on the DNA; therefore, the image intensities are not scaled equivalently at the different time points. (B) Average distributions of EGFP-MuB on lambda DNA were determined before disassembly (○) or 13 min after initiating the reactions (□). The data were averaged and fluorescence (arbitrary units) plotted as a function of position relative to the tethered end of the DNA molecules. Three regions of greater signal intensity were observed at 13 min, corresponding to the conserved positions of the MuB polymers. Note that the two curves have been normalized and are therefore not scaled equivalently, to allow the use of the same y axis. These data were superimposed on a base frequency plot (%GC, in red) of lambda DNA. (C) Lambda DNA (2.5 nM) tagged at the right end with a 32P-labeled oligo was reacted with MuA (200 nM) and Mu end oligo (100 nM) in the presence or absence of MuB (500 nM) and 2 mM ATP. Reactions were terminated with buffer containing SDS, and products were resolved on an agarose gel. Double-ended strand transfer produced DNA fragments corresponding to the insertion sites utilized by MuA. (D) Disassembly reactions were performed in the presence of 100 nM MuA (left panel) or with complexes assembled with 2 mM ATPγS (middle panel). In the right panel, two examples of target complexes are shown that were assembled in the presence of 50 nM EGFP-MuB. Molecular Cell 2002 9, 1079-1089DOI: (10.1016/S1097-2765(02)00514-2)

Figure 3 MuB Polymers Exhibit Variations in the Numbers of MuB Molecules within Each Polymer and in the Positioning of the Polymers on the DNA (A) The photon emissions from 78 polymers from the 13 min time point of the disassembly reaction were quantitated. A frequency distribution of the estimated number of molecules of EGFP-MuB per polymer is shown. (B) Five separate molecules from the 13 min time point of the disassembly reaction were aligned. The positions of the individual polymers within the left image are indicated. These images were processed equivalently so that direct comparisons can be made between fluorescence emission intensities for each of the individual polymers. Molecular Cell 2002 9, 1079-1089DOI: (10.1016/S1097-2765(02)00514-2)

Figure 4 Variations in the Size and Positions of the Polymers Are Due to Stochastic Behavior of MuB (A) Three consecutive disassembly reactions were performed on the same set of DNA molecules (chase #1, #2, and #3). As in Figure 2A, the image intensities are not scaled equivalently at the different time points. The DNA molecule shown here was the same molecule as in Figure 2A. (B) Images from the 8 and 13 min time points of the disassembly reaction were aligned relative to one another. The positions of the EGFP-MuB polymers are indicated at the left. The 8 min images were scaled equivalently relative to one another, and the 13 min images were also scaled equivalently relative to one another. Molecular Cell 2002 9, 1079-1089DOI: (10.1016/S1097-2765(02)00514-2)

Figure 5 MuB Polymers Exhibit a Broad Distribution of Disassembly Rates (A) The disassembly of the EGFP-MuB polymers located on a single molecule of DNA is shown at the top of the figure and the positions of the polymers relative to the end of the DNA molecule are indicated at the left. The disassembly data (bottom panel) are displayed as both fluorescence emission (counts, left y axis) and as the estimated number of EGFP-MuB molecules with each polymer (molecules of MuB, right y axis). The data points and the curves representing the best fit single exponential decay for the 6.5, 7.7, and 9.9 μm regions are shown. The decay rates (koff) for each polymer are indicated. As in Figure 2A, the image intensities within the top panel are not scaled equivalently at the different time points. (B) The target complex disassembly rates were determined for 171 separate regions on 39 different DNA molecules and displayed as a frequency distribution. Molecular Cell 2002 9, 1079-1089DOI: (10.1016/S1097-2765(02)00514-2)

Figure 6 MuB Catalyzes a Single Round of ATP Hydrolysis prior to Dissociating from DNA (A) Disassembly reactions were performed as in Figure 5A, with the exception that 2 mM ATP was included in the disassembly buffer. The images of the polymers on a single DNA molecule are shown at the top of the figure, and the positions of the polymers are indicated at the left. The signal decay data for four separate polymers is shown at the bottom of the figure. The disassembly rates for all of the individual polymers were determined by fitting the data to single exponential decays and are indicated. Note that the time frame differs slightly from that presented in reactions without nucleotide in the disassembly buffer because of inefficient mixing in the flow cell at the outset of the reaction. Molecular Cell 2002 9, 1079-1089DOI: (10.1016/S1097-2765(02)00514-2)

Figure 7 Assembly and Disassembly of the MuB Polymer At high protein concentration, in the presence of ATP, MuB binds to all DNA present in the reaction. The DNA-bound MuB can exist in at least two different conformational states, either high- or low-affinity polymers. The sequence of the DNA to which the polymer is bound influences whether the polymer will be in the high- or low-affinity state (step 1). When MuB is bound to a segment of DNA containing a preferred sequence (red), the polymer can undergo a conformation change to enter the high-affinity state. However, MuB bound to less preferred DNA sequences (blue) remains in the low-affinity state. Disassembly of the high-affinity polymers may occur via an end-dependent mechanism beginning with the dissociation of the terminal subunits and followed by the processive removal of additional subunits (step 2). Once the high-affinity polymer has been reduced to a sufficient size such that only the preferred DNA sequence is bound, the disassembly rate decreases, reflecting the greater affinity of the MuB monomers for the DNA in direct contact with the polymer (step 3). MuB in the low-affinity state may consist of a series of smaller units rather than a large single polymer. Molecular Cell 2002 9, 1079-1089DOI: (10.1016/S1097-2765(02)00514-2)