ClpX-Mediated Remodeling of Mu Transpososomes Briana M Burton, Tanya L Williams, Tania A Baker  Molecular Cell  Volume 8, Issue 2, Pages 449-454 (August 2001) DOI: 10.1016/S1097-2765(01)00307-0

Figure 1 ClpX Catalyzes Unfolding of MuA (A) Mass spectrum of native MuA (H-MuA, average mass of 75,073 Da) and deuterated MuA (D-MuA, average mass of 75,740 Da). The calculated masses are 75,003 and 75,816 Da (gray line), respectively. Deuterated MuA incubated with ClpX and ATP for 1 hr (D-MuA + ClpX) has a final most abundant mass of 75,159 Da. The dotted line indicates the maximum exchange possible (mass of 75,149 Da) since the reaction buffer contained 20% deuterium. (B) Time course of deuterium-hydrogen exchange for deuterated MuA incubated in ClpX and ATP (squares) or ClpX-buffer and ATP (circles). Each point is the average of three dataset collections, each from two independent preparations of the same deuterated sample. The dotted line represents the maximal theoretical exchange achievable given that the reaction buffer contained 20% deuterium. (C) Quantitation of a time course of transpososome destabilization. Complex remodeling was assayed by gel-mobility shift. A representative gel is shown (inset; C, complexes; D, free DNA). Data plotted are averaged from three experiments (circles). The dotted line represents the D/H exchange curve in the presence of ClpX from (B), superimposed here as remaining unexchanged (%) for comparison Molecular Cell 2001 8, 449-454DOI: (10.1016/S1097-2765(01)00307-0)

Figure 2 ClpX Unfolds Transposase Subunits during Remodeling Reactions (A) Assembled transpososomes on plasmids (lane 1) were challenged with high salt and were isolated by gel filtration. The resulting purified complexes (lane 2) were then treated with ClpX and ATP and again gel filtered. At each stage, samples were run (1) on a native agarose gel to visualize the DNA and asses the stability of the complexes and (2) on an SDS-PAGE to verify the presence of MuA in DNA-containing fractions by immunoblotting. The final panel shows that the ClpX-remodeled complexes are unstable to electrophoresis, but still contain most of the MuA protein (lanes 5-8). The (*) and (•) represent unreacted supercoiled and relaxed DNA, respectively. (B) GroELD87K was used to probe unfolding of MuA by ClpX. The immunoblot shows fractions from gradient sedimentation of intramolecular strand-transfer complexes composed of full-length MuA purified away from free MuA subunits and then treated with ClpX and ATP in the presence of GroELD87K. Greater than 60% of the MuA sedimented with GroELD87K. The anti-MuA antibody crossreacts with GroELD87K Molecular Cell 2001 8, 449-454DOI: (10.1016/S1097-2765(01)00307-0)

Figure 3 MuA Subunits Lacking a ClpX Recognition Signal Remain Native through the Remodeling Process (A) Schematic of the experimental procedure. (B) SYPRO Orange-stained gel (Molecular Probes, Eugene, OR) of gradient fractions. (C) Quantitation of the amount of 32P-MuA77–605 in each fraction. The ability of GroELD87K to efficiently trap chemically denatured MuA77–605 was verified Molecular Cell 2001 8, 449-454DOI: (10.1016/S1097-2765(01)00307-0)

Figure 4 A Single ClpX-Recognition Sequence Is Sufficient for Transpososome Destabilization (A) Schematic of the domain structure of Mu transposase. Divisions are based on protease-sensitive sites (Nakayama et al., 1987). The active site mutations in MuA77–605 DE/NQ are D269N and E392Q. (B) Schematic of the isolation procedure adopted from Williams et al. (1999). Complexes capable of cleavage off a neutravidin matrix must have the subunit with a functional active site subunit bound to the DNA fragment that is in trans to the biotinylated DNA fragment. (C) Wild-type complexes and complexes biased to carry a single recognition sequence were isolated as depicted in (B) and analyzed for destabilization by ClpX. Complexes cleaved off the neutravidin matrix are shown in lanes 1 and 3, and the same cleaved complexes incubated with ClpX and ATP are shown in lanes 2 and 4. Complexes composed entirely of MuA77–605 subunits were assembled and diluted to a similar concentration as those that were cleaved off the neutravidin beads for the other experiments (lane 5) and assessed for destabilization by ClpX (lane 6). Therefore, these complexes were not purified away from free DNA. Native agarose gels were run in the presence of heparin. (D) Aliquots of the samples from (C) lanes 1–4 were run on native gels lacking heparin to allow for visualization of weaker DNA binding. The intermediate bands (*) between the complexes and the free DNA correspond to a nonsynapsed band-shifted complex of MuA bound to the Mu end fragment Molecular Cell 2001 8, 449-454DOI: (10.1016/S1097-2765(01)00307-0)