DNA Looping and Catalysis Paul Crellin, Sven Sewitz, Ronald Chalmers  Molecular Cell  Volume 13, Issue 4, Pages 537-547 (February 2004) DOI: 10.1016/S1097-2765(04)00052-8 Copyright © 2004 Cell Press Terms and Conditions

Figure 1 Models for the Assembly and Unfolding of the Tn10 Transpososome (A) The Tn10 PEC was modeled by superimposing the DNA from the IHF cocrystal structure on the structure of the Tn5 PEC. Superimposition of the IHF-folded DNA was achieved by minimizing the RMS difference in the position of the atoms at bp 6–12 of the nontransferred strand of the transposon end (Crellin and Chalmers, 2001). This region most closely matches the structure of canonical B DNA. One transposon end, IHF, and flanking DNA have been omitted for clarity. Regions of transposase and IHF-mediated hydroxyl radical protection are shown in red and green, respectively. Every tenth nucleotide on the transferred strand is shown in white. The transposon end is seen embedded in the turquoise monomer of transposase. The subterminal transposase contacts located at bp+48–65 are at the top of the structure illustrated on the left. Note that only one IHF-folded transposon arm can be accommodated by the subterminal contacts because of steric hindrance as it passes across the dimer interface. (B) An illustration of the different transposon ends used in this paper. In this schematic representation, the target binding groove is at the top as also illustrated in the model on the left of (A). The large arrowhead is at the junction between the transposon and the flanking DNA. The vertical ticks are the subterminal transposase contacts determined by hydroxyl radical footprinting. These extend out to bp+80, beyond the contacts depicted in the model in (A). The 73 bp transposon end lacks the most distal set of contacts. The 52 and 56 bp transposon ends lack all of the contacts distal to the target binding groove. (C) IHF binds specifically to the outside end of Tn10 and activates assembly of the PEC. In the absence of divalent metal ion, competitor DNA or heparin treatment strips the IHF from the β side of the complex. On the α side of the complex, IHF remains locked in position until released by the addition of divalent metal ion. Further details are given in the text. Arrowhead, transposon end; hatched green oval, IHF; magenta and turquoise ovals, transposase (T'ase); ffbPEC, fully folded bottom PEC; sfbPEC, semifolded bottom PEC; tPEC, top PEC; Me++, divalent metal ion. Molecular Cell 2004 13, 537-547DOI: (10.1016/S1097-2765(04)00052-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 2 Reversal of the Metal Ion-Induced Conformational Change Requires IHF Hydroxyl radical footprints of the indicated complexes were performed as described in Experimental Procedures. Note that the bPEC can be footprinted in the presenceof Ca2+ because it is only converted to a tPEC in the presence of competitor DNA or heparin to absorb the free IHF. The footprints were resolved on a DNA sequencing gel and recorded on a phosphoimager. Autoradiograms of the sequencing gels are shown with traces above. The transferred strand contains the terminal 3′-OH group of Tn10; the nontransferred strand is the opposite strand. Position +1 is the terminal nucleotide of Tn10 and is indicated by the arrowhead. Positions with negative numbers are in the flanking DNA. In panels 3 and 5, the indicated complexes were prepared and subsequently treated with EGTA to chelate Ca2+ ions. The transposon ends used to form the complexes were prepared by restriction digestion of pRC98 with the enzymes shown. The asterisk indicates the location of the 3′ radioactive label. The arrowhead is the end of the transposon, and the lengths of the transposon arm and the flanking DNA are indicated. Molecular Cell 2004 13, 537-547DOI: (10.1016/S1097-2765(04)00052-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 3 Unfolding and Hypersensitivity of Short Transposon Ends Is Not Dependent on Divalent Metal Ions (A) bPEC was assembled and treated with heparin in the presence or absence of Ca2+. An autoradiogram of a gel shift assay is shown. The transposon end was generated by the indicated digestion of pRC173. The standard gel mobility shift assay, used here and in the other figures, does not resolve the ffbPEC and sfbPEC. However, since there is sufficient IHF present to shift all of the DNA, the bPEC indicated will be predominantly ffbPEC. IHF, IHF-bound transposon end; free DNA, unbound transposon end. Other details are as in Figures 1 and 2. (B) The tPEC formed in the absence of Ca2+ from (A) was footprinted with hydroxyl radicals. Other details are as given in Figure 2. Molecular Cell 2004 13, 537-547DOI: (10.1016/S1097-2765(04)00052-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 4 PEC Prepared Using a DDE Mutant Transposase Is Unlocked and Does Not Become Hypersensitive in Response to Ca2+ PEC was assembled using the transposase double mutant DA97/DA161. These residues are part of the DDE motif that coordinates the catalytic metal ion. (A) Footprints of the indicated complexes were generated as described in Experimental Procedures and in Figure 2. (B) PEC was assembled and visualized in a mobility shift assay. The concentration of IHF is indicated above the autoradiogram. The concentrations of other additions are given in Experimental Procedures. The transposon end was prepared by the indicated digestion of pRC98. Other details are as given in Figures 2 and 3. Molecular Cell 2004 13, 537-547DOI: (10.1016/S1097-2765(04)00052-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 5 Very Short Transposon Arms Inhibit Double End Cleavage The products of transposition reactions using a 56 bp transposon arm were analyzed on native and denaturing gels. Autoradiograms are shown. G+A, Maxam-Gilbert sequencing ladder. Transposon ends were prepared by digestion of pRC351. Other details are as given in Figure 3. (A) In reactions with a 56 bp transposon arm, only one transposon end is cleaved and the product is a SEB complex (details in text). In the wild-type situation, with full-length transposon ends, >95% of the PEC achieves double end cleavage and produces a DEB complex (see Figure 7, lane 2 for an example). (B) The SEB product from (A) was purified from the gel and deproteinated. The uncleaved transposon end was repurified from a second native gel and analyzed for the presence of nicks on a denaturing DNA sequencing gel. The arrowhead shows the position of the labeled fragment when it is nicked at the 3′ end of the transposon. Almost none (<1%) of the uncleaved transposon end from the SEB complex is nicked at this position. In the wild-type situation, with full-length transposon ends, >95% of the transposon ends achieve cleavage and would therefore appear at the position of the arrowhead. Molecular Cell 2004 13, 537-547DOI: (10.1016/S1097-2765(04)00052-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 6 Very Short Transposon Arms Inhibit Hairpin Resolution The products of transposition reactions using a 56 bp transposon arm were analyzed on native and denaturing gels. Other details are as given in Figure 5. (A) After incubation with Mg2+ (panel 1, 1.5 hr; panel 2, 5 hr), the SEB product of the reaction (as shown in Figure 5A) was purified from the gel using the crush and soak method, deproteinated, and analyzed on a DNA sequencing gel. In panel 2, the length of the flanking DNA is increased so that the hairpin intermediate is larger than the unreacted substrate. A significant proportion of the hairpin intermediate produced in these reactions achieves resolution and appears below the region of the gel shown. Note that in standard reactions with full-length transposon ends, the hairpin intermediate is resolved very quickly and is difficult to detect even at early time points (Kennedy et al., 1998). (B) The SEB product (as shown in Figure 5A) was purified from the gel and deproteinated. The cleaved transposon end was repurified from a second native TBE gel and analyzed on a two-dimensional gel for the presence of a hairpin end. The gel (3.5% MetaPhor + 0.5% Roche MP agarose) was native in the first dimension (TAE) and denaturing in the second (50 mM NaOH). (C) The transposon ends are represented as double-stranded DNA. The standard PEC is illustrated along with the SEB produced by the 56 bp transposon arm and the PEC and SEB produced in hypernicking situations. The ± symbol indicates that some of the hairpin intermediate achieves resolution. Other details are as in Figure 1. Molecular Cell 2004 13, 537-547DOI: (10.1016/S1097-2765(04)00052-8) Copyright © 2004 Cell Press Terms and Conditions

Figure 7 A Very Short Transposon Arm Rescues the Metal Ion-Sensitive RH119 PEC PECs were assembled using the standard protocol. The reaction was initiated by addition of Mg2+ and incubated for 3 hr. Transposase was either wild-type or the RH119 point mutation. The transposon arms were prepared by digesting pRC98 with the indicated enzymes. Cleavage of the very short transposon end by wild-type transposase was only 50% after incubation for 3 hr at 37°C with 5 mM Mg2+. Cleavage of the standard fragment is 95% complete after 1 hr. Bottom double end break (bDEB) is the IHF-folded product of the cleavage reaction. Other details are as given in Figure 3. Molecular Cell 2004 13, 537-547DOI: (10.1016/S1097-2765(04)00052-8) Copyright © 2004 Cell Press Terms and Conditions