1. The Mu Three-Site Synapse
Kerri Kobryn, Mark A Watson, Ron G Allison, George Chaconas 
Molecular Cell 
Volume 10, Issue 3, Pages (September 2002)
DOI: /S (02)

2. Figure 1 DNA Transposition by the Bacteriophage Mu
(A) DNA regions of Mu essential for the strand transfer reaction. More detailed structures of the ends and enhancer are shown in the enlargements. Modified from Allison and Chaconas (1992).
(B) Protein-DNA intermediates in the in vitro Mu DNA strand transfer reaction. The earliest characterized reaction intermediate to date is the three-site synaptic complex, or LER (Watson and Chaconas, 1996), in which the two Mu ends and the Mu enhancer are synapsed by the Mu transposase with the help of HU and IHF. Formation of the LER is reversible, and protein crosslinking is required for its stabilization. All subsequent protein-DNA intermediates are increasingly stable, irreversible, do not require protein crosslinking for their stabilization, and are referred to as transpososomes. The LER is rapidly converted to the type 0 (stable synaptic complex) in which the enhancer has been released and the active site of the transposase tetramer (Lavoie et al., 1991) has engaged the terminal base pairs. The first chemical step then results in specific nicking at each 3′ end of Mu with relaxation of the vector domain, forming the type 1 (cleaved donor complex) (Craigie and Mizuuchi, 1987; Surette et al., 1987). The second chemical step results in strand transfer into non-Mu target DNA upon addition of Mu B, ATP, and target DNA, forming the very stable type 2 (strand transfer complex). Target capture complexes, which can occur at the LER, type 0, or type 1 stage (Naigamwalla and Chaconas, 1997) are not shown (modified from Naigamwalla et al., 1998). The second phage encoded protein, Mu B, required for the full transposition reaction selects non-Mu target DNA to be recruited into a Mu transpososome.
Molecular Cell  , DOI: ( /S (02) )

3. Figure 2
Footprinting and Chemical Probing of the Mu Left End in the LER
(A) DNase I footprints of the Mu left end and the Mu-host junction at the left end. LER was captured by crosslinking a reaction after 10 s incubation at 30°C utilizing Mu AE392Q, a catalysis-defective transposase mutant still competent for tetramer formation (Baker and Luo, 1994). For comparison, footprints were also performed for Mu A binding to linearized Mu ends and for type 0 using Mu AE392Q (with crosslinking). “+1” indicates the position of the terminal Mu nucleotides, while the limits of the L1, L2, and L3 binding sites are indicated by the open boxes to the right of each sequencing gel panel. The stars denote positions where enhanced cleavage was observed.
(B) Hydroxyl radical footprint of the Mu-host junction at the left end. To accumulate sufficient levels of LER complex for this analysis, LER was assembled and crosslinked after a 10 min reaction in which Mu AT585D was used. This transposase mutant is blocked in the transition from LER to the type 0 (Naigamwalla et al., 1998).
(C) KMnO4 probing of the Mu-host junction at the left end. The reaction conditions were as described for (B). After exposure to KMnO4, the material purified from the segregation gel was treated with 0.5 M piperidine at 90°C for 30 min. Piperidine treatment alone produced a G-specific ladder and accounts for the bands seen in all the lanes, including the lanes without KMnO4.
Molecular Cell  , DOI: ( /S (02) )

4. Figure 3
Footprinting and Chemical Probing of the Mu-Host Junction at the Right End
(A) DNase I, (B) hydroxyl radical, and (C) KMnO4 probing of the Mu-host junction at the right end. Conditions and legend as reported for Figure 2.
Molecular Cell  , DOI: ( /S (02) )

5. Figure 4 Footprinting Analysis of the Transpositional Enhancer
The main panel presents the DNase I footprints for the enhancer in the LER complex assembled and crosslinked in a reaction with Mu AT585D compared with Mu A and IHF footprints on linearized enhancer, type 0 (Mu AE392Q), and type 1 (wt Mu A) transpososomes. The type 0 and type 1 were analyzed without glutaraldehyde crosslinking, since glutaraldehyde obliterated the enhancer footprint for these transpososomes. The inset presents the results of hydroxyl radical footprinting of the enhancer (IHF site profiled) in the LER, type 1, as well as on linearized enhancer bound only with IHF. The O1 and O2 operators of the enhancer are delimited by the open boxes to the right of the main panel sequencing gel. The endpoints for the enhancer regions removed in the deletion and substitution mutations, O1 and O2 proximal (P), middle (M), and distal (D) relative to the IHF site are noted. Stars indicate the positions of enhanced reactivity to DNase I. The noted nucleotide numbers are from the Mu left terminal nucleotide.
Molecular Cell  , DOI: ( /S (02) )

6. Figure 5
Formation of a Three-Site Synaptic Complex Using Donor Plasmids Carrying Deletions or Substitutions of Transposase Binding Sites
Reactions performed with mutant donor plasmids utilized Mu AT585D as in Figures 2B, 2C, 3B, and 3C and were crosslinked and digested with BstXI and MluI. BstXI cuts between the Mu right end and enhancer, yielding a product with a topologically induced bandshift that separates three-site synaptic complex from those synapsed only through the Mu ends. The MluI site in the enhancer is specifically protected in the LER and provides additional discrimination between three-site synapsis and two-site synapsis. “Δ” signifies deletion of a Mu A binding site; “S” denotes substitution of a Mu A binding site with a non-Mu sequence, while preserving wild-type spacing for the flanking sites. For the enhancer mutant plasmids, “D,” “M,” and “P” refer to distal, middle, and proximal repressor sites in each operator relative to the intervening IHF site (see Figure 4 for further details). The reactivity of each donor plasmid in a cleavage assay utilizing LER reaction conditions but with wild-type Mu A is noted under each gel.
Molecular Cell  , DOI: ( /S (02) )

7. Figure 6
Delivery of L1 to the Mu Three-Site Synaptic Complex Promotes Catalytic Commitment and Strand Transfer
(A) The schematic shows the substrates and products of the reactions employing Mu donors lacking the L1 or R1 transposase binding sites. A supercoiled donor plasmid with the L1 site deleted along with a linear, radioactively labeled, and precleaved L1 site and target DNA was incubated with Mu A, Mu B, IHF, and ATP as described in Experimental Procedures. The double-end strand transfer (DEST) product has nicked ΔL1 donor attached to linearized target DNA tagged at the other end with the L1 or R1 oligonucleotide. The single-end strand transfer products (SEST) between the Mu right end and target DNA are shown; both products are relaxed because of the nick present in each molecule. Reaction of the ΔR1 donor produces only SEST between target and the Mu left end, as the Mu right end has no cleavage site.
(B) Electrophoretic analyses of the strand transfer reactions with the ΔL1 and ΔR1 donor plasmids are shown. Reactions were split in half and run in the absence and presence of SDS, as indicated. The left panel presents an ethidium bromide stained gel and its accompanying autoradiogram for reactions with the ΔL1 plasmid. The L1 oligonucleotide used was 5′ end-labeled on the nontransferred strand overhang and was visualized by autoradiography (right panel) after drying the agarose gel. The unlabeled product in lane 4 that comigrates with the type 2 represents the product of a single-end strand transfer reaction between the right end of the donor plasmid and the target plasmid. The reciprocal single-end event between the L1 oligo and target plasmid, which would appear as labeled relaxed target, was not observed. The right panel presents an ethidium bromide stained gel and its accompanying autoradiogram for reactions with the ΔR1 plasmid. Production of SEST is HU-dependent and occurs without participation of the added L1 or R1 oligos. Aside from SEST, relaxed donor is also produced, indicating that some complexes released target without executing strand transfer. ST and SD are supercoiled target and supercoiled donor; RT and RD, relaxed target and relaxed donor.
Molecular Cell  , DOI: ( /S (02) )

8. Figure 7
Changes in DNA Structure during Transpososome Assembly and Proposed Pathway of Assembly
(A) A pathway for type 0 assembly is presented. An obligate order for initial three-site synapsis is not assumed (Watson and Chaconas, 1996). The LER−L1 is a three-site complex consisting of Mu A bound at L2, L3, the enhancer, and the right end sites. L1 delivery by HU-mediated DNA bending results in formation of a complete LER (LER+L1), which then allows tetramerization of Mu A, engagement of the active site, and other conformational transitions accompanying the production of the catalytically competent type 0 transpososome.
(B) An interpretive schematic of the changes in DNA structure that occur during LER formation and the transition from the LER to the type 0 is presented. Mu A bound to the strong L1 site is delivered into the assembling three-site synaptic complex by HU-induced DNA bending at the L1-L2 spacer (not shown) (Kobryn et al., 1999; Lavoie et al., 1996). L1 association with the LER is in equilibrium, resulting in only a partial protection of L1 in the LER footprint (Figure 2A). L1 association with the LER is characterized by an unusual hydroxyl radical and DNase I sensitivity (closed oval) at nucleotide position 24 (Figure 2B), likely the locus of a dramatic LER-stabilized DNA kink or bend. When the Mu A tetramer is formed in the type 0 transpososome, L1 becomes irreversibly tethered in the tetrameric Mu A complex, the +24 kink is removed, and the Mu-host junction becomes engaged (Lavoie et al., 1991; Mizuuchi et al., 1991, 1992) by the transposase active site (domain II of the Mu A monomer interacting at the R1 site, shown as a stippled half oval [Aldaz et al., 1996; Mariconda et al., 2000; Namgoong and Harshey, 1998; Savilahti and Mizuuchi, 1996; Williams et al., 1999]). This active site engagement is accompanied by an extended footprint into the donor DNA (Figures 2 and 3) (Lavoie et al., 1991; Mizuuchi et al., 1991, 1992), as well as by an enhanced sensitivity to KMnO4 (asterisk) and hydroxyl radicals (closed oval) at the positions indicated (nontransferred strand) near the Mu-host junction (see also Wang et al., 1996).
At the enhancer, Mu A monomers bind to the individual sites (not shown), and IHF binds to induce a dramatic planar bend in the DNA (Higgins et al., 1989; Surette and Chaconas, 1992; Surette et al., 1989). Formation of the LER (Figure 4) results in a dramatic hyperreactivity to hydroxyl radicals (closed oval) and DNase I (closed oval) in the IHF site (nucleotides 952 and 955 from the Mu left end). We interpret the enhancements as an indicator of additional stress resulting from the increased DNA bending necessary to establish a negative node in the enhancer region. Such a structure would facilitate the previously mapped complex circuit of end-enhancer interactions including O1-L3, O1-R1, O2-L1, and O2-R3 (Allison and Chaconas, 1992; Jiang and Harshey, 2001; Jiang et al., 1999; Lavoie and Chaconas, 1995). The presence of the node would assist the simultaneous interaction of the O1 and O2 regions with the left and right ends of Mu. The LER-specific enhancements disappear when the type 0 forms; hence, an opening of the node to restore a planar DNA bend at the IHF site is shown in the transition to the type 0. This change is expected to reduce the number of end-enhancer interactions by removing the architectural features in the DNA structure, which optimizes interactions in the LER.
Molecular Cell  , DOI: ( /S (02) )