The Mu Three-Site Synapse

Molecular Cell, Vol. 10, 659–669, September, 2002, Copyright 2002 by Cell Press

The Mu Three-Site Synapse: A Strained Assembly Platform in which Delivery of the L1 Transposase Binding Site Triggers Catalytic Commitment Kerri Kobryn, Mark A. Watson,2 Ron G. Allison, and George Chaconas1,3 Department of Biochemistry University of Western Ontario London, Ontario N6A 5C1 Canada

Summary The Mu DNA transposition reaction proceeds through a three-site synaptic complex (LER), including the two Mu ends and the transpositional enhancer. We show that the LER contains highly stressed DNA regions in the enhancer and in the L1 transposase binding site. We propose that the L1 site acts as the keystone for assembly of a catalytically competent transpososome. Delivery of L1 through HU-mediated bending completes LER assembly, provides the trigger for necessary conformational transitions in transpososome formation, and allows target capture to occur. Relief of the stress at L1 and the enhancer may help drive Mu A tetramerization and engagement of the Mu ends by the transposase active site. Introduction Phage Mu replicates its DNA by efficient DNA transposition. Since the early establishment of a defined in vitro strand transfer reaction (Craigie et al., 1985), Mu has served as a paradigm for systems with multiple proteins bound in complex arrangements involving DNA bending and/or wrapping. Such nucleoprotein “machines” are a recurring motif in many aspects of nucleic acid metabolism, including replication, transcription, translation, recombination, and splicing. The Mu in vitro reaction proceeds through a series of characterized nucleoprotein complexes referred to as transpososomes (for a recent review, see Chaconas and Harshey [2002]). Conceptually, the Mu reaction is often split into separate stages: synapsis, formation of the catalytically competent transpososome, DNA cleavage, target capture, and strand transfer. Each stage is typified by a distinct transpososome (see Figure 1). This study focuses on the first two stages of the reaction, which result in the formation of the catalytically committed type 0 transpososome. This process requires supercoiled Mu donor DNA, Mu transposase (Mu A), divalent metal ion, and two host-encoded DNA bending proteins, HU and IHF. At an early stage, a transient interaction of the two Mu ends with the transpositional enhancer results in the formation of the three-site synap1

Correspondence: [email protected] Present address: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, United Kingdom. 3 Present address: Department of Biochemistry and Molecular Biology, and Department of Microbiology and Infectious Diseases, The University of Calgary, 3330 Hospital Drive NW, Calgary, AB, T2N 4N1, Canada. 2

tic complex, the LER (Watson and Chaconas, 1996). The catalytically inert transposase monomers assembled in the LER are subsequently transformed into the stable, catalytically active Mu A tetramer (Lavoie et al., 1991) of the type 0 transpososome (Mizuuchi et al., 1992). The tetramer of Mu A molecules is bound to the Mu L1, R1, and R2 transposase binding sites at the Mu ends (Kuo et al., 1991; Lavoie et al., 1991; Mizuuchi et al., 1991). Of the four monomers, only the L1 and R1-bound Mu A monomers appear to catalyze the cleavage and strand transfer reactions, doing so in trans: the L1-bound transposase contributes its “DDE” motif for the reactions at the right end and vice versa at the left end (Aldaz et al., 1996; Mariconda et al., 2000; Namgoong and Harshey, 1998; Savilahti and Mizuuchi, 1996; Williams et al., 1999). The Mu ends are more complex than the standard inverted repeat arrangement of single transposase binding sites found for bacterial insertion sequences and transposons. Each Mu end has three transposase binding sites, all of which interact with the enhancer during transpososome formation. Adding to the complexity of the Mu ends is the asymmetric arrangement of the transposase sites (Figure 1A). Salient in this regard is the ⵑ80 bp spacer between the L1 and L2 sites at the left end. This is the binding site for the required hostencoded DNA bending protein, HU (Lavoie and Chaconas, 1993; Lavoie et al., 1996). The L1-L2 spacer site specifically recruits HU in a supercoiling-dependent interaction (Kobryn et al., 1999). A sharp DNA bend is induced that is essential for transpososome assembly. The role of the transpositional enhancer in building the type 0 complex, and hence the Mu A tetramer, is gradually being elucidated. The enhancer is ⵑ950 bp from the left end, is ⵑ100 bp in length, and is divided into two essential regions (O1 and O2) separated by an IHF binding site (see Figure 4). The enhancer is involved in two distinct levels of regulation. Aside from the direct role played in transposition through the LER, the enhancer region contains the operators O1, O2, and O3, which are binding sites for the Mu repressor (Krause and Higgins, 1986) and which regulate expression from the Mu early promoter along with IHF and H-NS (van Ulsen et al., 1996). The transpositional enhancer was first discovered as a sequence required for the in vitro transposition reaction (Leung et al., 1989; Mizuuchi and Mizuuchi, 1989; Surette et al., 1989) and as the site of IHF action as a supercoiling relief factor for the reaction (Surette and Chaconas, 1989; Surette et al., 1989). An elaborate network of Mu end enhancer interactions has been mapped in two studies (Allison and Chaconas, 1992; Jiang et al., 1999). Surprisingly, the transpositional enhancer was also discovered to be functional in trans when provided in 50fold molar excess (Surette and Chaconas, 1992). Topological specificity of the reaction and the “criss-crossed” L1-O2 and R1-O1 interactions are both maintained when the enhancer is provided in trans (Jiang and Harshey, 2001). Aside from the direct demonstration of the LER’s existence, data on the role of the enhancer has been inferred

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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.

indirectly from the effect on the reaction of end/ enhancer mutants, hybrid enhancers, trans-acting enhancers, and transposase mutants. Direct experimentation on the LER complex has been hampered by its normally reversible and transient nature. In order to study the details of the LER complex and the process of its conversion to the catalytically committed type 0, we combined three experimental approaches: (1) footprinting studies of all three sites in the LER synapse, (2) determination of the DNA site requirements for LER formation and donor plasmid reactivity, and (3) “add back” experiments (in trans) of missing transposase binding sites to assess the competence of alternate three-site synapses to complete the reaction. Results Footprinting the Mu Ends in the LER The unit structure of transposase bound at the Mu ends in the LER was assessed by DNA footprinting and chemical probing of the DNA. Mu A tetramerization and catalytic commitment are characterized by extension of footprints from the L1 and R1 transposase binding sites to include protection of the terminal Mu nucleotides and the Mu-host junction in the type 0 complex. Furthermore, tetramerization and catalytic commitment can be followed by assaying for the induction of the characteristic reactivity with KMnO4 in the Mu-host junction that accompanies the formation of the type 0 (Wang et al.,

1996). Stabilization of the LER with glutaraldehyde was necessary to obtain DNA footprints. The type 0 transpososome was also crosslinked, solely to give a footprint under directly comparable conditions. Crosslinker was not used for Mu A binding to linear DNA in any of the footprints because it eliminated the Mu A footprint, as glutaraldehyde-modified free transposase is unable to rebind DNA. Under these reaction conditions, all six transposase binding sites at the Mu ends were found to be occupied in the LER and type 0 (data not shown). The resulting footprints in linear DNA, the LER, and the type 0 differed significantly only at the L1 and R1 sites and in the vicinity of the Mu-host junction. DNase I protection of L1 and R1 in the LER was found to most closely resemble the binding of Mu A to linear Mu DNA (Figures 2A and 3A). Protection did not include the terminal Mu nucleotides nor the flanking host DNA as was the case for the type 0. An interesting feature of the DNase I protection pattern was the presence of an LER-specific DNase I enhancement in L1 at position 24 (compare LER versus no protein in Figure 2A, bottom panel). Enhanced susceptibility to DNase I was not observed at this position during Mu A binding to either linear DNA or in the type 0. However, enhancement was observed at the neighboring 23 position in all cases. The footprinting also revealed less DNase I protection of the L1 site in the LER compared to that seen for the L2, L3 (Figure 2A, top panel), and R1 sites in the LER (Figure 3A). This L1-

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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.

specific difference was not observed in binding to linear DNA or in the type 0. Two possible explanations exist for the weaker L1 footprint in the LER. The first is that Mu A has a decreased affinity for the L1 site in the LER. The second possibility is that the LER exists in two forms: one in which L1 is incorporated and another form where this site is not in the complex. In the latter case, Mu A binding to the L1 site would be in equilibrium. Protein crosslinking perturbs the binding equilibrium of Mu A on any site not in stable association with the complex, by virtue of the chemical modification of the protein. Mu A modified by glutaraldehyde while off the DNA is unable to rebind. Glutaraldehyde modification of free protein would shift the equilibrium toward the off state, resulting in a weakened footprint. Experiments described later indicate that the weakened footprint re-

sults from the existence of two LER forms: one with L1 (LER⫹L1) associated and one without (LER⫺L1). Transpososome formation was monitored with greater resolution by hydroxyl radical footprinting (Figures 2B and 3B). These results confirmed the DNase I observations that the Mu A footprint in the LER at L1 and R1 did not extend into the Mu-host junction. Also apparent was the reduced protection of the L1 site and a hydroxyl radical hypersensitivity in the L1 site at the same positions as the enhanced DNase I cleavage (⫹23, 24). A slightly enhanced hydroxyl radical sensitivity at ⫹23 was all that persisted in the type 0, as was seen with DNase I (Figure 2B). Hydroxyl radical enhancements specific to the type 0 were observed at the ⫺2 and ⫺3 positions at both ends, but more prominently at the right end (Figure 3B) as previously noted for the

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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.

type 1 (Lavoie et al., 1991). These junction enhancements were absent from the LER. To further probe possible differences between the LER and the type 0, we also used KMnO4 to examine the Mu-host junction; helix melting occurs here in the type 0, resulting in KMnO4 reactivity (Wang et al., 1996). KMnO4 is a chemical probe of DNA structure that only modifies unbase-paired nucleotides, most prominently at thymidines. Permanganate reactivity of the junction was found in the type 0, but not in the LER, at either the left (Figure 2C) or right end (Figure 3C). This is another indication that Mu A tetramerization and active site engagement with the Mu ends has yet to occur in the LER. Footprinting the Transpositional Enhancer in the LER The interaction of Mu A and IHF with the enhancer was examined in detail by footprinting of the purified proteins on linearized enhancer and in the LER, type 0, and type 1 with DNase I (Figure 4). The enhancer footprints in all three complexes were found to be similar to each other, yet distinct from the individual and additive footprints of IHF and Mu A on linear enhancer without glutaraldehyde crosslinking. The area of DNase I protection in the LER, type 0, and type 1 was smaller than that defined by Mu A binding to the linear enhancer (O2 distal was unprotected in the complexes). The LER enhancer footprint was further abrogated for the IHF distal portion of O1, indicating a smaller core of stable crosslinking-resistant interaction in the LER than defined by simple Mu A recognition of the enhancer. The most striking feature of the enhancer footprints was found in the IHF site of the LER complex, which appears to be under unusual stress, as evidenced by the dramatic DNase I sensitivity of this complex at nucleotide positions 952 and 955 (numbered from the Mu left end). The hyperreactivities were also observed in greater detail by hydroxyl radical probing (Figure 4, right inset) and correspond to the site of interaction of the IHF-␣ subunit at two (952) and five (955) nucleotides from the dramatic DNA kink induced by the intercalative interac-

tion in the DNA minor groove of its recognition site (Rice et al., 1996). We suggest that the LER-specific stress in the IHF site is caused by further deformation of the IHF site to form a negative node in the DNA, which is stabilized by Mu A monomers bridging the Mu ends and the O1 and O2 regions of the enhancer (see Discussion for further elaboration). It is noteworthy that while the type 0 and type 1 enhancer footprints in the absence of cross-linking (Figure 4) were similar to the crosslinked LER footprint, they did not show the hypersensitivities at positions 952 and 955; hence, these hypersensitivities are unique to the LER. Attempts to footprint the enhancer after crosslinking the type 0 and type 1 resulted in no protection of the enhancer (except for some residual IHF binding; data not shown). This indicated a weakened or disrupted set of end-enhancer interactions upon conversion of the LER into the more stable type 0 and type 1 transpososomes. Recent work by Pathania et al. (2002) has shown a continued association between the enhancer and the type 0 transpososome; our data indicate that the continued association differs from that in the LER. The L1 Site, Which Is Crucial for Catalysis, Is Not Required for Three-Site Synapsis Our interpretation of the weak protection of L1 in the LER (Figure 2) was that this site was not in stable association with the LER. A prediction from this interpretation was that a three-site synaptic complex might assemble in the absence of L1, a site whose presence is critical for transpososome formation, including Mu A tetramerization and catalytic activity. To test this, as well as the role of all of the Mu A binding sites at the Mu ends and the enhancer, we monitored the formation of three-site synaptic complexes in reactions using Mu end and enhancer mutants. The mutant mini-Mu donors contained precise deletions or substitutions of each Mu A binding site. For the enhancer mutants, deletion or substitution of the IHF proximal, middle, and distal regions defined in Figure 4 were used. The assay for LER formation (Watson and Chaconas, 1996) utilized Mu AT585D. This

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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.

transposase mutant accumulates LER because of a block in the transition from LER to the type 0 (Naigamwalla et al., 1998). The results of the LER reactions with the mutant miniMu’s are presented in Figure 5. Removal of any one of the three left end binding sites did not hinder LER formation. This was not surprising for L3 and L2, which are known not to be essential for substrate reactivity in the context of the cooperative transpososome assembly process (Allison and Chaconas, 1992; Lavoie et al., 199) (see DNA cleavage activity for each substrate noted under the gel profile in Figure 5). The observation that normal levels of a three-site synaptic complex formed in the absence of L1 was particularly important. This site is part of the active Mu A tetramer and is crucial

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. “⌬” 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.

for catalysis. Detailed examination of the three-site synaptic complex formed by the L1 plasmid revealed that besides being unable to proceed to the type 0, it was also unable to capture target DNA to form an LER target capture complex (TC-LER; data not shown). Mu target capture complexes contain target DNA in a noncovalent association with the Mu transpososome (Naigamwalla and Chaconas, 1997). In contrast to the findings at the left end, mutation of

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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 ⌬L1 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 ⌬R1 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 ⌬L1 and ⌬R1 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 ⌬L1 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 ⌬R1 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.

either the R2 or R3 binding sites blocked recovery of significant levels of three-site complex (Figure 5). This was expected for R2, which is required for transpososome formation, but not for R3, which is expendable (Allison and Chaconas, 1992; Lavoie et al., 1991). The ⌬R3 substrate, in spite of not making detectable levels of LER, was utilized in the cleavage reaction at about 40% wild-type levels (Figure 5A). We interpret our failure to recover three-site complex for the R3 deletion as a destabilization of the three-site complex that is severe enough to block crosslinking of the complex and detection in our assay, but not severe enough to eliminate donor plasmid reactivity. Interestingly, a small amount of LER was observed with the R1 deletion mutant, but this plasmid was unreactive in the cleavage assay. The severity of the right end effects indicate that the interaction between the right end and the enhancer is a primary determinant of LER stability. This agrees with the primacy of the O1-R1 interaction mapped previously in Jiang and Harshey (2001) and Jiang et al. (1999). Mutation of most of the Mu A binding sites in the O1 and O2 enhancer regions had quite severe effects on LER recovery. However, deletion of the O1 distal site,

which is not well protected in the LER footprint, had no effect on the recovery of LER complex. In contrast to the general decrease of three-site complex in our gel assay (with the exception of O1D), the individual enhancer mutations displayed only minor decreases in DNA cleavage in a type 1 assay. We again interpret these results as a destabilization of the three-site complex that is severe enough to block crosslinking of the complex and detection in our assay, but not severe enough to eliminate donor plasmid reactivity. The combined results to this point suggested that association of the left and right ends with the enhancer to form a three-site synaptic complex did not require the L1 site and that the L1 site was added subsequently to finalize LER assembly. The L1 Site Provides a Critical Signal for Transpososome Formation and Target Capture The robust yield of three-site synaptic complex obtained with the ⌬L1 plasmid prompted us to consider the possibility that “delivery” of the L1 site, mediated by the supercoiling-dependent site-specific action of HU in the L1-L2 spacer (Kobryn et al., 1999), was the trigger event

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for the transition from LER to the type 0 (i.e., the assembly of the Mu A catalytic tetramer). If L1 was not required for formation of the LER⫺L1 and was the last component recruited to the complex, it would be expected to demonstrate functional independence from the remainder of the end binding sites. We therefore provided L1 in trans as a precleaved oligonucleotide (see Experimental Procedures). Precleavage was required since it eliminates the need for DNA supercoiling at L1, bypassing the need for activation of the Mu-host junction by helix melting (Savilahti et al., 1995; Wang et al., 1996). Delivery of a 10- to 20-fold molar excess of precleaved L1 under normal reaction conditions (no DMSO or glycerol) triggered strand transfer and formation of a type 2 between the ⌬L1 donor and a target plasmid (Figure 6B, lane 3). Disruption of the complex with SDS (lane 4) released a double-end strand transfer product and lesser amounts of a single-end strand transfer product (migrating at the type 2 position) between the Mu right end and the target plasmid. Visualization of the 32P-labeled L1 oligonucleotide used in the reaction by autoradiography of the stained gel confirmed the presence of L1 in the type 2 and its incorporation into the double-end strand transfer product (lanes 3 and 4, respectively, of Figure 6B). The strict enhancer dependence of the reaction was maintained, as evidenced by the requirement for IHF in the L1 trans reaction (Figure 6B, lanes 5 and 6) and by the failure of the reaction when Mu A77-663, in which the enhancer binding domain is deleted, was used (data not shown). The L2 and L3 sites were also still required when L1 was provided in trans (data not shown). The persistence of the stringent enhancer and end requirement for the L1 trans reaction indicated that the reaction on the supercoiled ⌬L1 donor was not corrupted as in the presence of the stimulatory chemicals DMSO and glycerol, which allow promiscuous assembly of the Mu A tetramer (Baker and Mizuuchi, 1992; Mizuuchi and Mizuuchi, 1989). Unlinking L1 from the rest of the miniMu donor plasmid did, however, abolish the HU requirement of the reaction (reactions shown in Figure 6B are performed without HU unless otherwise indicated). This was not unexpected, since the only role of HU in a supercoiled substrate appears to be at the L1-L2 spacer (Kobryn et al., 1999; Lavoie and Chaconas, 1993; Lavoie et al., 1996). The reciprocal reactions with a ⌬R1 donor were not rescued by the addition of R1 in trans (Figure 6B, lanes 9–10). However, the ⌬R1 plasmid unexpectedly formed a type 2 when HU was also present in the reaction. The HU and IHF (data not shown) requirements implicated the need for both the L1 site and the enhancer, respectively. However, the resulting strand transfer products were all single-end events between the Mu left end and the target molecule. The R1 site was not required and could not be utilized when provided in trans, as evidenced by the lack of its incorporation into the type 2 or the single-end strand transfer product when radiolabeled R1 oligo was a component of the reaction (Figure 6B, lanes 11 and 12). The type 2 generated with the ⌬R1 donor was therefore an abortive end product. The ability of the ⌬R1 donor to undergo abortive single-end strand transfer (Figure 6) while being totally inactive in the DNA cleavage reaction (Figure 5) can be reconciled by the presence of Mu B, ATP, and target DNA for the strand

transfer reaction (Figure 6). The presence of these components is known to stimulate the reaction under suboptimal conditions (see Chaconas and Harshey, 2002). In summary, the ⌬L1 plasmid was totally inert in the absence of L1; however, addition of L1 in trans resulted in target capture, stable transpososome formation, and production of double-end strand transfer products. In contrast, the ⌬R1 plasmid could only enter an abortive assembly pathway that resulted in single-end strand transfers. Addition of R1, in trans, to the ⌬R1 plasmid was unable to rescue the reaction to produce bona fide double-end strand transfer events. The combination of these results highlights the importance of L1 and its usual delivery by HU in both transpososome assembly and target capture. In all cases, a strict dependence upon the presence of L1 for both of these processes was observed. Finally, L1, but not R1, demonstrated functional independence, as a ⌬L1 but not a ⌬R1 donor could be complemented in trans, as would be expected if L1 were normally the last component added in the transpososome assembly process. Discussion The L1 Mu A Binding Site Acts as the Keystone for Transpososome Assembly from the LER In the work described here, we present a biochemical characterization of the Mu LER synapse (Watson and Chaconas, 1996) and the process of its conversion to a catalytically competent transpososome. The LER is a critical intermediate leading to the assembly of the more stable Mu transpososomes (see Chaconas and Harshey, 2002). In footprinting studies, we found that the L1 transposase binding site was only partially protected in the LER (Figure 2), suggesting a mixture of complexes: one containing L1 (the LER⫹L1) and one in which L1 is not associated (the LER⫺L1). This suggested the possibility that a three-site complex could be formed in the absence of L1. This hypothesis was tested and we indeed found wild-type levels of three-site complex using a ⌬L1 mini-Mu, despite this site being essential for Mu A tetramerazition and donor reactivity (Figures 5 and 6). Finally, the ability of L1 to be delivered independently, in trans, was demonstrated (Figure 6). The information described above supports the assembly pathway depicted in Figure 7A. The early steps of LER assembly have been discussed (Watson and Chaconas, 1996), and we previously suggested alternative two-site synaptic complexes leading to the LER. Both the LR and RE complexes have been observed, although the LE has not. More recently, an LR assembly pathway has been proposed, but the data obtained using unlinked oligonucleotides in DMSO do not rule out other alternatives in supercoiled donor substrates (Mizuuchi and Mizuuchi, 2001). Similarly, the primacy of the enhancer/right end interaction (Jiang and Harshey, 2001; Jiang et al., 1999; and this study) is consistent with an RE pathway, but does not rule out others. Based upon the complexities of the Mu transposition system, it would not be surprising if all three two-site complexes were viable intermediates. The information in the assembly pathway contributed by this work is the existence of two distinct LER com-

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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 threesite 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.

plexes and the role of L1. The LER⫺L1, which contains the L2, L3, R1, R2, and R3 end sites and the enhancer, readily forms, but cannot proceed into the catalytically competent type 0, because it is missing L1 (Figure 5). The L1 site is subsequently delivered through HU-mediated bending of the ⵑ80 base pair L1-L2 spacer (see Figure 1A). In agreement with this is our ability to efficiently generate the three-site synaptic complex in the absence of HU despite this being a condition that completely blocks the formation of reactive transpososomes (Watson and Chaconas, 1996). Upon entry into the complex to establish the functional LER⫹L1, the L1 site undergoes additional bending (Figure 7B, discussed below), which would facilitate interactions with the enhancer and other end sites. The strain contributed to the LER from the DNA deformations may then either eject the

L1 site, establishing an equilibrium between the LER⫺L1 and the LER⫹L1, or may help trigger the conformational transitions required to generate the catalytically competent type 0. The choice between L1 dissociation from the LER versus type 0 formation may be critically modulated by the level of DNA supercoiling, which dramatically regulates HU binding to the L1-L2 spacer (Kobryn et al., 1999). The results presented in Figure 6 also highlight the requirement of L1 for target capture to occur, a process that involves recruitment of non-Mu DNA to the transpososome by the phage-encoded accessory protein, Mu B. Further experiments indicated that while necessary, the L1 site alone was not sufficient for target capture (data not shown). In contrast, target capture did occur in the absence of R1. More surprisingly, transpososome formation and single-end strand transfer were

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found to occur under certain conditions in the absence of R1 (see Figure 6B). Our explanation for these results is that the presence of MuB, ATP, and target DNA promoted cooperative assembly of the Mu transpososome (Mizuuchi and Mizuuchi, 2001; Mizuuchi et al. 1995) by helping to load a Mu A monomer next to R2 on the ⌬R1 plasmid. The resulting Mu A tetramer has no cleavage site at the right end and consequently could execute only single-end strand transfers. The R1 site added in trans was excluded from this altered transpososome either due to kinetic disadvantage during assembly or more trivially by steric exclusion of its delivery. The inability to utilize the R1 site added in trans to produce bona fide double-end strand transfer products indicates that the reaction observed with the ⌬R1 plasmid generates an abortive end-product. Changes in DNA Structure Associated with Transpososome Assembly Our DNA footprinting data indicate that several important changes in DNA conformation occur during transpososome assembly, as depicted in Figure 7B. The first is a DNA deformation resulting in enhanced reactivity to DNase I and hydroxyl radicals at position 24 of L1. Interestingly, L1 is the only Mu end binding site to display an LER-specific alteration of structure, a feature that may be related to its unique role in transpososome assembly. The L1 site has previously been reported to be the site of an approximate 90⬚ bend induced by Mu A binding to a solitary L1 site (Kuo et al., 1991). The LERspecific deformation that induces stress at position 24 must be an additional deformation, perhaps a “superbend” to the already bent DNA or a change in trajectory of one of the DNA arms to generate an “out-of-plane” bend. It is tempting to speculate that this additional stress plays a role in the apparent unstable association of L1 with the LER and/or with the proposed unique role of L1 as the trigger for the LER-type 0 transition. The LER-specific stress point in L1 is relieved upon conversion of the LER to the stable type 0. It should be emphasized that the observed enhancement is likely substantially greater than it appears in Figure 2, because the L1 site is likely associated with the LER in only a subpopulation of the footprinted complexes. Transition to the catalytically competent type 0 transpososome has been reported to be accompanied by an extended footprint of ⵑ15 base pairs into the Mu-host junction (Mizuuchi et al., 1992) as well as by destabilization of the host DNA adjacent to the cleavage site (Lavoie et al., 1991; Savilahti et al., 1995; Wang et al., 1996). We now know that neither of these features is present in the LER (Figures 2 and 3) and that they must occur during the transition of the LER to the type 0. They are therefore associated with engagement of the Mu ends by the active site of the stable transposase tetramer that characterizes the type 0 and later transpososomes. In agreement with this contention is genetic data that terminal base pair mutations at the Mu ends specifically block type 0, but not LER formation (Coros and Chaconas, 2001; Watson and Chaconas, 1996), arguing for a lack of engagement of the DNA cleavage site by the transposase in the LER. In addition to the changes in DNA structure noted at

the Mu ends, a dramatic enhancement to DNase I and hydroxyl radicals was noted at the enhancer, only in the LER (Figure 4). It was not observed with Mu A and IHF bound to a linear enhancer or in the type 0 or type 1 transpososomes. Once again, the LER, poised at a critical juncture of complex assembly, appears to contain transiently and excessively strained DNA. The LER-specific enhancements at positions 952 and 955 occur within the IHF site, which is believed to possess an IHF-induced planar bend approaching 180⬚ (Rice et al., 1996). We suggest that formation of the LER induces further bending at this locus, resulting in a ⬎180⬚ bend out-of-plane relative to the nearly planar IHF-induced bending. This arrangement would be the least costly energetically as a negative node in the negatively supercoiled DNA. We propose that this DNA deformation, along with the LER-specific alteration in structure at the L1 site, is necessary to optimize the complex circuit of interactions between the two Mu ends and the enhancer that are required to drive transpososome formation. Finally, it is possible that some of the energy of DNA deformation stored in the DNA “superbends” at L1 and the IHF site in the LER may be used to promote the LER-type 0 transition. In summary, the work described above establishes the role of two distinct three-site synaptic complexes (with and without L1) in the transpososome assembly process. We also demonstrate that the LER is a strained synaptic complex and propose that addition of the L1 site is the trigger for assembly of the catalytically committed transpososome and capture of target DNA. Experimental Procedures Proteins and Primers The Mu A, Mu B E. coli HU and IHF were all purified as reported previously (Chaconas et al., 1985; Coros and Chaconas, 2001; Lavoie and Chaconas, 1993). Primers used in this study were: P-6 (5⬘AATTCCACGTCATAGTAAAAATTGCTTTT-3⬘), P-25 (5⬘-AATACTCG AGAAAAAATAGTAAAAAATTGC-3⬘), P-27 (5⬘-TGAAAAGCTTTTTGT AAAGCTGCCCGATGTTTTCGGCC-3⬘), P-28 (5⬘-CAGTCTCGAGTGTA CTCCTTATTTATCAAC-3⬘), P-29 (5⬘-TACACTCGAGACTGTCTAACTT TATAGAAAAGAA-3⬘), P-36 (5⬘-AACCTCGAGACTGTCAATAATAC-3⬘), P-37 (5⬘-ATTGACAGTCTCGAGGTTACTTTTC-3⬘), #867 (5⬘-GGC CTTTTCGTTGGAACACA-3⬘), M-131 (5⬘-GGAAGCGGCTAAATACCA AAC-3⬘), M-132 (5⬘-CCCGCGGGATCCGAAAGCGTTTCACGATAAA TGCGA-3⬘). L1 oligonucleotides were M-157 (5⬘-CCTCCCGGTTTTT TTCGTACTTCAAGTGAATCAATACA-3⬘) for the transferred strand and M-158 (5⬘-CTAGTGTATTGATTCACTTGAAGTACGAAAAAAACC GGGAGG-3⬘) for the nontransferred strand. R1 oligonucleotides were M-159 (5⬘-CTAGTGAAGCGGCGCACGAAAAACGCGAAAG CGT-3⬘) for the transferred strand and M-160 (5⬘-ACGCTTTCGCGT TTTTCGTGCGCCGCTTCA-3⬘) for the nontransferred strand. Plasmid Constructions pBL07-NcoI for enhancer footprinting was constructed by NcoI linker insertion into the end-filled AflII site in pBL07 (Watson and Chaconas, 1996) located 1.15 kb inside the Mu left end. The ⌬L1 plasmid was constructed by excision of the small BglII-BamHI fragment of pBL03 (Lavoie et al., 1991) and religation of the blunt ended large fragment. This deleted half of the spacer, L1, and the terminal Mu nucleotide for the left end. ⌬R1 was constructed by PCR generation of R2R3 using primers M-131 and M-132 on pRA02 template. Digestion of the resultant PCR product with StyI and BamHI and ligation into the large StyI-BamHI fragment of pRA02 produced a mini-Mu in which two thirds of the R1 site is deleted (to maintain the overlapping part of R2) along with the terminal Mu nucleotide at the right end.

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The enhancer mutant plasmids were constructed as follows: substitution of the middle of the O1 enhancer region was performed by generating a PCR product using mutagenic oligonucleotides P-36 and P-25 on template of pRA01 (having a wild-type enhancer region). A second PCR product was generated with the mutagenic primer P-37 and the sequencing primer #867. The products were digested with NsiI plus XhoI and HindIII plus XhoI, respectively, and ligated into the large NsiI-HindIII fragment of pRA01 to generate pRA1M1 (SO1M). Substitution of the proximal region of the enhancer was accomplished by generating PCR products using the mutagenic primer P-29 and P-25 on a pRA1B5 template and mutagenic primer P-28 and primer #867 on a pRA02 template. The products were ligated into the large NsiI-HindIII fragment of pRA01 to generate pRAP1 (SO1P). Substitution of the region of O2 proximal to the IHF binding site was accomplished by using the primer P-27 and P-6 with pRA1B5 as template DNA. This PCR product was digested with HindIII and StyI and ligated into the large HindIII-StyI fragment of pRA01 to produce pRA1P2 (S02P). ⌬O1D is pRA1D4 and ⌬O2M is pRA1B73; they are described in Allison and Chaconas (1992).

Acknowledgments

Footprinting Reactions LER and type 0 complexes were assembled and crosslinked as reported previously (Watson and Chaconas, 1996). For footprinting the Mu left end, pBL03 was used (Lavoie et al., 1991) and digested and 3⬘ end-labeled at the StyI site inside the left end. Label was segregated, and two-site complexes separated from three-site complexes by digestion with SspI and NsiI, respectively. The NsiI digestion removed a large fragment of DNA from two-site complexes but not from the LERs. For the Mu right end, pRA02 was used (Lavoie et al., 1991) and digested and 3⬘ end-labeled at the StyI site inside the Mu right end. Label was segregated, and two-site complexes separated from three-site complexes by digestion with SspI and NsiI, respectively. For footprinting the enhancer, pBL07-NcoI was used. After complex assembly and crosslinking, the complexes were digested with NcoI and 3⬘ end-labeled. Label was segregated by digestion with SspI, and the LER reaction was further challenged with MluI, coincident with the SspI digestion. The LER reaction was dissociated in 0.5% SDS after treatment with footprinting reagent for application to the segregation gel (MluI-resistant NcoI-SspI fragments were purified). The uncrosslinked type 0 and type 1 complexes examined in the enhancer footprinting were isolated as complexes on the segregation gel as reported for the other figures. Processing of the segregation and sequence gel runs were as reported previously (Lavoie et al., 1991). The linear footprinting controls in Figures 2–4 were generated after binding of Mu A or IHF at 30 ␮g/ml and 1 ␮g/ml, respectively, at room temperature for 5 min, followed by treatment with footprinting reagent. The footprinting conditions were as follows: (1) DNase I treatment was at 0.04 ␮g/ml at room temperature for 4 min, (2) hydroxyl radical treatment was with 160 ␮M ammonium ferrous sulfate, 320 ␮M EDTA, 3 mM sodium ascorbate, and 0.0007% H2O2 at room temperature for 5 min, and (3) KMnO4 treatment was with 2 mM KMnO4 for 5 min at room temperature. All reactions were terminated by adding 1/10th volume of loading buffer (45% w/v glycerol, 250 mM EDTA). The segregation gel, processing, and markers are all as reported (Kobryn et al., 2000).

Baker, T.A., and Luo, L. (1994). Identification of residues in the Mu transposase essential for catalysis. Proc. Natl. Acad. Sci. USA 91, 6654–6658.

L1 and R1 in trans Strand Transfer Reactions The reactions were performed in buffer containing 25 mM HEPESNaOH (pH 7.6), 10 mM MgCl2, 100 mM NaCl, and 100 ␮g/ml BSA at 30⬚C for 30 min. Reaction components were 45 ␮g/ml ⌬L1 donor plasmid, 60 ␮g/ml pSD7 target plasmid (Surette and Chaconas, 1991), 24 ␮g/ml Mu A, 20 ␮g/ml Mu B, 2 mM ATP, ⫾1 ␮g/ml IHF, and ⫾6.6 ng/ml 5⬘ end-labeled L1 (annealed oligos M-157/8 5⬘ endlabeled with polynucleotide kinase on the BamHI overhang after annealing). As unincorporated L1 or R1 oligos run off the end of the assay gels, the specific activity of radiolabeling was confirmed independently. When used, HU was at 11.25 ␮g/ml. The oligo was added after a brief preincubation (ⵑ30 s) of the reactions at 30⬚C. 30 ␮l reactions were split and loaded onto agarose gels, either in normal loading buffer (see Footprinting Reactions section) or the same loading buffer with SDS at a final concentration of 0.5%.

We would like to thank Chris Brandl, Colin Coros, Brigitte Lavoie, and Yvonne Tourand for helpful comments on the manuscript, and Victor Zhurkin for helpful discussions. This work was supported by the Canadian Institutes of Health Research. G.C. was supported by a Distinguished Scientist Award from the Canadian Institutes of Health Research. Received: February 22, 2002 Revised: June 21, 2002 References Aldaz, H., Schuster, E., and Baker, T.A. (1996). The interwoven architecture of the Mu transposase couples DNA synapsis to catalysis. Cell 85, 257–269. Allison, R.G., and Chaconas, G. (1992). Role of the A protein-binding sites in the in vitro transposition of Mu DNA. A complex circuit of interactions involving the Mu ends and the transpositional enhancer. J. Biol. Chem. 267, 19963–19970.

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