©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Kinetic and Structural Probing of the Precleavage Synaptic Complex (Type 0) Formed during Phage Mu Transposition
ACTION OF METAL IONS AND REAGENTS SPECIFIC TO SINGLE-STRANDED DNA (*)

(Received for publication, April 20, 1995; and in revised form, January 10, 1996)

Zhenggan Wang(§)(¶) Soon-Young Namgoong (§) Xushao Zhang(§)(¶) Rasika M. Harshey(§)(**)

From the Department of Microbiology, University of Texas, Austin, Texas 78712

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In an earlier kinetic study (Wang, Z., and Harshey, R. M.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 699-703), we showed that supercoiling free energy was utilized during Mu transposition to lower the activation barrier of some rate-limiting step in the formation of the cleaved Mu end synaptic complex (type I complex). We report here results from kinetic studies on the assembled but uncleaved synaptic complex (type 0). Based on the estimated rate constants for the formation of type 0 and type I complexes, as well as their temperature and superhelicity dependence, we infer that the type 0 complex is an authentic intermediate in the pathway to Mu end cleavage. Our results are consistent with type 0 production being the rate-limiting step in the overall type I reaction. The conversion of type 0 to type I complex is a fast reaction, does not show strong temperature dependence, and is apparently independent of substrate superhelicity. We have explored the DNA structure within the type 0 complex using chemical and enzymatic probes. The observed susceptibility of DNA outside the Mu ends to single-strand-specific reagents suggests that a helix opening event is associated with type 0 formation. This structural perturbation could account, at least partly, for the high activation barrier to the reaction. There is a close correlation between the appearance of single strandedness near the Mu ends and the superhelicity of the DNA substrate. It is possible that supercoiling energy is utilized in effecting specific conformational transitions within DNA. We have found that Zn and Co ions, like Mg and Mn ions, can efficiently cleave the type 0 complex. However, unlike Mg and Mn ions, Zn and Co ions cannot support assembly of type 0. We discuss the implications of our findings for the mechanism of Mu transposition.


INTRODUCTION

Mu DNA transposition occurs by cleavage (nicking) of the left and right ends of Mu DNA (attL and attR), followed by joining (strand transfer) to target DNA, a reaction carried out by Mu transposase (A protein) (reviewed in Mizuuchi(1992)). The functional configuration of Mu A protein is a tetramer (Lavoie et al., 1991). Assembly of the active Mu A tetramer is a complex process requiring negatively supercoiled DNA substrate, a set of six att subsites, a pair of internal enhancer sites, the Escherichia coli HU protein, and divalent metal ions (Mg or Mn) (see Mizuuchi(1992), Lavoie and Chaconas(1993), Wang and Harshey(1994), and Yang et al. (1995a, 1995b)).

After the cleavage or nicking step of transposition, the cleaved Mu ends can be located within a stable high-order DNA-protein assembly called a ``type I complex'' (Fig. 1) (Surette et al., 1987). In the presence of Ca, an uncleaved synaptic complex (type 0 complex) accumulates in a reaction that also requires DNA supercoiling, the internal enhancer sequence, and HU protein for its formation and resembles the type I complex in harboring the tetramerized form of Mu A (^1)(see Fig. 1) (Mizuuchi et al., 1992; Surette and Chaconas, 1992).


Figure 1: The Mu end nicking step of transposition. Mu ends (L and R, heavy line) on a negatively supercoiled plasmid (left) are brought together (synapsed) in the presence of Mu A protein, E. coli HU protein, and Ca (Me, metal ions) to generate a presumed intermediate (type 0 complex, middle). An internal enhancer DNA element (dumbell) is required for the formation of this complex. In the presence of Mg, Mu ends are cleaved (single-stranded nick at each 3` end) by Mu A protein to generate a type I complex (right; non-Mu sequences are relaxed). The Mu A protein is found as a tetramer in both complexes (see Introduction for details).



During a kinetic investigation of the role of DNA supercoiling in type I complex formation, we observed a dependence of the reaction rate on superhelical density as well as on the length of DNA outside Mu in the donor plasmid (Wang and Harshey, 1994). A strong temperature dependence (in the range of 15-35 °C) was also observed, and a rather high activation enthalpy (67 ± 15 kcal/mol) was calculated for the reaction. These results could be well explained by a model in which the free energy of supercoiling is used directly to lower the activation barrier of a rate-limiting step. Our results implied the existence of an intermediate in which Mu ends have come together to allow domain closure, i.e. separation of Mu and non-Mu domains, the supercoiling energy from the non-Mu domain being utilized to promote the rate-limiting step of the reaction. We speculated that free energy from DNA supercoiling might be used to drive conformational changes of DNA, such as melting of the ends, and/or conformational changes of Mu A protein.

In this study we show first that the uncleaved type 0 complex assembled in the presence of Ca is likely a bona fide intermediate in formation of the type I complex. We present evidence suggestive of the presence of single-stranded DNA just outside the Mu ends in the type 0 complex and its dependence on DNA supercoils.


EXPERIMENTAL PROCEDURES

Reagents

Chloroquine phosphate, KMnO(4), and MES (^2)were purchased from Sigma, P1 nuclease from Boehringer Mannheim, topoisomerase I from Life Technologies, Inc., and DeltaTaq and Sequenase DNA sequencing kits from U.S. Biochemical Corp. Mu A and E. coli HU proteins were purified as described (Kuo et al., 1991).

Construction of Plasmids

Plasmid pRA170 has been described (Leung et al., 1989; Wang and Harshey, 1994). pZW140 was derived from pRA170 by deleting a PvuII fragment in the trp-lac region within mini-Mu. The lengths of the Mu and non-Mu domains in this plasmid are similar (2 and 2.2 kilobases, respectively).

Preparation of Topoisomers

Topoisomers of mini-Mu plasmids were prepared and analyzed as described earlier (Wang and Harshey, 1994).

Kinetic Assay of the Type 0 Complex and Its Conversion to Type I Complex

Kinetic assays for formation of the type I complex have been described (Wang and Harshey, 1994). Mu A and HU proteins were added to the mini-Mu donor plasmid in 25 mM Tris, pH 7.5, 130 mM NaCl, and 10 mM MgCl(2), at indicated temperatures. Formation of the type 0 complex was assayed indirectly as follows. The complex was formed in the presence of 10 mM CaCl(2) instead of MgCl(2), and the reaction was stopped by the addition of 0.5 µg/ml heparin. The complex was purified over a Bio-Gel P-100 spin column (spin dialysis) and cleaved by the addition of 20 mM Tris-HCl, pH 7.5, 130 mM NaCl, and 10 mM MgCl(2), followed by a 20-min incubation at room temperature. Cleavage reactions were terminated by the addition of either 20 mM EDTA or 0.2% SDS (final), and electrophoresed on 1.2% agarose gels in 1 times TAE buffer with 0.3 µg/ml EtBr. Conversion of type 0 to type I complex was assayed the same way, except that type 0 complexes were formed at 30 °C for 10 minutes and the cleavage reaction was carried out for various times at the required temperature.

In experiments using different metal ions, type 0 complexes were directly assayed according to the procedure recently described by Kim et al.(1995), where treatment of type 0 reaction mixtures with 10 µg/ml heparin before electrophoresis results in a distinct type 0 complex band, migrating just above the supercoiled donor plasmid.

P1 Nuclease Cleavage

Type 0 and type I complexes that were passed over a Bio-Gel P-100 spin column were incubated with 50 ng/ml P1 nuclease in 20 mM MES, pH 6.5, for 20 min on ice. P1 digestion was stopped by the addition of 100 mM Tris-HCl, pH 8.5. In primer extension experiments designed to map P1 cleavages (see below), 25 ng/ml heparin was used to stop the reactions before spin dialysis. The mixtures were then extracted twice with phenol, and DNA was precipitated with 70% ethanol. Control DNA missing one or more of the reaction components in the initial kinetic reaction was treated in the same way.

KMnO(4) Reactions

Approximately 2 µg of type 0 complex DNA was reacted with 4 mM KMnO(4) at 20 °C for 15 min in 5 mM Tris-HCl, pH 8, 0.5 mM EDTA. DNA was spin dialyzed over a Sepharose CL-6B column pre-equilibrated with buffer, precipitated twice with ethanol, and dissolved in 100 µl of 1 M piperidine. Samples were heated at 90 °C for 30 min and dried in vacuum with two to three changes of water. 0.2-0.5 µg of DNA was used for primer extension reactions.

Primer Extension

The sequences of the primers used are as follows: R3, AATGAATAAAAAGCAGT; RB, GGTTAATGTCATGATAATAA; L2, AGCTTCTTTCGCGTTTCATTGATTAACG; LT, CAGGAAGGCAAAATGCCGCA.

Primer extension was carried out in two alternate ways. 1) Primers were first labeled with [-P]ATP to a specific activity of 1 Ci/µmol. Reaction mixtures containing 1 µl (0.1 µg) of template DNA, 0.5 µl (0.5 µM) of labeled primer, and 1.9 units of DeltaTaq polymerase in 30 mM Tris, pH 9.5, 7.5 mM MgCl(2), were incubated at 90 °C for 2 min and immediately cooled on ice. Next, 2 µl of extension mix (15 µM dNTP) was added, and incubation continued at 50 °C for 20 min. DNA chain extension was stopped by the addition of 3.5 µl of formamide stop solution. The resulting DNA fragments were resolved on 6% polyacrylamide sequencing gels. 2) Primers were extended using the Sequenase sequencing kit, with [S]dATP to detect the extended primer chains. Reactions were stopped and analyzed as in method 1.

Quantitation

DNA band intensities were quantitated using the NIH Image, version 1.55 software, as described by Wang and Harshey (1994).


RESULTS

Kinetic Characterization of Type 0 Complex

The type 0 complex has been implicated as an early reaction intermediate in the pathway leading up to Mu end cleavage and formation of the type I complex (Mizuuchi et al., 1992) (Fig. 1). In order to verify this postulate, we followed the time course of type 0 and type I complex formation and of the conversion (cleavage) of type 0 to type I. Note that, in these experiments, only the type I complex was assayed directly by measuring the fraction of the supercoiled DNA substrate cleaved by Mu A in presence of Mg. Formation of the type 0 complex and the conversion of the type 0 complex to type I were measured indirectly by generating the DNA-protein complex in the presence of Ca and following DNA cleavage upon replacing Ca with Mg. Details of the assays are described under ``Experimental Procedures.''

Data from a typical kinetic experiment at 25 °C are shown in Fig. 2. Under these assay conditions, where the DNA substrate was the only limiting reaction component, a pseudo-first-order reaction with respect to DNA is expected (Wang and Harshey, 1994). When the kinetic data were fitted with a pseudo-first-order equation (see Fig. 2legend), we obtained an apparent rate constant of 0.38/min and 0.75/min for type 0 formation and for conversion from type 0 to type I, respectively. These two rate constants correspond to a half-time of 1.8 and 0.92 min for the two reactions, respectively.


Figure 2: Kinetics of formation and conversion of type 0 and type I complexes at 25 °C. Mini-Mu donor plasmid pZW140 was assayed for formation of type 0 (circle), conversion from type 0 to type I (bullet) and type I (down triangle) from 0 to 16 min at 25 °C, as described under ``Experimental Procedures.'' The final stages of all reactions were stopped by the addition of 0.2% SDS followed by electrophoresis on agarose gels. DNA band intensities were quantified as described under ``Experimental Procedures,'' and the yield of cleaved product (relaxed DNA species) was plotted as a percentage of the total DNA against time. Superimposed on the kinetic data for type 0 complex formation and its conversion are the best fitting traces (thick and thin lines, respectively) for an exponential rise ([product] = S(0) (1 - exp(-kt)), from which the rate constants were obtained. The dotted line is the computed time course for two irreversible, consecutive reactions (), using k(1) = 0.38/min and k(2) = 0.75/min.



From the two rate constants calculated above, we computed a theoretical trace (Fig. 2, dotted line) for the time course of type I formation using the following standard equation for two irreversible, consecutive reactions of first order,

where S(0) is the starting concentration of substrate. The experimentally obtained kinetic data (open triangles) for the overall type I reaction showed a fairly good fit to the theoretical curve.

It should be pointed out that the DNA substrates employed here, as isolated from E. coli, consist of different topoisomers and are thus kinetically heterogeneous. A rigorous quantitative analysis of the reaction scheme is therefore not strictly meaningful unless each individual topoisomer population is analyzed. Although large differences in the reaction rates among the DNA substrates is not expected here, given the average superhelicity of the plasmids (estimated to be -0.06), we still caution against the literal interpretation of the kinetic constants observed.

Temperature Dependence of Type 0 Complex

We showed earlier that formation of the type I complex has a strong temperature dependence (Wang and Harshey, 1994). We were therefore interested in deciphering whether one of the two steps, formation of type 0, its conversion to type I (cleavage of type 0), or both exhibit temperature dependence. Fig. 3shows the kinetics of one set of experiments at 20 °C, analyzed as described above for the kinetics at 25 °C (Fig. 2). The average half-times for type 0 formation were 6.3 ± 3.1 min (the number of experiments (n) = 8) at 20 °C and 1.7 ± 0.8 min (n = 3) at 25 °C, respectively; and those for conversion from type 0 to the type I complex were 2.9 ± 0.5 min (n = 5) at 20 °C and 1.3 ± 0.3 min (n = 3) at 25 °C. The average values for the overall type I reaction were 32 ± 7 min (n = 6) at 20 °C and 5.0 ± 2.2 min (n = 3) at 25 °C.


Figure 3: Kinetics of formation and conversion of type 0 and type I complexes at 20 °C. Experiments were performed at 20 °C and analyzed as in Fig. 2. The best fitting curves for the data points were obtained as described in Fig. 2. Symbols are as in Fig. 2.



We caution the reader against possible misinterpretation of the type 0 to type I conversion data in Fig. 3. The plateau of the kinetic curve represented by the filled circles denotes 100% conversion of type 0 to type I. The 40% value on the ordinate corresponds to the total yield of the type 0 complex (prior to cleavage) in this experiment. Recall that our assays measure type 0 indirectly following DNA cleavage. The validity of the interpretations based on this indirect estimate of type 0 has been verified by the subsequent development of a direct gel mobility assay for type 0 (Kim et al.(1995); see also Fig. 10).


Figure 10: Zn and Co ions can substitute for Mg in cleavage but not assembly of type 0. In a two-step cleavage reaction, type 0 complexes of pZW140 were first assembled (lane 2) and then incubated with 10 mM MgCl(2) (lane 3), 2 mM MnCl(2) (lane 4), 1 mM ZnCl(2) (lane 5), or 1 mM CoCl(2) (lane 6) at 30 °C for 20 min. The addition of 10 µg/ml heparin to the reaction mixtures before electrophoresis in an agarose gel allowed a small but significant separation of the type 0 complex from unreacted supercoiled donor substrate plasmid (Kim et al.(1995); see ``Experimental Procedures''). Lanes 7-10 are as 3-6 except that the assembly-cleavage reactions were carried out in one step with the indicated metal ions. Lane 1 is a control reaction with no proteins added. The band migrating below type I in lanes 4 and 8 is the product of intramolecular strand transfer. The band above R is a supercoiled dimeric form of the plasmid.



We note that the kinetics of type 0 formation (in the presence of Ca) or type I formation (in presence of Mg) show greater variability from experiment to experiment than the conversion of type 0 (formed in the presence of Ca) to type I in the presence of Mg. The reasons for this are not clear. One potential contributing factor is the nonuniform recovery of the type 0 complex from individual reaction mixtures using the ``spin dialysis'' protocol (see ``Experimental Procedures''). Nonetheless, the data from Fig. 2and Fig. 3reveal important rate-determining aspects of the strand cleavage reaction by the Mu A protein. First, the conversion of type 0 to type I is a fast reaction, showing only a modest rate acceleration with temperature (ratio of t values at 20 and 25 °C is approximately 2). On the other hand, Ca-mediated formation of the type 0 complex or Mg-mediated formation of the type I complex are slower reactions that show markedly higher temperature-dependent rate enhancements (ratio of t values at 20 and 25 °C is approximately 4 for the type 0 and approximately 6 for the type I reaction). Second, the significantly lower t for the type 0 reaction (Ca-mediated) compared with the type I reaction (Mg-mediated) at both 25 and 20 °C suggests that the assembly of the precleavage synaptic complex (type 0) has a lower activation barrier in the presence of Ca than in the presence of Mg. Thus we conclude that the strong temperature dependence of the type I reaction is contributed mainly by the step of assembling a complex equivalent to type 0. It should be noted that, despite the drop in the rate of type 0 formation upon temperature shift from 25 to 20 °C, there was little change in the final yield of type 0 (^3)(compare Fig. 2with Fig. 3). All of the above results are consistent with a model in which the formation of a type 0 equivalent is the rate-limiting step of the overall strand cleavage reaction, at least for the DNA substrate tested here.

Superhelicity Dependence of Type 0 and Type I Formation

The rate of type I formation varies with the superhelical density of the mini-Mu donor plasmid (Wang and Harshey, 1994). To see how the kinetics of type 0 production is influenced by superhelical density, we generated topoisomers of mini-Mu plasmid pZW140 as described previously (Wang and Harshey, 1994). Fig. 4follows the kinetics of type 0 and type I formation with increasing superhelical density. The similarity between the two curves (open circles or open triangles) suggests that the dependence of type 0 formation on superhelicity parallels that of type I formation (notice the nearly identical slopes of the linear portions of the curves). It should be clarified that we do not expect the two curves to be identical. From the results shown in Fig. 2and Fig. 3, we already know that the kinetics of type 0 and type I reactions have inherent differences. Furthermore, it is possible that the degree of superhelicity of the DNA substrate may have an influence on the equilibrium of the reaction (see ``Discussion'').


Figure 4: Superhelical density dependence of type 0 and type I formation. Topoisomers of mini-Mu plasmid pZW140 were assayed for type 0 and type I formation at 30 °C as described in Fig. 2. Type 0 reactions were incubated for 15 min followed by spin dialysis and cleavage with Mg, and type I reactions were incubated for 20 min. The reactions were stopped with 20 mM EDTA before electrophoresis. DNA bands were quantified as described in Fig. 2, and the percentage of type I (cleaved product) formed was plotted against superhelical density, after normalizing to the product yield at = -0.069. Drawn through the data points is a 5th order spline function. Symbols are as in Fig. 2.



Since we measure type 0 after converting it into type I, one might argue that the apparent dependence of type 0 formation on superhelicity is due to the requirement of supercoils during conversion of type 0 to type I complex. However, as shown below, supercoils appear not to be required for this conversion step.

Thus, the demonstration that the kinetic dependence of the formation of type 0 on superhelicity parallels that of type I lends further support to the inference that the type 0 complex is a real intermediate in the type I reaction pathway.

Are Supercoils Needed for Conversion of Type 0 to Type I?

The results thus far strongly support the notion that the requirement for DNA supercoiling is crucial for type 0 complex formation. To test whether supercoils are also needed for the cleavage reaction, we used P1 nuclease to nick mini-Mu pZW140 DNA and thus release the supercoils in the Mu or non-Mu domain of the type 0 complex. (The choice of P1, as opposed to S1, was dictated by the observation that Zn ions required for S1 activity were efficient at cleaving the type 0 complex (see below).) Although the optimal pH for P1 activity toward the denatured DNA is around 5.3, it has significant activity at neutral pH (Fujimoto et al., 1974). We first established that the kinetics of type I formation is not detectably different in the pH range of 6.5-8.0 (data not shown) and then chose pH 6.5 for P1 cleavage. Fig. 5shows the results of experiments with type 0 using amounts of P1 just sufficient to relax supercoiled DNA. Under these conditions most of the type 0 complexes migrated at the relaxed position (lane 2), suggesting that type 0 complexes are not stable upon removal of supercoils. However, this instability appears to occur during electrophoresis within the gel rather than in the reaction mixture prior to fractionation. When P1 cleavage was followed by incubation with Mg, a significant fraction of the type 0 complexes could be converted to stable forms labeled TL (twin loop), L* (linear, single loop) and type I (lane 3). The TL band could be converted to a relaxed form upon SDS treatment and was found to carry specific nicks at both Mu ends as determined by primer extension experiments (not shown). We surmise that in the TL band the Mu ends are held together in a complex in which both the Mu and the non-Mu regions (of equivalent length in pZW140) are relaxed. This assignment is supported by the observation that relaxing the Mu domain of type I complexes with P1 produces a band that co-migrates at the TL position (lane 4). Thus, the TL species most likely resulted from Mu A protein cleavage of type 0 complexes in which the Mu domain was relaxed due to a P1 nick. (On naked supercoiled DNA two preferential P1 cleavage sites were found, one between Mu attL and enhancer region and the other in the vector plasmid sequences (data not shown). Neither of these cleavages was close to the ends of Mu (see Fig. 7)). The L* band could be converted to a linear form upon SDS treatment, and the restriction enzyme pattern of this species was consistent with a double strand cut near the left end of Mu (not shown). This species most likely resulted from Mu A protein cleavage (in the presence of Mg) of type 0 complexes in which P1 had nicked the non-Mu or vector domain close to the left Mu end, on the continuous strand (opposite the strand cleaved by Mu A) as confirmed in primer extension experiments described below.


Figure 5: Removal of supercoils destabilizes the type 0 complex but allows Mu end cleavage. Type 0 complexes formed with pZW140 (lane 1) were treated with P1 nuclease alone (lane 2) or P1 followed by Mg (lane 3). The type 0 complexes migrated at the same position as the supercoiled (S) donor substrate under these experimental conditions. When P1 cleavage was followed by the addition of Mg, forms labeled TL (twin loop) and L* (linear, single loop) and type I were generated. Lane 4 was a control reaction in which type I complexes were treated with P1. See text for details. R and L refer to the relaxed and linear forms of the plasmid.




Figure 7: Superhelicity dependence of generation of the P1-sensitive region outside the left end of Mu in type 0 complexes. Type 0 complexes generated with plasmid pZW140 substrates of varying superhelicity were treated with P1 nuclease. Primer extension using the L2 primer was performed as in Fig. 6. A, C, G, and T, dideoxy sequencing lanes; lane 1 (unlabeled), control plasmid DNA (superhelical density, about -0.06); lanes 2-13, type 0 complexes with DNA of increasing superhelicity (-0.020, -0.024, -0.027, -0.032, -0.036, -0.041, -0.046, -0.051, -0.055, -0.060, -0.065, -0.069), as described in Fig. 4.




Figure 6: P1 cleaves the top strand outside the left end of Mu in type 0 complexes. A, four different primers (LT, L2, R3, RB) were used to probe the top and bottom strands near the left and right ends of Mu in type 0 complexes of pZW140 cleaved with P1. Thick lines, Mu DNA; thin lines, non-Mu DNA; diamonds, Mu A protein cleavage sites. L and R, left and right ends of Mu. B, for each primer, dideoxy sequencing reactions are shown first (A, C, G, and T). Primer extensions were carried out on the following substrates as described under ``Experimental Procedures'' (method 1): untreated type 0 complexes (lane 1); type 0 complexes treated with P1 nuclease (lane 2); type 0 complexes treated first with P1 and next with Mg (lane 3); type I complexes (lane 4); type I complexes treated with P1 (lane 5). Arrowheads indicate Mu (black)/non-Mu (white) DNA junctions.



In summary, although removal of supercoils from the type 0 complex with P1 nuclease destabilizes the complex under gel electrophoresis conditions, cleavage with Mg (prior to gel electrophoresis) retains a significant fraction as stable forms that are consistent with having arisen from type 0 substrates lacking supercoils in either the Mu or the non-Mu domains of the mini-Mu plasmid. This result shows that substrate supercoiling is required only for assembling the cleavage-competent protein-DNA architecture (type 0) and not for the execution of the cleavage reaction per se.

Mapping P1 Cleavages in Type 0 Complex

In order to determine the cleavage sites of P1 in the type 0 complexes, primer extensions were carried out by annealing appropriate primers to the template DNA. Fig. 6shows the reactions. Primers L2 and LT were used to probe the top and bottom strands, respectively, at the left end of Mu, while RB and R3 were used to probe these at the right end (Fig. 6A). Specific P1 cleavage bands (Fig. 6B) were seen only in the type 0 complexes probed with primer L2 (lanes 2 and 3), on the continuous (top) strand outside the left end of Mu. Similar cleavages were not observed with this primer on naked DNA (not shown), on type 0 complexes untreated with P1 (lane 1), or in type I complexes (lanes 4 and 5). These cleavages extend from within Mu (+2 nucleotides) to 8 nucleotides outside Mu. Thus P1 cleaves on the strand opposite to the one destined to be cleaved by Mu A protein, outside the left end. No specific P1 cleavages were seen on strands probed with LT, R3 or RB primers (see lanes 2 in these panels). The only other cleavages observed in the type 0 complexes were those produced by Mu A protein subsequent to P1 treatment (bottom strand at the left end and top strand at the right end; lane 3 in LT and RB). Observe, however, that after P1 cleavage of type 0 complexes, only a fraction of the complexes could be cleaved by Mu A on the bottom strand at the left end (compare Mu A cleavage intensity in lane 3 versus lane 4 in panel LT). In contrast, cleavage at the right end by Mu A after P1 treatment was almost as efficient as seen in type I complexes (RB, compare band intensities of Mu A nick in lane 3 versus lane 4). This suggests that type 0 complexes treated with P1 nuclease were stable enough to complete Mu A cleavage, which occurred predominantly at the right end. We also observed that addition of HU protein to purified type 0 complexes inhibited P1 cleavage at the left end (data not shown), presumably because HU binds preferentially to this region to block P1 access to the site.

Dependence of the P1 Nick on Superhelical Density of Type 0 Complexes

P1 cleavage at the left end of Mu DNA is, presumably, due to the unpairing of the base pairs in this region. If the free energy of DNA supercoiling is utilized to promote this conformational transition, we should expect P1 cleavage at the left end to depend on superhelicity. Indeed, P1 cleavage, as revealed by primer extension experiments, showed the same degree of dependence on supercoils as formation of type 0 complex (Fig. 7; see also Fig. 4). Note that control DNA (unlabeled lane) did not show any specific cleavage in the region immediately outside the left Mu end. The dependence of P1 cleavage on superhelicity of type 0 complexes, which are not as yet cleaved by Mu A protein, is fully consistent with a model in which the formation of the type 0 complex is the rate-determining step in type I production.

Cleavage of Type 0 Complexes with KMnO(4)

If P1 nuclease is indeed sensing the presence of single-stranded DNA outside the Mu ends in a type 0 complex, other single-strand specific probes should also detect such regions. We chose KMnO(4), a smaller probe than P1 nuclease, which is reported to be a pyrimidine-specific reagent that oxidizes unpaired bases in DNA (Hayatsu and Ukita, 1967; Sasse-Dwight and Gralla, 1988). More recently, KMnO(4) has also been found to modify purines in denatured DNA (Akman et al., 1990). Type 0 complexes were treated with KMnO(4), the DNA cleaved at the modified sites, and the cleavage sites mapped by primer extension. In these experiments, KMnO(4) cleavage sites are identified as polymerase stops across all four sequencing lanes (see ``Experimental Procedures''). The results comparing KMnO(4) cleavage of control DNA versus type 0 complexes are shown in Fig. 8. Cleavages were detected outside both ends of Mu, on both the top and bottom strands. However, the most extensive cleavages were detected on the continuous strands (top strand at left end (L2 primer), and bottom strand at right end (R3 primer)). At the right end, these extended from within Mu DNA (+2 nucleotides) to 8 nucleotides outside Mu, analogous to the P1 cleavages on the continuous or top strand outside the left end as seen in Fig. 6. At the left end, the KMnO(4) cleavages overlapped and extended beyond the cleavages produced by P1.


Figure 8: KMnO(4) cleaves the continuous strands outside both ends of Mu in type 0 complexes. Type 0 complexes made with plasmid pZW140 were treated with KMnO(4), followed by DNA strand breakage at the modified sites as described under ``Experimental Procedures.'' Primer extension reactions were carried out as described under ``Experimental Procedures'' (method 2). LT, L2, RB, and R3 refer to primers used in sequencing reactions to probe both strands at the two ends of Mu (see Fig. 6A). For each primer, the first four lanes are reactions with KMnO(4)-treated naked supercoiled DNA, and the next four lanes those with type 0 complexes. All other symbols are as in Fig. 6.



Fig. 9summarizes the cleavage sites of P1 outside the left end ( Fig. 6and Fig. 7), and KMnO(4) outside both ends of Mu (Fig. 8) in type 0 complexes. We interpret the Mu end cleavages seen with these two single-strand specific probes as indicators of the generation of single-stranded DNA at both ends of Mu in the type 0 complex.


Figure 9: Summary of P1 and KMnO(4) cleavages outside Mu ends in type 0 complexes. A, sequence spanning the Mu (highlighted area)/non-Mu junction at the left (L) end of Mu. The diamond indicates the cleavage site of Mu A, arrowheads the cleavage sites of P1 nuclease, and ovals the cleavage sites of KMnO(4) in the type 0 complexes. The heights of these symbols roughly correlate with the relative cleavage intensities. A 5-base pair stretch directly duplicated outside each Mu end is underlined. B, sequence spanning the Mu/non-Mu junction at the right (R) end of Mu. Symbols are as in A.



Type 0 Complexes Are Cleaved with Zn and Co

While experimenting with removal of supercoils in the type 0 complex using S1 nuclease, we discovered that Zn ions can cleave the type 0 complex. We therefore tested other transition metal ions Co, Ni, Cu, and Fe for their ability to support cleavage of type 0 complexes. Of these, only Co ions were seen to be effective. Fig. 10compares the activity of Zn, Co, Mg, and Mn ions using a recently described gel assay, which directly monitors the formation of type 0 complexes (Kim et al.(1995); see ``Experimental Procedures''). In a two-step cleavage reaction, where type 0 complexes were first assembled, purified over a gel filtration column (lane 2), and then incubated with the four indicated metal ions (lanes 3-6), all four were seen to convert type 0 to type I. Co (lane 6) ions were as effective as Mg (lane 3), while Zn ions (lane 5) were less so. Mn ions (as has been seen before), promote intramolecular strand transfer (lane 4, faster migrating band below the type I complex). The inter- or intramolecular strand transfer step of transposition was similarly supported by both Zn and Co (not shown). However, when type I complex formation was monitored directly in a one-step reaction, Zn and Co ions could not support either type 0 or type I formation (lanes 9 and 10), while Mg and Mn ions were efficient at type I (and hence type 0) formation (lanes 7 and 8).


DISCUSSION

In this report, we have presented kinetic studies on the type 0 complex, its formation and conversion to type I complex. The kinetics of type 0 formation in the presence of Ca fits well with the expectation that it precedes type I in the Mu A mediated DNA cleavage reaction (Fig. 2). The rate of production of type 0 and type I show close parallels in their dependence on temperature (Fig. 3) and on the superhelicity of the DNA substrate (Fig. 4). The simplest explanation for these observations is that type 0 complex is a bona fide intermediate in the pathway leading to the type I complex. Our results also suggest that the formation of the pre-cleavage synaptic complex (type 0 or type 0 equivalent) has a lower activation barrier in presence of Ca than in presence of Mg Furthermore, we find that the conversion of type 0 to type I complex is a relatively rapid reaction, and is fairly insensitive to temperature. Overall, these observations suggest that type 0 formation is the rate-limiting step in type I formation, at least for a DNA substrate with superhelicities at or below -0.06.

Given that the formation of the type 0 complex is the rate-limiting step in generating the type I complex, the observed dependence of type 0 production on DNA superhelicity likely reflects a true rate dependence on supercoils. However, we cannot rule out the possibility that supercoiling may determine the equilibrium of the reaction as well. Indeed, if supercoils are used to elevate the energy level of the substrates, instead of lowering the energy level of the transition state, supercoiling will influence both the rate and equilibrium of type 0 complex formation. If formation of type 0 complex is already overwhelmingly favored, as seems to be the case for plasmids with a superhelicity near -0.06, further increase in superhelicity would result in no detectable difference in the yield but could lead to higher reaction rates.

We had proposed that in the Mu transposition reaction, the free energy of supercoils might be used to promote energetically costly events such as the structural transition of DNA (e.g. melting) and/or of proteins (Wang and Harshey, 1994). It may well be that the target for Mu nicking is single- rather than double-stranded DNA. If Mu DNA ends must melt before they can be cleaved, then the supercoiling, in principle, could facilitate this event. We have now shown that DNA at the ends of Mu in the type 0 complex is susceptible to cleavage by single-strand-specific agents (nuclease P1 and KMnO(4); Fig. 6Fig. 7Fig. 8Fig. 9). Furthermore, P1 cleavage near the left end of Mu is dependent upon supercoils, and this dependence is correlated with formation of the type 0 complex (Fig. 4Fig. 5Fig. 6Fig. 7). Cleavage of type 0 by P1 does not abolish cleavage by Mu A protein at the DNA ends (Fig. 5, 6), although we do not know whether the conformational changes in DNA, as detected by P1 nuclease and KMnO(4), are removed along with the presumed removal of supercoils upon P1 cleavage. Since Mu A protein contacts DNA outside Mu in the type 0 complex (Mizuuchi et al., 1992), it is possible that the structural distortions within the type 0 remain after P1 cleavage, even though they may not be exactly the same.

Both single strand DNA probes used in this study were seen to preferentially cleave the continuous strands of DNA (the strands not cleaved by Mu A) in type 0 complexes (see Fig. 9). These cleavages extended from 2 nucleotides within Mu to 8 nucleotides outside Mu. It is very likely that this specificity for the continuous strand is a result of protection of the complementary strand by Mu A protein. We note that although footprinting of type I complexes with DNase I and methidiumpropyl-EDTAbulletFe(II) showed protection of 12-13 base pairs outside Mu ends (Lavoie et al., 1991; Mizuuchi et al., 1991), dimethyl sulfate modification experiments failed to reveal methylation protection of the continuous strands outside Mu (Kuo et al., 1991). It is not clear why P1 did not cleave DNA outside the right end of Mu, a region readily detected by KMnO(4). This preference may be a result of preferential access of P1 to only the left end of Mu. Alternatively, the single-stranded features at the two ends are a result of structural distortions that are not equivalent. Of interest in this regard are results of hydroxyl radical footprinting of type I complexes, which showed enhanced cleavages 2 base pairs outside the Mu ends on the continuous or unnicked strands (Lavoie et al., 1991). However, the hydroxyl radical hyperreactivity was higher at the right end. The asymmetric DNA conformation outside the two ends of Mu as detected by the various DNA structure probes should not be surprising in view of the fact that, except for the two terminal base pairs at the ends of Mu, the sequence and arrangement of the att subsites (Mu A protein binding sites) are quite different at attL and attR (see Fig. 9) (Craigie et al., 1984; Zou et al., 1991). Also, the sequences outside the two Mu ends (in their integrated state on the host genome) are normally unrelated (except for the 5-base pair target duplication bordering each end). The asymmetry of DNA conformation outside the Mu ends may be of significance to the biology of phage Mu replication, which is thought to proceed asymmetrically, from the left to the right end of Mu (see Harshey(1988)). Further experiments are needed to explore the DNA structure in this region. We note that KMnO(4) reacts not only with bases in denatured DNA but has also been reported to selectively react with B-Z or Z-Z junctions in supercoiled plasmid DNA (Jiang et al., 1991). Similarly, P1 nuclease is capable of recognizing a range of non-A/non-B/non-Z conformations in double-stranded DNA (see Pulleybank et al.(1988)).

We conjecture that the single-stranded DNA regions in a type 0 complex may have specific interactions (such as hydrogen bonding and hydrophobic interactions) with Mu A protein to stabilize the melted/distorted structure. This could account for the sequence preference exhibited by Mu A beyond the cleavage sites at the Mu ends (see Wu and Chaconas(1992)). However, considerable flexibility must exist to accommodate a variety of base combinations. We note that a change of even one hydrogen bonding interaction within the type 0 complex could have an apparently dramatic consequence for its stability. The type 0 complex formed with pZW140 has an apparent half-time of decay of 16 min at 30 °C (expected to be around 90 min at room temperature; the decay rate was estimated by timed incubation of type 0 complex in the absence of divalent metal ions followed by quantitating the amount of type I complex generated after the addition of Mg). The average free energy for a hydrogen bond (or more precisely, the average free energy change for a hydrogen bond exchange), is about 1.2 kcal/mol (Fersht, 1987). Thus, change of a single hydrogen bond within the type 0 complex could extend its half-life to 12 h or shorten it to just 11 min at room temperature, on average. (^4)If the rate-limiting step of type 0 formation is the opening of the DNA helix outside the Mu ends, then it is also reasonable to expect that the sequence of this DNA will influence the activation barrier to the transition state, and therefore the rate of type 0 formation (see Cantor and Schimmel(1980) and Murchie et al.(1992)).

We have shown in this study that Zn and Co metal ions (in addition to Mg and Mn) can support cleavage of the type 0 complex (Fig. 10). Divalent metal ions play a central role in phosphoryl transfer in nucleic acids. Structural analysis of E. coli alkaline phosphatase (Kim and Wyckoff, 1991) and of the Klenow polymerase (Beese and Steitz, 1991) complexed with a deoxynucleoside monophosphate and a single-stranded DNA substrate strongly suggest that two metal ions are liganded to specific amino acids. One metal ion is proposed to function as a Lewis acid and to form the attacking hydroxide ion, whereas a second metal ion is proposed to coordinate the bridging 3`-oxygen atom at the cleavage site, stabilizing the transition state and facilitating the leaving of the 3`-OH group. It is likely that metal ions play several roles in the Mu transposition reaction, including one similar to that postulated for the nuclease activity of the Klenow fragment (see Baker and Luo(1994) and Kim et al.(1995)). For example, the inability of Ca ions to support cleavage of the type 0 complex suggests at least two roles for divalent cations, one in assembly of the complex and the other in cleavage (Mizuuchi et al., 1992). The ability of Zn and Co to support only cleavage but not formation of type 0 (Fig. 10), lends further credence to this notion. Metal ions such as cobalt, zinc, and copper can have differential, sequence-specific effects on DNA (see Kang and Wells(1994) and references cited therein). It is likely that during assembly, metal ions may play a role in promoting and/or stabilizing the altered DNA conformation in the type 0 complex. We have recently described the isolation of a mutant of Mu A that shows a differential response to different divalent cations (Kim et al., 1995). Mu A(G348D) was seen to assemble a type 0 complex only in the presence of Ca (and not Mg or Mn) and cleave a preassembled type 0 only in the presence of Mn (and not Mg), but strand transfer precleaved Mu ends in the presence of either Mg or Mn. All of these results suggest not only a multiple role for metal ions in Mu transposition, but also a different role in each of the three observable stages of transposition: assembly, cleavage, and strand transfer. The combination of variant proteins and metal ions should provide tools for the dissection of the role of metal ions in the various steps of the transposition reaction.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM33247. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present Address: Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94704.

**
To whom correspondence should be addressed. Tel.: 512-471-6881; Fax: 512-471-5546; rasika{at}uts.cc.utexas.edu.

(^1)
To avoid possible confusion, we refer to the first step in the kinetic scheme (Fig. 1) as type 0 complex formation, the second step as cleavage or conversion from type 0 to type I, and the overall reaction as type I complex formation.

(^2)
The abbreviations used are: MES, 2-(N-morpholino)ethanesulfonic acid; TL, twin loop; L*, linear, single loop.

(^3)
The kinetics of type 0 formation at 30 °C is too fast to be manually resolved. We estimate at least a 2-fold reduction in the rate constant when the temperature is lowered to 25 °C. No change in the yield can be detected.

(^4)
The change in the decay rate due to changes in interaction free energy within the complex, if contributing entirely to stability, can be calculated by the equation, f = exp(DeltaG/RT), where DeltaG is the change in free energy, R is the gas constant, and T is the absolute temperature. For DeltaG = 1.2 kcal/mol, f approx 8 at room temperature.


ACKNOWLEDGEMENTS

We thank M. Jayaram for comments on the manuscript.


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