Role of DNA topology in Mu transposition: Mechanism of sensing the relative orientation of two DNA segments

Cell, Vol. 45, 793-800,

June 20, 1988, Copyright

0 1986 by Cell Press

Role of DNA Topology in Mu Transposition: Mechanism of Sensing the Relative O rientation of Two DNA Segments Robert Craigie and Kiyoshi Mizuuchi Laboratory of Molecular Biology National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases Bethesda, Maryland 20892

Summary DNA strand transfer at the initiation of Mu transposition normally requires a negatively supercoiled transposon donor molecule, containing both ends of Mu in inverted repeat orientation. We propose that the specific relative orientation of the Mu ends is needed only to energetically favor a particular configuration that the ends must adopt in a synaptic complex. The model was tested by constructing special donor DNA substrates that, because of their catenation or knotting, energetically favor this same configuration of the Mu ends, even though they are on separate molecules or in direct repeat orientation. These structures are efficient substrates for the strand transfer reaction, whereas appropriate control structures are not. The result eliminates tracking or protein scaffold models for orientation preference. Several other systems in which the relative orientation of two DNA segments is sensed may utilize the same mechanism. introduction An early step in transposition of bacteriophage Mu is a DNA strand transfer reaction (Craigie and Mizuuchi, 1985a). The resulting branched DNA structure is an intermediate in both the simple insertion and cointegration transposition pathways (Craigie and Mizuuchi, 1985a). Generation of this intermediate in vitro requires a supercoiled mini-Mu molecule, containing the Mu left and right ends in inverted orientation, as the transposon donor (Craigie et al., 1985). For high efficiency the reaction requires Mu A protein, Mu B protein, HU protein, ATP, and Mg2+. The Mu B protein together with ATP serves to increase the efficiency of the reaction; in the absence of one or both of these factors the reaction product is formed, but at a much reduced efficiency (unpublished data). The structural requirement that the two Mu end DNA segments participating in the Mu strand transfer reaction be on the same molecule and in only one of the two possible relative orientations is common to a group of closely related site-specific recombination systems. These systems include reslresolvase of transposons Tn3 and ~6 (reviewed by Heffron, 1983; Krasnow and Cozzarelli, 1983) phage Mu gixlgin (Plasterk et al., 1983) and phage Pl cixlcin (lida et al., 1984), which control host range, and the Salmonella hixlhin flagellin switching system (Scott and Simon, 1982; Johnson et al., 1984; Johnson and Simon, 1985). When a circular DNA molecule that contains a pair of recombination sites is used as the substrate, site-

specific recombination might be expected to generate, with similar efficiencies, a deletion if the sites are in tandem orientation and an inversion if they are in inverted orientation. However, in each of the systems mentioned above, there is a strong bias in the efficiency of the reaction in favor of one specific orientation of the recombination sites. The orientation specificity of these recombination reactions implies that the DNA linking the two recombination sites is involved in sensing their relative orientation. It has been proposed (Kitts et al., 1983; Krasnow and Cozzarelli, 1983) that this is achieved by one of the required protein factors initially binding to one site and then, while still bound to this site, tracking along the DNA until the second site is encountered. This class of model can also account for the observation that the products of the reslresolvase reaction are singly interlinked catenanes (Krasnow and Cozzarelli, 1983): if active complexes of two res sites are formed purely by random collision, a variable number of interlinks should be trapped at the recombination step, as with phage I. recombination (Mizuuchi et al., 1980; Spengler et al., 1985). However, direct experimental support for a tracking mechanism in these recombination reactions is lacking; a direct test with the reslresolvase system (Benjamin et al., 1985) was inconsistent with a simple tracking model, but a modification of this model was suggested to account for the data. We propose an alternative model, in which orientation specificity is the consequence of a well defined wrapping, in a DNA/protein complex, of two recombination sites within a supercoiled DNA molecule. We report the results of experiments that unambiguously distinguish between this model and a tracking mechanism for the Mu DNA strand transfer reaction. Results The Model Prior to the DNA strand transfer step the two Mu ends must be synapsed in a DNA/protein complex. Figure 1 illustrates the general case in simplified form. Negatively supercoiled DNA exists predominantly as a right-handed superhelix. The arrows represent the two Mu end DNA segments. In Figure 1A they are in inverted repeat orientation and are therefore most easily juxtaposed in a parallel configuration in the right-handed helix (parallel and antiparallel configurations are defined in the legend to Figure 1). If the Mu end DNA segments within an active complex resemble such a right-handed helix with the ends in parallel configuration, the formation of this complex will be energetically favored when the ends are in inverted repeat orientation in the negatively supercoiled molecule. Conversely (Figure lB), Mu ends in direct repeat orientation are most easily juxtaposed in an antiparallel configuration in a negatively supercoiled molecule, and an energy barrier must be overcome to bring them together in parallel configuration. We suggest that this accounts for the orientation specificity of the Mu DNA strand transfer reaction.

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Figure 1. Relationship between Negative Supercoiling cally Favorable Configurations of the Mu Ends

and Energeti-

(A) A negatively supercoiled molecule containing the Mu ends in inverted repeat orientation. Negatively supercoiled DNA exists predominantly as a right-handed interwound helix; therefore parallel configuration of the Mu ends (indicated by arrows) in a right-handed helix is energetically favored. We propose that an active complex between the Mu ends and protein factors (denoted by the shaded area) requires such a configuration of the Mu ends as is favored by negative supercoiling. This diagram shows the simplest possible geometry. (B) A negatively supercoiled molecule with the Mu ends in direct repeat orientation. In this case, antiparallel configuration of the Mu ends in a right-handed helix is energetically favored; an energy barrier must be overcome to form the active complex shown in (A). Similarly, an energy barrier must be overcome to form this complex if the Mu ends are located on different molecules that are not topologically linked. We will use the following conventions to describe the geometry of the synaptic complex. To distinguish parallel from antiparallel configurations of the Mu ends in the interwound helix, the helix can be considered as a twisted ribbon: the Mu ends are parallel if they form a pair of parallel lines on the flat surface generated by untwisting the ribbon; conversely, they are antiparallel if they form a pair of antiparallel lines on this surface. The term “configuration” is used to describe the local three-dimensional geometry of the Mu ends, distinguishing it from the one-dimensional “orientation” of the ends along a continuous length of DNA. The illustration of a synaptic complex as a segment of an interwound right-handed helix, with the Mu ends in parallel configuration (A), is purely heuristic. We claim only that the actual configuration of the Mu ends in an active complex is favored by negative supercoiling; the drawing presented (A) is the simplest possible example. In particular, “parallel orientation” does not imply any particular alignment of the sites of DNA strand transfer, which constitute only a small fraction of the two interwound Mu end DNA segments within the synaptic complex.

We malte no assumptions about the exact path of the two DNA segments within the complex, except that the configuration is energetically favored by negative supercoiling if the ends are in inverted repeat orientation. The righthanded helix with the Mu ends juxtaposed in parallel (Figure 1A) is presented only as the simplest possible example that meets these criteria. Construction of Test DNA Substrates According to the above model, an active substrate need not have the Mu ends in inverted repeat orientation on the same molecule, provided the topology of the substrate makes their configuration in an active complex energeti-

cally favorable. For simplicity we shall continue to assume that the Mu ends must form a right-handed helix with the ends in parallel orientation. We used the phage h in vitro recombination system to make mini-Mu DNA substrates that meet these topological criteria, even though the Mu ends are not in inverted repeat orientation on the same molecule. Appropriate control substrates were also made. The phage h attachment sites aff 6 and att P were inserted into mini-Mu plasmids such that the Mu left end and right end were each flanked by att B and aff P (Figure 2). With the core sequences of att B and aff P in direct repeat orientation, the products of h integrative recombination are two multiply catenated circles, each containing a single Mu end (Figures 2A and 28); catenation results from trapping of supercoils at strand exchange of att B and att P (Mizuuchi et al., 1980). In Figure 2A the Mu ends are in inverted repeat orientation in the mini-Mu prior to h recombination and are thus in parallel configuration in the righthanded helix formed by the catenated circles (Figure 2A, part b). This substrate is therefore expected to work efficiently in the Mu DNA strand transfer reaction. In Figure 28 the Mu ends are in direct repeat orientation prior to h recombination and are thus in antiparallel configuration in the right-handed helix of the catenated product (Figure 28, part b). With att B and aff P in inverted repeat orientation, the product of L recombination is a knotted circle with an inversion (Figures 2C and 2D). In Figure 2C the unrecombined mini-Mu has Mu ends in inverted repeat orientation (part a); therefore the recombined product (part b) has the Mu ends in direct repeat orientation. A simple supercoiled molecule with the Mu ends in this relative orientation is not a substrate for the Mu DNA strand transfer reaction (Craigie et al., 1985). However, knotting, which occurs by trapping of negative supercoils at strand exchange of aff B and atf P favors juxtaposition of the Mu ends in parallel configuration in the right-handed helix (Figure 2C, part b). The product is therefore expected to be a good substrate for the Mu DNA strand transfer reaction, even though the Mu ends are in direct repeat orientation. As a control, the knotted product (Figure 2C, part b) was cloned to generate the same molecule in unknotted form (Figure 2C, part c). In Figure 2D the unrecombined mini-Mu (part a) has Mu ends in direct repeat orientation. The knotted product with an inversion (part b), generated by X recombination, contains the Mu ends in inverted repeat orientation. Although an unknotted molecule with the Mu ends in this relative orientation is a good substrate for the Mu DNA strand transfer reaction, knotting favors juxtaposition of the ends in antiparallel configuration in the right-handed helix; therefore this product is expected to be a poor substrate for the Mu DNA strand transfer reaction. However, some residual reaction is expected because it may adopt the alternative geometry shown in Figure 2D (part c), in which the Mu ends are in parallel configuration in a right-handed helix. Novel DNA Structures Are Efficient Substrates for the Strand Transfer Reaction The Mu DNA strand transfer reaction (Craigie and Mizuuchi, 1985a) is illustrated in Figure 3. In this reaction

DNA Topology 795

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of Mu End Orientation

B’ OB

P’ OB

B’ OB

Figure 2. Construction

B’OB

8’ OP

P’ OP

P’ OB

P’ OB

of Novel Substrates

for the Mu DNA Strand Transfer Reaction

(A) The negatively supercoiled plasmid pMK390 (part a) has the Mu left and right ends in inverted repeat orientation. The plasmid also contains the phage h attachment sites atf B (BOB’) and aff P (POP’) in direct repeat orientation. In vitro recombination between a8 B and a8 P, mediated by proteins Int and IHF, generates a multiply interlinked catenane product (part b). Although the Mu left and right ends are located on different molecules in this product, they are in parallel configuration in the right-handed helix formed by catenation. (B) The negatively supercoiled plasmid pMK395 (part a) differs from plasmid pMK399 in that the Mu ends are in direct repeat orientation; they are therefore in antiparallel configuration in the right-handed helix of the catenated recombination product (part b). (C)The negatively supercoiled plasmid pMK389 (part a) differs from pMK390 in that a8 P is in the opposite orientation. The product of recombination (part b) is knotted, with an inversion about att B and att F! Although the Mu ends are in direct repeat orientation in this’product, knotting energetically favors their juxtaposition in parallel configuration in a right-handed helix. E. coli was transformed with the knotted product, and the unknotted form (part c) was isolated. (0) The negatively supercoiled plasmid pMK393 (part a) differs from plasmid pMK389 in that the Mu ends are in direct repeat orientation. Although the 3 recombination product (part b) contains the Mu ends in inverted repeat orientation, knotting energetically favors their juxtaposition in antiparallel configuration in a right-handed helix. The topological conformation shown in part b is interconvertible with that shown in part c. The topology of the molecules is shown in the drawing at the top of each panel, with a simplified drawing below. Mu ends that form a right-handed helix with the ends in parallel configuration are drawn within a shaded area to indicate the formation of an active DNA/protein complex.

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a

c d

b

e

f

IntermedIate

Figure 3. Structure Product

of the Intermolecular

Mu DNA Strand Transfer

(A) A mini-Mu plasmid is the donor and rpXRF DNA is the target. The Mu part of the mini-Mu plasmid is denoted by thick lines; DNA flanking the mini-Mu part of the mini-Mu plasmid, by wavy lines; qXRF DNA, by thin lines. (6) A staggered cut in the target molecule and nicks, on opposite strands, at each end of the Mu DNA, coupled with a pair of single-strand DNA transfers, generate the transposition intermediate shown in (C). Half-arrows denote the 3’ end of each strand.

the free 3’ends of the transposon on the donor molecule, which are generated by nicks made on opposite strands at each end of the transposon, are transferred to the 5’ ends made by a double-strand cut at the target site (Figures 36 and 3C). When the donor molecule is neither catenated nor knotted, the Mu ends must be on the same molecule and in inverted repeat orientation for a detectable reaction to occur (Craigie et al., 1985). The novel substrates described above, which do not fulfill these two criteria, were tested for their ability to function as the donor DNA in this strand transfer reaction. Figure 4 shows the results with the catenated substrates depicted in Figures 2A and 2B. In lanes a and b the substrate was the unrecombined mini-Mu shown in Figure 2A (part a). As expected, the intermediate (labeled I) was formed efficiently (lane a; lane b is a control in which Mu A protein is omitted from the reaction). When the multiply catenated substrate with the Mu ends in parallel orientation (Figure 2A, part b) was the donor, a new product (labeled Ica) was generated efficiently (lane c; the corresponding Mu A- control is shown in lane d). However, when the multiply catenated substrate with the Mu ends in antiparallel orientation (Figure 28, part b) was the donor, no product was formed (lane e; lane f is the corresponding Mu A- control). The knotted structures shown in Figures 2C and 2D were also tested as substrates for the Mu DNA strand

Figure 4. Efficiency Transfer Reaction

of Catenated

Substrates

in the Mu DNA Strand

The products of the Mu DNA strand transfer reactions, with the donor substrates described below and oXRF DNA as the target, were electrophoresed in 0.7% HGT agarose, in TBE buffer, for 2 hr at 5 V/cm and stained with ethidium. The donor DNA was the structure shown in Figure 2A, part a (lanes a and b); the structure shown in Figure 2A, part b (lanes c and d); the structure shown in Figure 28, part b (lanes e and f). The Mu A protein was omitted from the reactions of lanes b, d, and f. The following bands are labeled: donor DNA, D; (pXRF DNA, rpX. In lanes a and c the Mu DNA strand transfer products are labeled I and Ica. The lowercase letter “s” denotes the supercoiled form of both the catenated donor substrate, generated by the A recombination step, and the unrecombined donor substrate remaining after )i recombination, which comigrate; approximately SO% to 70% of the substrate was in the recombined form. The lowercase letter “0” denotes the unrecombined nicked circle substrate. The nicked forms of the recombined substrate migrate as a series of bands between band Ds and band Do.

transfer reaction. The results are shown in Figure 5. The unrecombined mini-Mu with the ends in inverted repeat orientation (Figure 2C, part a) is, as expected, an efficient substrate (lane a; lane b is the corresponding Mu A- control). With the knotted structure depicted in Figure 2C (part b), which has the Mu ends in direct repeat orientation, a new product (labeled Ikn) is generated (lane c; lane d is the Mu A- control). However, the unknotted form of this molecule (Figure 2C, part c) is not a substrate for the strand transfer reaction (lanes e and f). The knotted molecule with the Mu ends in inverted repeat orientation (Figure 24 part b) is a poor substrate; a new product is barely detectable with ethidium staining (lanes g and h). Structure of Products Ica and Ikn The results presented above show that a multiply interlinked catenane, with the Mu ends in parallel configuration

DNA Topology 797

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ab

of Mu End Orientation

Figure 5. Efficiency of Knotted Substrates the Mu DNA Strand Transfer Reaction

Cd

The products of the Mu DNA strand transfer reaction, with the donor substrates described below and qXRF DNA as the target, were electrophoresed in 0.7% HGT agarose, in TBE buffer, for 2 hr at 5 V/cm and stained with ethidium. The donor DNA was the structure shown in Figure 2C, part a (lanes a, b); that shown in Figure 2C, part b (lanes c, d); that shown in Figure2C, part c (lanese. 1); and that shown in Figure 2D, part b (lanes g. h). The Mu A protein was omitted from the reactions of lanes b, d, f, and h. The Mu DNA strand transfer products rn lanes a and care labeled I and Ikn. Lowercase “s” denotes the supercoiled forms of the knotted donor substrate, generated by the h recombination step, and the unrecombined donor substrate remaining after h recombination, which comigrate; approximately 60%-70% of thesubstrate was in the recombined form. Lowercase “0” denotes the unrecombined nicked circle substrate. The nicked forms of the recombined substrate migrate as a series of bands between bands Ds and Do. Other labeling is as in Figure 4.

Ik

I @X0 Do @Xs Ds

I

Pvul

Figure 6. Predicted

Structures

in

BamHl

D

of the Mu DNA Strand Transfer Products

‘I

Ica and Ikn

(A) The expected product (lea) of the Mu DNA strand transfer reaction with the catenated structure depicted in Figure 2A (part b) as the donor DNA. (B) The product shown in (A) after cleavage by Pvul. (C)The expected product (Ikn) of the Mu DNA strand transfer reaction with the knotted structure depicted in Figure 2C (part b) as the donor DNA. (D) The product shown in (C) after cleavage by BamHI. The topology of the structures is not shown. In (B) and (D) continuous single strands are labeled a through j, with their lengths in parentheses. The locations of BamHl (solid arrows) and Pvul (line arrows) restriction sites are indicated. Thick lines are the donor DNA and thin lines are the rpXRF target DNA.

in a right-handed helix, is an efficient substrate for the Mu DNA strand transfer reaction. A mini-Mu containing the Mu left and right ends in direct repeat orientation is also a good substrate, provided the molecule is knotted so that the parallel configuration of the ends in a right-handed helix is favored. To confirm that Ica and Ikn, the products obtained with these novel substrates, resulted from precisely

the same pair of single-strand DNA transfers depicted in Figure 3, we determined their structures. The expected structures of the products (Ica and Ikn respectively) when the catenated (Figure 2A, part b) and knotted (Figure 2C, part b) structures are used as donor substrates for this reaction are depicted ‘in a topologically simplified form in Figure 6.

2nd r-+

2nd cd

ab

e

h

1st c

1st

IlPvu

I kn/Bam

i

i

I /Barn Ica I Pvu

3!2 Figure 7. Two-Dimensional Gel Electrophoresis DNA Strand Transfer Product Ica

117 Analysis

of the Mu

The products of the Mu DNA strand transfer reaction with the structure shown in Figure 2A (part b) as the donor DNA were digested with Paul and electrophoresed in HGT agarose, in TBE buffer, for 2 hr at 5 Wm. The gel was then equilibrated in alkali and electrophoresed, in alkali, perpendicular to the first dimension for 2.5 hr at 2.5 V/cm. The DNA was then transferred to a hybridization membrane and hybridized with probe specific for the donor DNA. A row of spots in the second dimension (labeled IcalPvu) is as expected for the Pvul-digested structure shown in Figure 6B. The spots are labeled a through e to indicate their correspondence with the fragments predicted in Figure 68. A second row of spots (labeled I) is derived from the Pvul-digested strand transfer product with unrecombined substrate (Figure2A, part a), remaining after the 1 recombination step, as the donor DNA. The lengths of these fragments are indicated on the second dimension in kilobases.

The structure of the product Ica was determined by twodimensional gel electrophoresis. The products shown in lane c of Figure 4 were digested with Pvul and electrophoresed in a native state in the first dimension. The DNA was then denatured with alkali in situ and electrophoresed, in alkali, perpendicular to the first dimension. After transfer to a hybridization membrane the DNA was hybridized with a probe specific for the donor molecule shown in Figure 2A. The autoradiogram is shown in Figure 7. A row of spots (labeled IcalPvu) in the second dimension corresponds precisely with those expected (see Figure 6B) for the Pvul-digested products of the strand transfer reaction with the catenated substrate (Figure 2A, part b) as the donor. A second row of spots (labeled I) corresponds to the single-strand components of the Pvul-digested strand transfer product with residual unrecombined mini-Mu (Figure 2A, part a), present in the substrate, as the donor; between 30% and 40% of the DNA remained unrecombined after the h recombination step. A two-dimensional gel, with an in situ BamHl digestion of the undigested product after the first dimension, confirmed that the product labeled Ica in lane c of Figure 4 is the expected product (data not shown). The structure of product Ikn was determined by a similar two-dimensional gel electrophoresis analysis. The

Figure 6. Two-Dimensional Gel Electrophoresis DNA Strand Transfer Product Ikn

Analysis

of the Mu

The experiment was the same as in Figure 7, except that the donor DNA was the structure shown in Figure 2C (part b) and the products were cleaved with BamHI, instead of Pvul, prior to electrophoresis. A row of spots in the second dimension (labeled IknlBam) is as expected for the BamHI-digested structure shown in Figure 6D. The spots are labeled f through j to indicate their correspondence with the predicted fragments in Figure 6D. Fragment k is not visible in this gel. Other labeling is as in Figure 7.

product was digested with BamHI, instead of Pvul, prior to the first dimension. The row of spots in the second dimension, labeled IknlBamHl in Figure 8, agrees precisely with those expected (Figure 6D) for the BamHIdigested product of the strand transfer reaction with the knotted substrate (Figure 2C, part b) as the donor. The structure of this product was confirmed by a two-dimensional gel analysis with an in situ BamHl digestion after the first dimension (data not shown). Discussion Orientation Specificity Is Determined by the Wrapping of the Mu Ends within a DNA/Protein Complex and by the Topology of the DNA Substrate With a negatively supercoiled mini-Mu molecule that is not catenated or knotted, the Mu ends must be located on the same molecule and be in inverted repeat orientation for an efficient DNA strand transfer reaction (Craigie et al., 1985a). We have demonstrated that this is not a requirement, provided the topology of the DNA energetically favors the same configuration of the pair of ends as is favored in a negatively supercoiled, but otherwise topologitally simple, molecule with the ends in inverted repeat orientation. Tracking mechanisms, which require a continuous DNA path between two sites, are therefore eliminated. Instead, we propose that the topology of the DNA determines which relative orientation of the ends energeti-

DNA Topology 799

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caky favors, or alternatively presents a barrier to, the formation of an active complex between the Mu ends and protein factors. We suggest the term “plectosome” to describe such a DNA/protein complex in which two doublestranded DNA segments are associated with a specific superhelical geometry. Role of Negative Supercoiling in the Mu DNA Strand Transfer Reaction Under the in vitro reaction conditions described previously (Craigie et al., 1985) and used in this study, a miniMu that is topologically simple must be negatively supercoiled for a detectable strand transfer reaction. The above model predicts that if a detectable reaction can occur with linear or nicked circular DNA, there will be no orientation specificity. We have previously noted (unpublished data) that inclusion of dimethyl sulfoxide in the strand transfer reaction enables linear or nicked circular mini-Mu to work, albeit at reduced efficiency; the reaction products have not been analyzed rigorously, but appear to be mostly intramolecular. Orientation specificity is lost with these linear or nicked substrates (data not shown). Under our standard in vitro strand transfer conditions, and probably also in vivo, negative supercoiling may be required only to facilitate the formation of an appropriate DNA/protein complex prior to strand transfer. Applicability of the Model to Other Recombination Systems Several prokaryotic site-specific recombination systems share the apparent requirement that the two DNA segments participating in the reaction be on the same DNA molecule in one specific relative orientation (see Introduction). Their mechanism of sensing the orientation of the sites is likely to be the same as that described here for the Mu DNA strand transfer reaction. Some systems such as Tn3 reslresolvase (reviewed by Heffron, 1983; Krasnow and Cozzarelli, 1983) work efficiently only with the DNA sites in tandem orientation, to produce deletions; others, such as Salmonella hixlhin (Scott and Simon, 1982; Johnson et al., 1984; Johnson and Simon, 1985) require the sites to be in inverted orientation and produce inversions. Excisive h recombination, in the absence of Xis protein, also has a required orientation for the aft sites (Craig and Nash, 1983). Since the different bias of these systems is observed with topologically identical substrates, it is likely to be due to a different (i.e. parallel rather than antiparallel) configuration of the two sites within the plectosomes they form at the initial stage of the recombination reaction. Whether this is the case can be most easily tested by constructing special DNA substrates such as we have described above for the Mu DNA strand transfer reaction. In particular, we expect that for reactions requiring tandemly repeated sites in a negatively supercoiled molecule, such as Tn3 reslresolvase, a multiply interlinked catenane, forming a right-handed helix with one site on each DNA circle, should be a good substrate provided the sites are in antiparallel configuration; reactions like Salmonella hixlhin, which have natural substrates with inverted repeat

sites, should require the sites to be in parallel configuration in the catenane for activity. The products of the Tn3 reshesolvase reaction have been analyzed in detail (Krasnow and Cozzarelli, 1983) and found to be singly interlinked catenanes. Such a topological constraint is expected if the two res sites align within a plectonemically supercoiled molecule, prior to recombination, as shown in Figure 1B. In this conformation, no supercoils are accidentally trapped between the DNA segments that are to form the two circular products; therefore any catenation of the products must result from the fixed geometry of the recombination reaction, and hence the topology of the products should be unique. N. Boocock, J. Brown, and D. Sherratt (personal communication) have independently proposed a similar model for the recognition of the relative orientation of two res sites by Tn3 resolvase, based on the observation that, under modified reaction conditions which allow nonsupercoiled molecules to recombine, linear DNA with two res sites in either relative orientation is a substrate for recombination.

Experimental Procedures Enzymes Restriction enzymes were purchased from New England Biolabs. Mu A protein (Craigie and Mizuuchi, 1985b) and E. coli HU protein (Craigie et al., 1985) were purified as previously described. Mu B protein was purified as described by Chaconas et al. (1985). Purified In! and IHF proteins were provided by Howard Nash (NIMH, Bethesda, MD). DNAs The vector used for constructing the mini-Mu plasmids was pMM306 (Mizuuchi and Mizuuchi, 1985). A Hindlll fragment containing the Mu left end was inserted at the BamHl site of pMM306 with BamHl linker, and a Haelll fragment containing the Mu right end was inserted at the EcoRl site with EcoRl linker; in the resulting plasmid (pMK108), the Mu ends are in inverted repeat orientation, with the 1 atf B site from pMM306 outside the Mu part of the molecule. A unique Bglll site was created in pMK108 by inserting a Bglll linker at the Ahalll site at position 3251 of the pBR322 sequence in pMK108. The Bamlil fragment of pPA259 (Mizuuchi and Mizuuchi, 1980) containing atiP was inserted at this Bglll site with aft P in a direct orientation (pMK390) and in an inverted orientation (pMK389) with respect to att B. The EcoRl fragments of pMK390, and pMK389 containing the Mu right end were inverted to generate pMK395 and pMK393 respectively. In Vitro A Recombination Reactions Reaction conditions were as described by Kitts et al. (1984). except that 40 )II reactions contained 7.5 &ml In! protein, 6 Kg/ml IHF, and 5 pglml DNA and the incubation time was 1 hr. Mu DNA Strand man&r Reactions Reaction conditions were as previously described (Craigie et al., 1985). All DNA substrates were treated with Pronase and extracted with phenol, as were the products prior to gel electrophoresis. Two-Dimensional Gel Electmphomsis Methods were as previously described

and Southern Transfers (Craigie and Mizuuchi, 1965a).

Acknowledgments We thank Martin Gellert and Howard Nash for helpful discussions on this work and critical reading of the manuscript. Purified Int and IHF proteins were generous gifts from Howard Nash. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

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marked hdvwtisement” in accordance solely to indicate this fact. Received

with 18 U.S.C. Section

1734

March 17, 1988.

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