Binding of the Tn3 transposase to the inverted repeats of Tn3

J. Md. Biol. (1988) 201, 5899599

Binding of the Tn3 Transposase to the Inverted Repeats of Tn3

James H. New, Angela IL Eggleston and Michael Fennewald

Department of Biological Sciences University of Notre Dame

Notre Dame, IN 46556, U.S.A.

(Received 27 August 1987, and in revised form 27 November 1987)

The transposase protein and the inverted repeat sequences of Tn3 are both essential for Tn3 cointegrate formation and transposition. We have developed two assays to detect site- specific binding of transposase to the inverted repeats: (1) a nitrocellulose filter binding assay in which transposase preferentially retains DNA fragments containing inverted repeat sequences, and (2) a DNase 1 protection assay in which transposase prevents digestion of the inverted repeats by DNase 1. Both assays show that transposase binds directly to linear, duplex DNA containing the inverted repeats. The right inverted repeat of Tn3 binds slightly more strongly than the left one. Site-specific binding requires magnesium but does not require a high energy cofactor.

1. Introduction

Transposons are discrete segments of DNA that can move from one genomic site to another. The bacterial transposon Tn3, which confers penicillin resistance on its host, is the best understood member of a family of transposons that carry antibiotic genes in both gram( +) and gram( -) bacteria (for recent reviews, see Heffron, 1983; Grindley & Reed, 1985). Transposition of Tn3 occurs primarily by a two-step, replicative process which requires the participation of two proteins encoded by Tn3, the transposase and the resolvase. The first step is the formation of a cointegrate in which the donor DNA and the recipient molecule have been joined with a copy of Tn3 at each boundary (Gill et al., 1978). In this step, which absolutely requires the transposase, a 5 bpt (base- pair) duplication of the target sequence, char- acteristic of Tn3, is created. The resolvase then carries out site-specific recombination at the res sites to produce the complete transposition products (Kostriken et al., 1981; Reed, 1981a,b).

Also necessary for efficient transposition are the 38 bp inverted repeats (IR) at the ends of Tn3. Models of transposition call for the inverted repeats to be recognized and bound by transposase (Shapiro, 1979; Galas & Chandler, 1981; Arthur & Sherratt, 1979). The inverted repeats are considered

7 Abbreviations used: bp, base-pair; IR, inverted repeat; DMSO, dimethyl sulphoxide; SDS, sodium dodecyl sulphate; BME, fl-mercaptoethanol.

to be the site of transposase action because they are, exclusive of other Tn3 sequences, sufficient for cointegrate formation if transposase is supplied in trans (Huang et al., 1986). Furthermore, deletions in the inverted repeats block cointegrate formation and cannot be complemented in tram (Heffron et al., 1977; Gill et al., 1979). The inverted repeats of Tn3 show considerable homology to those of other transposons of the Tn3 family. The transposon TnlOOO (gamma-delta) has repeats of 35 bp and shares 27 of these with Tn3. Despite this close similarity, the respective transposases complement each other poorly or not at all (Heffron et al., 1977).

Tn3 displays immunity in that molecules with an inverted repeat of Tn3 are poor recipients of a second Tn3 (Robinson et aZ., 1977; Lee et al., 1983; Huang et al., 1986). This is known to be an effect on the recipient molecule alone because Tn3 transposes normally to other target DNAs in the same cell (Robinson et al., 1977). The inverted repeats and a functional transposase gene are absolutely required for immunity, but other regions of Tn3 may be involved as well. Models for immunity also propose that the inverted repeats are bound by transposase (Lee et al., 1983).

The transposase of bacteriophage Mu is known to bind to the ends of the Mu phage DNA. DNase 1 protection experiments have shown that the MuA protein, required in the initial stages of transposition (Faelen et aZ., 1978; Craigie et al., 1985) and replication of Mu (Waggoner & Pato, 1978), binds to each end of Mu DNA (Craigie et al., 1984).

0022%2836/88/l 10589-11 $03.90/O 589

0 1988 Academic Press Limited

590 J. H. New et al.

It has been reported that the Tn3 transposase binds site-specifically to the ends of Tn3 in the presence of high concentrations of ATP (Wishart et al., 1985). Using transposase purified to homo- geneity, we have demonstrated, by both nitrocellulose filter binding and DNase 1 protection experiments, that the binding of transposase is localized to the inverted repeats, and that this occurs efficiently without ATP.

2. Materials and Methods

(a) Bacterial strains and plasmids

DS410 is an Escherichia oli mini-cell strain which has been described (Adler et al., 1966; Roozen et al, 1971). Plasmids pMC1211 (Casadaban et al., 1982), and RSF1050 and RSF1596 (Heffron et al., 1977) have also been described. pBR38-6 is a derivative of pBR322 which carries a mini-Tn3 (Huang et al., 1986). pDVN-1 was constructed from pBR38-6 by cutting at the unique PvuII site, blunt-end ligation of BamHI linkers, cutting with BarnHI, and ligation of overlapping BamHI ends. The resulting plesmid is deleted for the 1691 bp sequence located between bp 375 and 2066 of pBR322.

(b) Enzymes and nucleotides

Restriction enzymes were purchased from New England BioLabs or United States Biochemicals. The Klenow fragment of DNA polymerase I was obtained from Pharmacia, calf intestinal alkaline phosphatase from Boehringer, phage T4 polynucleotide kinase from USB, and DNase 1 from Worthington. ICN was the source for [a-32P]dCI’P, [a-32P]dATP and [y-32P]ATP. Nucleotides and calf thymus DNA were from Sigma.

(c) Labelled fragments

RSF1050 or RSF1596 was digested with HpaII. The digested DNA was then labelled by a fill-in reaction with [a-3zP]dcTP and the Klenow fragment of DNA polymerase I. The reaction was stopped by addition of EDTA to 20 mM and applied to a 1 ml G-50 spin column to remove unincorporated nucleotides. The DNA was subsequently extracted with phenol, precipitated by addition of 0.5 vol. 7.5 M-ammonium acetate and 2 vol. absolute ethanol, sedimented, and resuspended in 15 mna-Tris . HCl (pH 7.5), 15 mm-Nacl, 2 mmEDTA.

The source of Tn3 inverted repeat DNA used in the DNase 1 protection experiments was the 415 bp AccI- SapI fragment of pDVN-1. The 3’ end-labelled fragment was obtained by digestion with AccI and SspI. The AccI site was labelled with a fill-in reaction with [a-32P]dATP (3000 Ci/mmol) and the DNA pol I Klenow fragment. 5’ End-labelling was performed by cutting with AccI and XmnI, removing the 5’ phosphates with calf intestinal alkaline phosphatase, labelling with [Y-~~P]ATP and T4 polynucleotide kinase, and then cutting with SspI. In both cases, the labelled DNA was isolated on a 5% (w/v) polyacrylamide gel crosslinked with bis-acrylylcystamine (Bio-Rad) (Hansen, 1981), and located by autoradiography. After solubilization of the desired band with #?-mercaptoethanol, the fragment was recovered by purification on a Schleicher & Schuell Elutip. The fragment was precipitated with ethanol and resuspended in 15 mm-Tris. HCI (pH 7.5), 15 mM-NaCl, 2 mM-EDTA.

(d) Transposase purijcation

Transposase was purified by a modification of existing procedures (Wishart et aE., 1985; Fennewald et al., 1981). 18 1 of LB (pH 7), supplemented with kanamycin (10 pg/ml), were inoculated with 5 ml of an overnight culture of DS410 harbouring pMC121 I. and incubated at, 30°C for 19 h with vigorous shaking. The remaining steps were done at 0 to 4°C. Cells were harvested, washed in 10 mM-Tris.HCl (pH 8), 1 mM-EDTA, and resuspended in 100 mmTris. HCI (pH 8), IO mM-BME, 1 mM-EDTA to produce a 20% (w/v) suspension. The suspension was then sonicated, using a Branson sonifier, with 15 30-s bursts separated by 1 min intervals for cooling or passed twice through a French press at 14,000 lbf/in2 (1 lbf/in’ zzz 6.9 kPa). Phenylmethylsulfonyl fluoride (BRL) was added to 1 mM after the first burst or the initial French press treatment.

The lysate was centrifuged at 7000 revs/min (SS34 rotor) for 10 min, and the supernatant was then centrifuged at 38,000 revs/min (Ti45) for 30 min. Polymin P (BRL) (pH 7.5) was added dropwise with stirring to the supernatant to a final concentration of 0.75%. Stirring was continued for 30 min after which the suspension was spun at 5000 revs/min (SS34) for 30 min. The pellet was resuspended in 1 M-ammonium sulphate. 10 mM-BME, and 10% (v/v) glycerol for 30 min and then spun at 10,000 revs/min (SS34) for 30 min. The supernatant was brought to 45% saturation by addition of solid ammonium sulphate and stored on ice.

The suspension was centrifuged at 20,000 revs/min (SS34) for 30 min. The pellet was resuspended in 1 M-ammonium sulphate in HBEG (25 mw-Hepes (pH 7+), 10 mM-BME, 1 mM-EDTA and 10% (v/v) glycerol), dialysed against the same, and the dialysate was then spun at 20,000 revs/min (5534) for 20 min. These steps were followed for successive dialyses against 500 mM, 250 mm, and 50 mw-ammonium sulphate in HBEG. After centrifugation of the final dialysate, the supernatant was loaded onto a phosphocellulose (Whatman, P-11) column (2.5 cm x 12 cm) which was washed with 2 column volumes of 50 mmKP, (pH 7.5) in HBEG (with EDTA at 0.1 mM) and developed with 10 column volumes of a 50 mM to I M-KP, (pH 7.5) gradient in HBEG.

Transposase purification was monitored using 4 to 8% (w/v) SDS/polyacrylamide gels (Laemmli, 1970). Peak fractions, containing transposase which was greater than 90% pure, were either frozen in liquid N, and stored at - 70°C or made 40% in glycerol and stored at -20°C. Site-specific binding activity was stable for several months in both cases.

In some cases, transposase was further purified as described (Fennewald et al., 1981). Briefly, peak transposase fractions were pooled, and the protein w&s precipitated by addition of ammonium sulphate to 75% saturation. The pellet was resuspended in 2.5 ml of buffer A (25 mnil-Tris. HCl (pH 7.5), 250 mM-NaCl, 0.5 mM- dithiothreitol, 0.5 mM-EDTA, 10% glycerol) and applied to a Sephacryl S-200 column (2 cm x 75 cm) equilibrated with the same buffer. Transposase eluted with a K,, of 0.16. It was diluted with an equal volume of buffer B (25 mM-Tris HCI (PH 7.5), 5 mM-Mgcl,, 0.5 rnM- dithiothreitol, 0.5 mm-EDTA) and loaded onto a column of gapped DNA-cellulose (1 cm x 3 cm) (Low et al.. 1976). After washing with 2 column volumes of 80-mM NaCl in buffer B. the column was developed with 8 column volumes of an 80 mM to 1 M-NaCl gradient in this buffer. Transposase containing fractions eluted at 360 m&r-Nacl.

The data in Fig. 2 were obtained using phosphocellu-

Site-speci$c Binding by Tn3 Transposase 591

lose-purified transposase which was then chromatographed on hydroxylapatite (Bio-Rad, HTP) as follows. Peak transposase-containing fractions were pooled, dialysed against buffer C (30 mM-KP, (pH 68), 30 mM-KCl, 10 mM-BME, 0.1 mM-EDTA, 10% glycerol), and loaded onto a hydroxylapatite column (25 cm x 4 cm). After washing with 2 column volumes of buffer C, transposase was eluted with 8 column volumes of a 50 mM to 1 M-KC] in buffer C. Active fractions eluted at 325 mM-KCl.

(e) Nitrocellulose filter binding assay

Binding reactions were composed in binding buffer (10 m&r-sodium succinate (pH 5), 100 mM-KCl, 10 m&i- QC], 1 0.1 mM-EDTA, 0.1 mmdithiothreitol, 50 pg bovine serum albumin/ml, 5oj dimethyl sulphoxide (DMSO)) with 400 ng sonicated calf thymus DNA, in a volume of 250 ~1 and containing 12-5 ng end-labelled RSF1050 or RSFl596 HpaII fragments. The HpaII restriction patterns for these 2 plasmids differ only in that RSF1050 has a fragment which migrates between the IRR and IRL fragments. RSF1596, which is otherwise isogenic with RSF1050, carried a deletion that removes this fragment. DMSO is not required for binding but enhances site-specificity by reducing non-specific binding. Transposase was added to initiate binding, and after incubation for 5 min at, 28”C, heparin (Sigma) was added to 32 fig/ml and incubation was continued for another 5 min. The reaction was diluted with 750 ~1 of binding buffer and slowly filtered over nitrocellulose (Schleicher & Schuell BA85) which had been washed in 400 mM-KOH, rinsed in distilled water, and stored in binding buffer. The filter was washed with 3 ml of binding buffer and the DNA was eluted in 300~1 of 20 mM- Tris.HCl (pH 7.5). 0.5% SDS. The DNA was precipitated with ethanol, resuspended, and electro- phoresed on a 5 y0 (w/v) polyacrylamide gel (100 V. 5 h) using TBE buffer (89 m&r-Tris, 89 mM-boric acid, 2 mM- EDTA). The gel was dried and exposed to Kodak X-omat film for analysis,

(f) DBase 1 protection

DNase 1 protection experiments (Galas & Schmitz, 1978) were performed in binding buffer, without DMSO, in a volume of 100~1: 20ng of end-labelled AccI-SspI fragment and 0.06 to 4 ,ug of transposase were added, and the reaction was incubated at 28°C for 5 min. After addition of 1 pg of sonicated calf thymus DNA, incubation continued for an additional 5 min: 10 ~1 of freshly diluted DNase 1 was added to a final concentration of 1 /*g/ml, and digestion proceeded at room temperature for 35 s. The reaction was stopped by the addition of phenol, and after precipitation with ethanol, the digestion products were resuspended in 95 ‘$& deionized formamide, 10 m&r-EDTA, 0.1% (w/v) xylene cyanol, 0.1% (w/v) bromphenol blue, boiled for 3 min, and loaded onto an 8% (w/v) polyacrylamide, 8 M-urea sequencing gel for analysis. Electrophoresis was performed at 60 W using TBE buffer. Maxam & Gilbert chemical sequencing reactions were performed as described (Maniatis et al., 1982).

(g) Protein concentration determination

Protein concentrations were determined either by laser scanning densitometry, using an LKB Ultroscan 2202, of Coomassie-stained gels with Bio-Rad high molecular

weight standards or with the Bio-Rad protein assay using bovine serum albumin as the standard.

3. Results

(a) Puri$cation of transposase and site-speci$c binding

Using the overproducer plasmid pMC1211 (Casadaban et al., 1982), Tn3 transposase was purified by procedures that are modified from previously published methods (Fennewald et al., 1981; Wishart et al., 1985). The presence of transposase was monitored by polyacrylamide gel electrophoresis during the purification. Cells were lysed by sonication or with a French press, and the lysates centrifuged at high speed. The soluble fraction contained almost all the transposase. Polyethyleneimine was added to the cleared lysate

123456789

TnpA 0 6 3 1.2 O-6 0.24 0.12 O-05 0.02 (Ml

IR R- IR L-

Figure 1. Site-specific binding as a function of transposase concentration. Site-specific binding was measured using a nitrocellulose filter-binding assay. Binding reactions were constructed in 10 mM-sodium succinate (pH 5), 100 mmKC], 0.1 mmdithiothreitol, 0.1 mM-EDTA, 50 fig bovine serum albumin/ml, 5% DMSO, 10 mm-&Cl,. The reactions also contained 12.5 ng of “P end-labelled fragments of HpaII-digested RSF1596, and the indicated levels of transposase were added to initiate binding. After 5 min incubation at 28°C heparin was added to 32 pg/ml ,and incubation continued for 5 min. The reactions were filtered over nitrocellulose, and after elution, the DNA was subjected to electrophoresis on a 5% (w/v) polyacrylamide gel. The gel was dried and autoradiographed using Kodak X-omat film. The fragments which contain the right and left inverted repeats are indicated as IRR and IRL, respectively.


IO

‘f Q 6 s L .- c 3 a 4

L% k-

2

C

600 z

iz 400 L-8

16 I8 20 22 24 26 28

Fraction

Figure 2. Elution profiles of transposase and site-specific binding activity. Transposase was chromatographed over a hydoxylapatite column as described in Materials and Methods. Site-specific binding experiments were done as described in the legend to Fig. 1 with 5 ~1 of a 0.1 dilution of each fraction added to each reaction. The amount of transposase was measured by scanning densitometry of an SDS/polyacrylamide gel and is indicated by --+-+-- [KC11 is indicated by the straight line. Results of the filter binding assays for fractions 16 to 23 are presented in the inset. The DNA substrate for these reactions was RSF1050 which was digested with HpaII and end&belled.

and transposase was eluted from the precipitate with 1 M-ammonium sulphate. The ammonium sulphate concentration was reduced by successive dialyses against 500 mM, 250 mM, and 50 mM- ammonium sulphate buffers, and the supernatant obtained following the last dialysis was applied to a phosphocellulose column. Transposase eluted between 250 and 300 mM-KPi and is greater than 90% pure at this point. Most of the experiments described here used transposase purified up to this step. For some experiments, transposase was further purified by chromatography on Sephacryl 5200 and gapped DNA-cellulose or on hydroxylapatite.

Site-specific binding activity was measured using a nitrocellulose filter binding assay in which transposase-DNA complexes are retained on the filter under conditions that allow non-complexed DNA to pass through. The substrate is labelled, HpaII-cut RSF1050 DNA, which carries a copy of Tn3 and produces approximately 25 fragments when digested with HpaII. Site-specific binding is measured as preferential retention of the two fragments containing the Tn3 inverted repeats on nitrocellulose filters. After filtration of the binding reaction, the DNA is eluted from the filters with SDS, precipitated with ethanol, resuspended, and

then subjected to polyacrylamide gel electrophoresis and autoradiography.

Transposase binds tightly to linear, duplex DNA, and detection of transposase-IR specific interaction requires heparin as a competitor. Transposase binding to the IR sequences is shown in Figure 1. No DNA is retained without the addition of transposase (Fig. 1, lane 1). At higher levels of transposase, the competitive effects of heparin are overcome and non-specific binding occurs, with larger fragments bound first as expected (Fig. 1, lane 2). Preference for the IR sequences is at least 20-fold greater than other DNA sequences. The inverted repeat at the right end of Tn3 (nearest to the ampicillin resistance gene) is bound slightly better than the fragment with the other repeat at the left end of Tn3 (Fig. 1, lane 5). Binding is proportional to the amount of transposase added once a level of 0.6 pg of transposase per reaction is achieved and is approximately linear over the concentrations of protein and DNA used in Figure 1. Little or no binding is detected at lower levels of transposase (Fig. 1, lanes 6 to 9).

This binding is due to the transposase because the site-specific binding activity and transposase copurify. A preparation of transposase that had been purified on phosphocellulose, and which was

Site-speci$c Binding by Tn3 Transposase 593

I2345678

TnpA +-tt+ttt

ATPpH7 3.2 0 0 0 I.6 3.2 4.6 6.4

Buffer pH 7.5 -5-

IR R-

IR L -

Figure 3. Site-specific binding at pH 5 and the effect of ATP. Filter-binding experiments were performed using either Tris. HCl (pH 7.5) (lane l), sodium succinate (pH 5) (lanes 2 and 3) or sodium phthalate (pH 5) (lanes 4 to 8) at 10 mM. Reactions contained 0.6 c(g of trensposase (lanes 1 and 3 to 8) and neutralized ATP at indicated levels (concentrations are in mM). The substrate used in these reactions was the same as that used for the experiments in Fig. 2.

greater than 90% pure was further chromatographed through hydroxylapatite. The DNA binding activity was measured along with the protein. The elution profile of the site-specific binding activity matched the profile of the transposase protein monitored by gel electrophoresis (Fig. 2). The IR site-specific binding activity also copurifies with the transposase protein, as measured with polyacrylamide gels, through Sephacryl 5200 and DNA-cellulose columns (data not shown).

(h) Optimal conditions for binding

The binding reactions used differ significantly from previous investigations (Wishart et al,, 1985) in that we used reactions with a pH of 5. Binding at this pH requires Mg2+ and is optimal at 100 mM-NaGl. ATP is not a necessary cofactor for site-specific binding. Binding reactions were constructed using either sodium succinate (pK, = 5.61), sodium phthalate (pK,=5.51), or sodium cacodylate (pK, =6*27) (data not shown) at a concentration of 10 mM. Figure 3 (lanes 3 and 4) shows that, in the absence of ATP, specific binding occurs when either succinate or phthalate, respectively, is used. The same results were seen with cacodylate. Neutralized ATP, titrated over the

range of 1.6 mM to 8 mM, did not affect binding specificity in either phthalate (Fig. 3, lanes 5 to 8) or succinate (Fig. 4(a), lanes 3 to 5) buffered reactions. We have detected very little site-specific binding at pH 7.5 using Tris buffers (Fig. 4(b), lanes 3 to 5), and addition of ATP does not improve site-specificity (Fig. 4(b), lanes 4 and 5). The only conditions which promote site-specific binding at pH 7.5 are very weak Tris buffers (10 mM) with unneutralized solutions of ATP (Fig. 5, lanes 2 and 4). Under these conditions, the pH of the reactions is below 6. Addition of neutralized ATP solutions at pH 7.5 gives little site-specific binding (Fig. 4(b), lanes 4 and 5; Fig. 5, lanes 1 and 3).

(c) DNase 1 protection of the inverted repeat by transposase

To more precisely position the binding site of transposase at the nucleotide level, we measured the ability of transposase to protect IR sequences from DNase 1 digestion. The 415 bp AccI-SspI fragment of pDVN-1 was end-labelled at the AccI site using either the Klenow fragment of DNA polymerase I or T4 polynucleotide kinase. The 3’ end-labelled fragment was used for analysis of the upper strand as depicted in Figure 10, and 5’ end- labelled fragment served as substrate for lower strand analysis. Standard binding conditions were those determined by the filter-binding studies to optimize site-specificity. Reactions were performed at pH 5, using a succinate buffer, without ATP. As neither DMSO nor heparin stimulated protection (data not shown), both were omitted. First, transposase was incubated with the end-labelled DNA and then the endonuclease DNase I was added and the reaction continued until the DNA molecules were cleaved once on average. The DNA was recovered from the reactions and subjected to polyacrylamide gel electrophoresis under denaturing conditions and then autoradiography. The location of the inverted repeat was determined either by parallel Maxam & Gilbert sequencing reactions or by cleavage with EcoRI and BamHI because the inverted repeat is bounded by an EcoRI site on the inside end and a BamHI site on the outside end.

Transposase binding to the lower strand (lower in Fig. 10) protects a region of 55 bases, including the entire inverted repeat, from three bases adjacent to the inside border to 14 bases away from the outside border (Fig. 6). Titration of the level of transposase does not reveal any clear differences in protection at lower transposase concentrations. This suggests that there is only one binding site or a series of sites of equal strength. Transposase binding to the upper strand produces the protection pattern pictured in Figure 7 along with Maxam & Gilbert sequence reactions used to identify protected nucleotides. As seen in Figure 7, lane 2 and diagrammed in Figure 10, a 29-base region within the IR is strongly protected. This region extends from an enhanced cleavage site five nucleotides away from


STDS I 2 3 4 5 I 2 3 4 5

TnpA + + + - - + + +

-lRL

Figure 4. Effect of pH and ATP on site-specific binding. Binding reactions were performed using either (a) succinate buffer (pH 5) or (b) Tris.HCl buffer (pH 75) and 0.6 yg transposase ((a) and (b), lanes 3 to 5). Neutralized ATP was added to each reaction to the indicated level, and site-specificity was measured using the filter-binding assay. The substrate for the binding reactions is the same as that used for the reactions in Fig. 1 and is in STDS lane.

the inside border of the inverted repeat (near the EcoRI site) to the thymidine 15 bases away from the outside border. This area contains a weak enhanced cleavage site and is flanked by short stretches of weak protection. Weak protection on both strands is also consistently seen about 25 bp away from the inside end of the inverted repeat. This protected region overlaps the protected region on the complementary strand.

indicating that ATP neither stimulates transposase binding nor detectably affects the transposase-IR configuration. This result confirms the filter-binding result that ATP is not required for site-specific binding.

4. Discussion

The binding of transposase to the IR as measured The transposase protein and the inverted repeats by DNase 1 protection occurs at both pH 5 and 7.5 of Tn3 are both necessary for cointegrate formation (Fig. 8). The pattern observed is not changed with by Tn3. Our results show that the transposase respect to sequence or strength of protection except binds directly to inverted repeat sequences. This that at higher levels of transposase complete binding occurs with linear, duplex DNA and does protection of the DNA is seen at pH 7.5 (Fig. 8, not require two inverted repeats to be on the same lane 2). This result is in contrast to the filter DNA molecule. The right inverted repeat binds a binding results where there is a difference in site- little more tightly than the left inverted repeat specificity at varying pHs. The addition of ATP has (Fig. 1, lane 5). Since the sequences are the same little or no effect on the protection pattern by within the inverted repeat itself, the differences in transposase. The binding site (Fig. 9, lane 3) binding are most likely due to differences in the remains unaffected by the presence of 8 mM or bases surrounding the inverted repeat. This idea is 2 mM-ATP (Fig. 9, lanes 1 and 2, respectively), supported by the results from DNase 1 protection

Site-specijk Binding by Tn3 Transposase 595

IR R-

IR R-

Figure 5. Effect of ATP solution pH on site-specific binding at pH 7.5. Binding reactions were performed at pH 7.5 and analysed using the filter-binding assay. Neutralized (pH 7) ATP solutions (lanes 1 and 3) or unneutralized (pH 3) ATP solutions (lanes 2 and 4) were added to give a final concentration of 3.2 mM. RSF1596 which was digested with HpaII and end-labelled was the substrate for the reactions and is in STDS lane.

Figure 6. DNase 1 protection of the inverted repeat (lower strand). The 415 bp AccI-SspI fragment of pDVN- 1 was 5’ end-labelled at the AccI site and subjected to DNase 1 protection analysis as described in Materials and Methods. Lanes 1 and 2 are the C+T and G+ A Maxam $ Gilbert sequencing reactions, respectively. Lane 6 is the protection pattern produced in the absence of transposase, and lanes 3 to 5 are the cleavage patterns obtained in the presence of the indicated levels of transposase. The 5’ end-labelled DNA corresponds to the lower strand in Fig. 10. EcoRI and BarnHI designate the EcoRI and BarnHI cleavage sites, respectively. The inverted repeat sequence is enclosed by the bracket with the terminal base-pairs designated as + 1 and +38. IN and OUT indicate the inside and outside borders, respectively, of the IR relative to the element. A designates the region outside of the IR which is protected on both strands.

I 2 3 4 5 6

CtT G+A

TnpA (pgl 1.8 l-2 0.6 0

596 J. H. New et al

I 2 3 4 5

C C+T G+A

OUT +I

BarnHI -

Figure 7. DNase 1 protection of the complementary strand. Lane 1 shows the DNese 1 cleavage pattern in the region of an unprotected inverted repeat. Lane 2 is the digestion pattern of the same region in the presence of 3.6 pg of transposase. Approximately 20 ng of 32P- labelled DNA are present in the reaction. Lanes 3 to 5 are the C, C+T, and G+A Maxam t Gilbert sequencing reactions, respectively. The DNA is the same fragment used in Fig. 6, labelled at the 3’ end, and corresponds to the upper strand in Fig. 10. The EcoRI and BarnHI designate the EcoRI and BamHI cleavage sites, respectively. The inverted repeat sequence is enclosed by the bracket with the terminal base-pairs designated as + 1 and +38. IN and OUT indicate the inside and

experiments, which show transposase binding extends beyond the 38 bp of the inverted repeat. The finding that the strength of transposase binding to the inverted repeat depends, in part, upon the surrounding DNA sequences may explain the differences detected in Tn3 immunity. Current models for immunity call for the transposase and the inverted repeat to be primarily responsible for immunity (Lee et al., 1983), but there are many differences in the level of immunity conferred by inverted repeats in different locations or with differing surrounding DNA sequences (Heritage & Bennett, 1984). Thus, differences in the ability of the various inverted repeats to bind transposase is a possible explanation for the variability in the resulting levels of immunity.

DNase 1 protection experiments define the binding site more precisely than the filter-binding assay. On one strand (the top strand of Fig. lo), the strongly protected region starts at the inside end of the inverted repeat and continues until 10 bp from the outside end. A strong DNase 1 enhancement site is seen proximal to the inside end of the repeat, and a weaker DNase 1 enhancement site is located 13 bp away from it. The bottom strand shows a much larger region of protection extending from the inside end to 10 bp beyond the outside end of the inverted repeat, The sequence, RCGAAAR, which is conserved between the ends of Mu and Tn3, and which is present in all six MuA binding sites (Craigie et al., 1984), is strongly protected by transposase on both strands.

We also observe a small protected region (labelled A in Fig. 6 and Fig. 7) on both strands approximately 25 bp away from the inside end of the repeat. One possible explanation relating the DNase enhancement sites and the non-continuous binding sites is that transposase binds to both the IR and this distant sequence simultaneously, distorting or bending the helix in the process. Such a distortion could result in the DNase enhancement sites we see.

The conditions for binding by transposase are similar to many other DNA binding proteins with a requirement for a divalent cation such as magnesium and a salt optimum of approximately 100 m&I-Na.Cl. The unusual feature for DNA binding by transposase is the broad pH optimum. Using the DNase 1 protection assay, site-specific binding is seen from pH 5 through pH 8. The protection assay itself does not work well beyond this pH range. Binding is about the same at a pH of 5 as at 7.5, although the DNase 1 enhancement sites are more clearly evident at a pH of 5. The filter binding assay shows DNA binding at both a pH of 5 and 7.5, but good site-specific binding is detected

outside borders, respectively, of the IR relative to the element. A designates the region outside of the IR which is protected on both strands. Sites of DNase 1 enhancement are marked with an arrow whose size is proportional to the degree of enhancement.

Site-specific Binding by Tn3 Transposase 597

I 2 3 4 5 6 7 8

TnpA (ino) 0 4-2 I.8 0.6 4.2 I.8 0.6 0

Figure 8. Effect on pH on pattern of trensposase-IR binding. DNase 1 protection experiments were performed at pH 5 (succinate buffer) (lanes 5 to 8) or pH 7.5 (Tris HCl buffer) (lanes 1 to 4). Transposase was added to the indicated levels and the 3’ end-labelled substrate, corresponding to the upper strand in Fig. 10, was used.

I 2 3 4

ATP (mu) 6 2 0 0

Figure 9. Effect of ATP on pattern of transposaseIR binding. DNase 1 protection experiments, using 3’ end- labelled DNA, were performed with 0.6 fig transposase (lanes 1 to 3) and ATP concentrations as indicated. Lane 4 is the protection pattern observed in the absence of transposase.


INSIDE OUTSIDE

I IR I

+ +

- TTTCGTCTT AGCG~C&GMXCCGt,jGGATCCGGC~GGATCGGCT$CCTCGC- -AAAG-TTCT GGAGCG-

+l * I 1

ECORI RtYmHI

Figure 10. DNA sites protected by transposase. The sequence of the inverted repeat region contained on the AceI- SapI fragment of pDVN-1 is shown along with a summary of data from DNase 1 protection experiments. Filled bars indicate strong protection; striped bars indicate weak protection. Sites of DNase 1 enhancement are marked with an arrow whose size is proportional to the degree of enhancement. + 1 denotes bp 1 of pBR322. INSIDE and OUTSIDE indicate, respectively, the inside and outside borders of the IR relative to the element.

only at pH 5. The reasons why site-specificity is not evident at pH 7.5 with the filter binding assay are not clear. While both assays measure site-specific binding, the filter binding assay measures binding as a rate, especially as an off-rate because competitor DNA and heparin are added before filtration and washing on the filter (Bailey, 1979). The DNase 1 protection assay, however, measures steady-state binding to the DNA (Senear et al., 1986). Site-specific binding by transposase does not require ATP or other high energy cofactor, and this is true for both the filter binding and DNase 1 protection assays.

The Tn3 transposase shares a number of properties with the MuA protein (or Mu transposase). Both bind to the termini of the elements, although Mu does not have a structure like a 38 bp inverted repeat. Mu transposase also has more than one distinct binding site at each end (Craigie et al., 1984), but Tn3 transposase may have only one site in the inverted repeat. In addition, the Mu transposase does not seem to protect DNA adjacent to the Mu ends.

In summary, our results demonstrate that the Tn3 transposase binds to the inverted repeats of Tn3. This is the result predicted from genetic experiments with deletions of the inverted repeat. These results are likely to be true for other members of the Tn3 family of transposons that have homologous inverted repeats. We are now testing whether the inability of transposases in the Tn3 family to complement each other is due to inability of the transposases to bind to slightly different inverted repeats.

We thank F. Heffron and M. Caaadaban for kindly giving us strains and plasmids, and J. Yarger and M. Gorman for assistance with the Maxam & Gilbert sequencing reactions. We also thank D. Nissley for providing pDVN-1. This work was supported by NSF grant PCM83-03222.

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Edited by J. Miller

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Binding of the Tn3 transposase to the inverted repeats of Tn3