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J Biol Chem, Vol. 273, Issue 13, 7367-7374, March 27, 1998
From the Department of Biochemistry, University of Wisconsin,
Madison, Wisconsin 53706
This communication reports the development of an
efficient in vitro transposition system for
Tn5. A key component of this system was the use of
hyperactive mutant transposase. The inactivity of wild type transposase
is likely to be related to the low frequency of in vivo
transposition. The in vitro experiments demonstrate the
following: the only required macromolecules for most of the steps in
Tn5 transposition are the transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be
able to self-dissociate from product DNAs; Tn5 transposes
by a conservative "cut and paste" mechanism; and Tn5
release from the donor backbone involves precise cleavage of both 3'
and 5' strands at the ends of the specific end sequences.
Transposition is a process that gives rise to a variety of genomic
rearrangements such as insertions, deletions, inversions, and
chromosome fusions (1). Because transposition can play a profound role
in genome evolution and in a variety of genetic diseases, how this
process occurs and how its frequency is determined is of considerable
interest. Bacterial transposons, such as Tn5, are convenient
tools for studying these two questions.
The transposition process can be understood in the context of the
following model (2, 3). The first step in transposition involves the
sequence-specific binding of the transposon-encoded transposase
(Tnp)1 to the specific end
sequences that define the ends of the transposable element. The second
step involves formation of a synaptic complex in which the ends of the
transposable element are brought together through Tnp oligomerization.
In some well studied cases such as Tn10 (4, 5), synaptic
complex formation is facilitated by one or more host proteins. The
third step involves a Tnp-mediated nucleolytic attack on the
phosphodiester bonds adjacent to the ends of the transposable element.
The precise nature of these cleavages (whether just the 3' ends of the
transposon are liberated or both the 3' and the 5' ends and the exact
location of the cleavage in the case of 5' end release) is a special
property of the system being studied. The fourth step involves Tnp
binding to the target DNA sequence (target capture). Different
transposons demonstrate various degrees of sequence specificity at this
step. The fifth step involves a concerted nucleophilic attack by the
transposon 3'-OH ends on phosphodiester bonds in both strands of the
target sequence leading to strand exchanges in which the transposon
3'-OH ends are covalently linked to the target 5'-PO4
groups. The sixth step involves a release of Tnp from the complex (6).
Finally, the transposition process is completed by a patch repair or a replicative resolution process.
It is clear that transposition is a complex multistep process and that
Tnp participates in an intimate fashion in almost all of the steps.
This means that Tnp is a multifunctional protein and that the
activities of Tnp are likely to be manifested in a sequential fashion
controlled by a variety of allosteric phenomena. Understanding the
molecular details of the various steps is an important goal of current
research efforts. To that end, a variety of systems have been studied
in vitro. Some bacterial transposable elements (such as Mu,
Tn7, and Tn10) have been particularly useful in
this effort although, with the cases of Mu and Tn7 in
particular, the systems are quite complex in regard to the protein and
DNA participants (2, 7, 8). A simpler system involving essentially only
one protein (Tnp) for most of the steps and simple DNA sequences might
aid in the effort to relate structure to function. As we shall
demonstrate in this communication, Tn5 is such a simple system.
Another critical feature of transposition is the frequency with which
it occurs. For some transposable elements, a high frequency is
allowable or desirable. Examples include the site-specific transposition process for Tn7 (9) and the lytic/replicative transposition process for Mu (2). However, many transposons have
evolved mechanisms that facilitate the establishment of long term
stable associations with the host genome. In these cases a very low
frequency of transposition would be necessary. How can a low
transposition frequency be ensured? In the case of Tn5, this
low frequency appears to be the result of multiple features, two of
which we have exploited in the development of an in vitro system. First, the low frequency of Tn5 transposition
results from Tnp having suboptimal properties; it is a protein with a low level of activity. At least three classes of Tnp mutants have been
isolated that enhance Tnp activity relative to different steps in the
transposition process (10-12). Second, the activity of Tnp is further
down-regulated by a trans-dominant negative variant of Tnp, the
inhibitor (Inh) (13, 14). Inh synthesis can be blocked without
impairing Tnp function by destroying the Inh initiator codon (10).
Tn5 is a composite transposon in which three genes encoding
antibiotic resistance are flanked by two IS50 elements (see
Fig. 1A for a schematic
presentation). Tn5 has been described in detail in two
reviews (3, 15). For the purpose of understanding the Tn5
transposition process, it is important to know the following. One of
the IS50 elements (IS50R) encodes the cis-active
476-amino acid Tnp and the trans-dominant negative Inh. The inactive
C-terminal truncated versions of Tnp and Inh encoded by
IS50L and the antibiotic resistance functions in the central
region play no role in the transposition process. The second critical
component in transposition are the inverted 19-bp sequences that define
the ends of Tn5, the so-called outside end (OE) sequences
(Fig. 1C). There is a second type of 19-bp sequence that can
also be recognized by Tnp, the inside end (IE) sequence. IE is involved
in IS50 transposition (an OE-IE event) but not
Tn5 transposition (an OE-OE event). Thus Tn5
transposition involves Tnp and the two appropriately spaced and
oriented 19-bp OE sequences.
Tn5 in Vitro Transposition*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (27K):
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Fig. 1.
Tn5. A, the features
critical for Tn5 transposition include the transposase
(Tnp), the inhibitor of transposition (Inh), and
the 19-bp end sequences (the outside end, OE, and the inside end, IE). The P3 and P4 proteins are nonfunctional versions
of Tnp and Inh. A more detailed description of Tn5 can be
found in Refs. 3 and 15. B, hyperactive Tnp mutations used
in this study. C, 19 bp outside end sequence.
In this communication we report the development and characterization of an in vitro transposition system for Tn5 using purified Tnp. We used hyperactive Tnp that contained three mutations (Fig. 1B) to accomplish this goal. EK54 enhances OE binding activity of Tnp (12). MA56 blocks the synthesis of Inh (10), thus removing its possible inhibitory activity from the reaction. LP372 enhances Tnp activity possibly by altering the dimerization potential for Tnp (11). The results of these studies show that most of the steps in the Tn5 transposition process involve three macromolecular components as follows: the 476-amino acid long Tn5 Tnp, the 19-bp OE sequences that define the ends of Tn5, and a target DNA sequence. In addition we demonstrate, among other features, that Tn5 transposition occurs by a conservative cut and paste mechanism in which the Tn5-donor backbone cleavage reaction involves a precise cleavage at the end of the OE.
It is quite likely that many other wild type transposase enzymes manifest a similar low level of activity. Thus the strategy that we have employed in developing the Tn5 in vitro transposition system should have general applicability. Finally, the high efficiency and simplicity of the Tn5 in vitro transposition plus the existence of many Tn5-related constructs (16) should make this system useful for many investigators.
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EXPERIMENTAL PROCEDURES |
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Abstract
Introduction
Procedures
Results
Discussion
References
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Bacterial Strains, Plasmids, and Reagents--
Escherichia
coli strains for overexpression of hyperactive transposase were
BL21(DE3)pLysS (17) and BL21 OmpT (18). T7 RNA polymerase was encoded
by 
DE3 phage (17) or by a plasmid, pT7pol26 (which provides tighter
control of polymerase expression) (19). For all DNA manipulations and
electroporation of reaction products, E. coli DH5
(20)
was used.
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used for the ligation-mediated PCR experiment is
similar to pFMA187 (21) but with the IE of Tn5 substituted by OE (12) and the entire transposase gene deleted by cutting with
BspHI, followed by ligation.
Bacteria were cultured in Luria broth (LB). Antibiotics were purchased
from Sigma, and the final concentrations were 100 µg/ml ampicillin,
15 µg/ml tetracycline, 20 µg/ml chloramphenicol, and 40 µg/ml
kanamycin. Isopropyl-1-thio-
-D-galactopyranoside was purchased from Diagnostic Chemical. Restriction enzymes were purchased from New England Biolabs and Promega and were used following the manufacturers' recommendations. Radionucleotides were purchased from
Amersham Pharmacia Biotech. Oligonucleotides were purchased from
Research Genetics.
DNA Preparation-- pRZTL1 plasmid DNA for the experiment described in Fig. 3 was prepared as follows. After standard alkaline lysis purification (20), the DNA was run on a preparative 1% agarose gel, and supercoiled monomer DNA was cut out of the gel and was prepared with a Geneclean II kit (BIO 101). This protocol avoided the presence of multimeric forms of the plasmid and chromosomal DNA contaminants that would complicate the interpretation of the agarose gel patterns. Control DNA plasmids were prepared in the same fashion but were subjected to sufficient vortexing during the Geneclean II procedure to produce linear and relaxed forms of the plasmids. For the rest of the experiments, pRZTL1 was prepared with the Qiagen Plasmid Maxi Kit, followed by an additional alkaline treatment, neutralization, and ethanol precipitation to enrich for supercoiled DNA. Minor amounts of multimers were present after this procedure.
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Protein Purification--
Hyperactive mutant Tn5
transposase was purified in general as described previously (22) with a
few modifications. Cells for induction were grown starting from a
single colony after transformation of E. coli BL21(DE3)pLysS
with plasmid pRZPET2. The colony was chosen for the inoculation that
gave the highest extent of killing on agar containing 0.1 mM isopropyl-1-thio--D-galactopyranoside. This test is based upon the lethal effect of transposase overproduction (23). One liter of cells was induced at A600 = 0.6 by the addition of 0.5 mM
isopropyl-1-thio--D-galactopyranoside followed by
incubation for 1.5 h at 37 °C. Cells were collected by
centrifugation and washed once with TEGX (20 mM Tris-HCl, 1 mM EDTA, 10% glycerol, 0, 1% Triton X-100, pH 7.5) buffer
modified to contain 0.1 M NaCl and 0.1 mM
phenylmethylsulfonyl fluoride and resuspended in 20 ml of the same
buffer. The cells were lysed by two passages through a French pressure
cell at 16,000 lb/in2. The lysate was centrifuged for 20 min at 20,000 × g and then the supernatant was brought
to 0.25% polyethyleneimine (Sigma), and the mixture was immediately
centrifuged for 10 min at 10,000 × g. The supernatant
was brought to 47% saturation with ammonium sulfate, and the pellet
was collected by centrifugation at 10,000 × g for 15 min. The pellet was resuspended in 5 ml of 0.3 M NaCl/TEGX buffer containing 10% glycerol, dialyzed against this buffer, and
applied to a 10-ml heparin-agarose (Sigma) column equilibrated in the
same buffer. After washing with 3 column volumes of buffer, Tnp was
eluted with a 100-ml linear (0.3-1.2 M) NaCl gradient. Tnp
was eluted at approximately 0.6 M NaCl. Fractions with
higher activity (earliest in the peak) were pooled and stored at
70 °C in the same buffer containing 10 mM CHAPS;
Pierce). Tnp of this quality (86% pure, see Fig.
4, lane 2) was used in most
experiments described in this paper. More highly purified Tnp was
obtained, in addition, by applying a Reactive Yellow No. 3 (Sigma)
column chromatography step2
followed by chromatography on a second heparin-agarose column. Protein
of this quality (96% pure, Fig. 4, lane 3; purified by D. York) gave essentially the same reaction efficiency. Estimation of the
protein purity was accomplished by analyzing the Coomassie Blue-stained
gel shown in Fig. 4 with a Personal Densitometer (Molecular Dynamics)
set at the highest sensitivity.
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In Vitro Reaction Conditions--
For a typical reaction, 2 µl
(approximately 0.2 µg of protein/µl) of Tnp was added to 18 µl of
pRZTL1 plasmid (approximately 1 µg of DNA) in the transposition
reaction buffer (0.1 M potassium glutamate, 25 mM Tris acetate, pH 7.5, 10 mM Mg2+
acetate, 50 µg/ml bovine serum albumin, 0.5 mM
-mercaptoethanol, 2 mM spermidine, 100 µg/ml tRNA;
final concentrations). The reaction was incubated for 1 h at
20 °C and then diluted 2-3-fold in the same buffer and transferred
to 37 °C. This procedure was performed to facilitate binding in the
presence of CHAPS present in the reaction mixture as a component of the
Tnp storage buffer (TEGX, 10 mM CHAPS). Dilution increased
the cleavage reaction presumably due to dilution of CHAPS. CHAPS can be
eliminated from the reaction (and storage buffer), and a simple
incubation at 37 °C with no dilution is satisfactory if
Qiagen-purified DNA is used. We nevertheless used the above two-step
procedure for all experiments to synchronize the start of cleavage.
Analysis of Reaction Products-- Reaction products were analyzed by agarose gel electrophoresis followed by staining with SYBR Green II (Molecular Probes) following the manufacturer's recommendations, and pictures were taken with a Fluorimager S1 (Molecular Dynamics) and analyzed with software provided by the same company.
DNA Sequencing-- Sequencing of transposition products and PCR fragments was accomplished with a modified dideoxy chain termination procedure with use of 10% dimethyl sulfoxide (Sigma), boiling, and snap-cooling (24), Sequenase 2.0 (U. S. Biochemical Corp.), terminating mixtures (U. S. Biochemical Corp.), and as a sequencing buffer, KGB (20).
Ligation-mediated PCR--
Ligation-mediated PCR was used to
define the 5' end exposed after transposon excision during the in
vitro reaction. Ligation-mediated PCR was performed essentially as
described (25, 26). After a standard reaction with plasmid
pFMA187OO, 2 µl of the reaction mixture was added to 20 µl of a
primer extension mixture (1× KGB, 1 mM primer S30:
5'ACCTCGGTTCAAAGAGTTGG 3'). After heating in a boiling bath for 3 min
and annealing at 37 °C for 15 min, 1 mM dNTPs and 2 units of Sequenase 2.0 (U. S. Biochemical Corp.) were added, and
the reaction was incubated for 20 min at 37 °C. Then Sequenase was
heat-inactivated, and after a 2-fold dilution in water, ATP (10 mM final), 1 unit of ligase (Promega), and the anchor
linker (5 µM final) were added. The anchor linker was
assembled from the following oligonucleotides: S28, 5'
GGCTCGGGACCGTGGCTAGCATTAGT 3'; and S29, 5' ACTAATGCTTAG 3' (26). After
overnight ligation at 16 °C, chloroform extraction, ethanol
precipitation, and resuspension in 20 µl of H2O, 5 µl
was added to 100 µl of a standard PCR reaction with 0.2 µM primers S28 and S31 (S31, 5' GGTAGCTCAGAGAACCTTCG 3').
After 30 cycles (93 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min) products of the reaction were run on a 1.5% agarose gel,
and after staining with ethidium bromide, the DNA band corresponding to
expected fragment (250 bp) was recovered from the agarose using the
Geneclean procedure and directly sequenced as described above. The
primer for sequencing was S32, 5' CGAAAAACCGCCCTTGCAAGG 3'.
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RESULTS |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Experimental System-- The four critical components of an in vitro transposition system are the DNA substrate, the DNA target, the protein component(s), and the reaction solution conditions. These are discussed below in the context of our studies on Tn5.
Extensive in vivo experimentation has shown that two properly oriented 19-bp OE sequences (or for IS50 transposition, one OE sequence, and one 19-bp IE sequence) are the only substrate DNA sequences required for Tn5-specific transposition (27-30). Thus we constructed as a substrate DNA molecule plasmid pRZTL1 in which the only Tn5-related sequences are two inverted 19-bp OEs (Fig. 2). These two inverted 19-bp OE sequences flank an origin of replication, a chloramphenicol resistance gene, and a "silent" tetracycline resistance gene that can only be expressed following a DNA rearrangement placing this gene downstream of an active promoter. In the experiments to be reported in this communication, pRZTL1 was also used as a target molecule. Transposition was assayed biologically by electroporating the plasmid DNA preparation following the indicated treatment into E. coli DH5 and quantifying
the number of tetracycline-resistant colonies. The structures of the resulting plasmid DNAs in representative tetracycline-resistant cells
were analyzed. In addition, the reaction products were directly analyzed following phenol extraction by agarose gel electrophoresis (see Fig. 3, A and B).
The only protein component added to the Tn5 transposition
reactions was purified, hyperactive mutant Tnp. Three purification procedures for preparing Tnp were utilized with equivalent results. In
most experiments we utilized Tnp purified by a procedure similar to
that described previously (22). These preparations were approximately 86% pure as judged by Coomassie stain analysis of SDS-polyacrylamide electrophoresis gels (see Fig. 4, lane 2). Two additional
purification steps were imposed on some preparations (reactive dye
Yellow No. 3 chromatography and a second heparin agarose
chromatography) to yield a preparation that was over 95% pure (see
Fig. 4, lane 3). Finally, one preparation of a GST-Tnp
fusion protein that had been purified by affinity chromatography was
used. Since all preparations gave essentially identical results (data
not shown), these experiments suggest that Tnp is the only protein
required for Tn5 transposition; however, we cannot rule out
that a low level host protein contaminant is also required for the
reaction. Moreover, the electrophoretic mobility of many of the
reaction products is dependent on a prior phenol extraction (see below) implying that Tnp release from these products has not been effected in
our defined system.
The Tnp concentration dependence of the transposition reaction has been
examined in the experiment shown in Fig.
5. Various amounts of transposase, from 0 to 3.8 pmol, were added to a standard 20-µl reaction containing 0.26 pmol of pRZTL1 and incubated for 4 h at 37 °C. The products
were then analyzed by agarose gel electrophoresis. The yield of donor
backbone (DBB) product, which is easiest to quantify, is shown in Fig.
5B. It is obvious that the reaction is dependent upon the
amount of Tnp added and that this dependence is slightly sigmoidal.
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Analysis of Reaction Products-- Efficient in vitro transposition was shown to have occurred both by transformation and by direct electrophoretic analysis of phenol-extracted products. As noted above, tetracycline resistance can only arise in this system if the transposable element is transposed to a site that is downstream of an active promoter. In a typical experiment utilizing purified hyperactive Tnp (containing mutations EK54, MA56, and LP372), the frequency of transformed cells that were tetracycline-resistant (the indicator product of transposition events) was 0.5% of the chloramphenicol-resistant cells (having received pRZTL1 DNA). However, this number underestimates the total transposition frequency because the detection system limits the target to 1/18 of the plasmid DNA (i.e. the chloramphenicol resistance gene was the only available target for intramolecular events which represented at least 95% of the products in our standard system (see below)), and this gene is 660 bp long relative to the plasmid length of 5838 bp. In addition only inserts of one orientation will yield transcription of the tetracycline resistance gene.
Plasmid DNA was analyzed from over 300 independent tetracycline-resistant transformants.4 Two types of final products representing intramolecular events and intermolecular events were recovered. An analysis of representative DNAs is shown in lanes 2 and 3 of Fig. 3A. As expected, the relative abundance of intermolecular versus intramolecular events is related to the DNA concentration in the transposition reaction. For instance, a reaction performed at 0.05 µg of DNA/ml yielded 5% intermolecular and 95% intramolecular products, whereas a reaction performed at 0.005 µg of DNA/µl yielded only intramolecular products. The transposon-target junctions on these plasmids have been sequenced and proven to be the products of true Tn5 transposition events with 9-bp repeats flanking the OEs.4 When the same reaction was performed with purified MA56 Tnp, no tetracycline-resistant transformants were found (in contrast to over a thousand transformants generated in a standard reaction using EK54, MA56, LP372 Tnp) suggesting that the EK54 + LP372 changes have increased the in vitro activity of Tnp by at least 103-fold. Phenol-extracted products of two independent reactions utilizing supercoiled substrate DNA and hyperactive Tnp were directly analyzed by agarose gel electrophoresis (Fig. 3A, lanes 4 and 5). The DNA was recovered from different positions in the gel and studied by restriction digestion analysis and by transformation into competent cells, followed by restriction digestion and sequence analysis of the resulting plasmids. These analyses led to the following interpretations (see Fig. 3B). The DNA in band 1 has an apparent molecular weight (as judged by its electrophoretic mobility) and a restriction digest pattern consistent with these molecules being released donor backbone DNA. This conclusion is verified by the results shown in Fig. 3A, lane 6, in which the reaction substrate was pRZTL1 linearized at the XhoI site within the donor DNA backbone. In this case the resulting product is the expected donor backbone subfragment. Band 2 is excised Tn5 DNA. This was determined from its apparent molecular weight (notice that its mobility is identical to that of the linearized intramolecular transposition product in lane 2 that should be the same length as the excised Tn5 DNA) and its restriction digestion pattern (data not shown) (see also the experiment described in Fig. 7B). The bands in region 3 contain knotted and relaxed forms of single intramolecular inversion transposition events. These structures were determined by analyzing the effect(s) of digesting these species with NcoI that cuts once in the transposon sequence. This digestion gave rise to a single molecular weight species whose electrophoretic mobility was that of a linear transposon sequence. Intramolecular transposition events of supercoiled DNA is expected to yield knotted transposon length circular products if Tnp is keeping the transposon topologically closed after cleavage but prior to strand transfer. Region 3 may also contain catenated deletion circles (alternative products of intramolecular transposition events), but these would not have yielded uniformly sized products upon NcoI digestion. Band 4 contains the products of single cleavage events (next to one or the other of the OE sequences). This was determined from its apparent molecular weight and its restriction digestion pattern (data not shown). The bands in region 5 contain products of double transposition events (one intramolecular and one intermolecular). These products were identified by analyzing the plasmids found following transformation. The presence of multiple bands is presumably due to the presence of various numbers of knots in the products. The DNA in band 6 contains intermolecular transposition products (single events) as determined by analyzing product DNAs following transformation. The efficient production of the 1256-bp released donor backbone DNA fragment, the appearance of the excised Tn5 DNA fragment, the production of transposition products in the apparent absence of replication machinery, and the absence of DNA products that migrate more slowly than intermolecular transposition products lead us to the conclusion that Tn5 transposes via a "cut and paste" conservative mechanism. This conclusion is supported by the kinetic analysis of the transposition reaction.Transposition Products (but Not Released Donor Backbone DNA) Have Associated Proteins-- One of the final steps in the transposition process is release of the transposase from the rearranged DNA molecules. We have examined whether this process occurs in our system by studying the agarose gel mobility of reaction products either directly from the reaction mixture, after chloroform extraction, after SDS treatment, or after phenol extraction. An equivalent amount of released donor backbone DNA is apparent in all four preparations (see Fig. 3C), but many other product DNAs are not apparent in the untreated preparation as compared with the phenol-extracted or SDS-treated preparations. CHCl3 treatment appears to give a mixture of product mobilities. Since Tnp and bovine serum albumin are the only proteins present in stoichiometric quantities in these reactions, these results suggest that Tnp is tightly associated with Tn5 containing products after the transposition reaction.
Transposition Reaction Time Course-- Considerable information regarding the transposition reaction mechanism can be deduced by studying the time course for the generation of various key reaction products. Preliminary experiments indicated that the hyperactive Tn5 Tnp will bind to OE sequences (and presumably form synaptic complexes) but will not catalyze transposition when incubated with pRZTL1 DNA at 20 °C (data not shown). We used this observation to perform a synchronized transposition reaction. A standard reaction system was incubated at 20 °C for 1 h, and then the temperature was brought to 37 °C. Samples were removed at various times between 0 and 9 h, phenol-extracted, and analyzed by agarose gel electrophoresis.
The results are shown in Fig. 6, A-C. In Fig. 6A we have marked the three product molecules that can be unambiguously quantitated, the excised transposon fragment (ETF) which should be an intermediate in a cut and paste transposition event, the released donor backbone (DBB), which should be a dead-end product, and the intermolecular transposition product (Inter). These products were quantitated using a fluorimager, and the appearance of products with time is shown in Fig. 6B. The ratio of the abundance of ETF/DBB and ETF/Inter is presented in Fig. 6C. All products appear early, but the ETF appears to be chased into other products as would be expected for a cut and paste transposition reaction.
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Linear DNA Acts as an Efficient Substrate-- Fig. 3A, lane 6, displays the result of an experiment in which pRZTL1 DNA linearized in the donor backbone was used as a substrate. This result clearly demonstrates that linear DNA containing two inverted OE sequences can undergo Tnp-mediated cleavage and strand transfer. We have also performed reactions with pRZTL1 linearized by cleavage within the transposable element and reactions with pRZTL1 cut within both the donor backbone and the transposable element (thus breaking any covalent linkage between the two OE sequences). In all cases Tnp mediates DNA rearrangements (data not shown).
Tn5 Tnp Produces a Precise Cleavage of the OE Donor Backbone
Junction--
We have used ligation-mediated PCR analysis of a typical
reaction mixture to define the 5' cleavage product generated by Tnp at
one end of the Tn5 substrate. After a 3-h transposition
reaction the DNA was phenol-extracted, denatured, and hybridized to a
primer located 250 bp away from and facing one OE sequence. The primer was extended using Sequenase. A pre-existing nick in the template strand will give rise to a blunt end to which a double-stranded linker
was ligated. A PCR reaction was then performed utilizing the
appropriate two primers (one from the linker and the other adjacent to
the original primer), and the resulting molecule was sequenced. As
shown in Fig. 7A, there is one
ligation-mediated PCR product generated in the vicinity of the OE, and
this corresponds to a cleavage event precisely between the +1 position
of the OE and the 1 position of the DBB. This defines the 5' end of
the Tnp-cleaved molecules. If we assume that the 3' end that is
transferred in the overall reaction represents the position of the 3'
cleavage reaction (which is true for other transposition systems) then we conclude that Tn5 Tnp generates a double strand cleavage
precisely between the OE +1 position and the 1 position of the donor
DNA backbone. This conclusion is supported by the analysis of the excised transposon fragment described below.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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A critical requirement for studying transposition is the availability of a defined in vitro transposition system. In this communication we describe such a system for the bacterial transposon Tn5, and we use this system to address several key mechanistic questions. Initially we attempted to develop such an in vitro system utilizing purified wild type Tn5 Tnp. The resulting experiments demonstrated occasional, barely detectable levels of transposition as measured by a transformation assay (data not shown). We reasoned that perhaps the wild type Tnp was functionally inefficient because it had evolved to be largely inactive as a mechanism for ensuring that transposition occurred at a very low frequency in vivo. Based upon this assumption, we turned to hyperactive mutants of Tnp. Purified triple EK54/MA56/LP372 mutant Tnp proved to catalyze efficient in vitro Tn5 transposition that could be assayed either biologically or by direct gel analysis. Furthermore, the products of these transposition events were similar to those catalyzed by wild type Tnp in vivo in two key respects as follows: the transposition events utilized the 19-bp OE sequences, and the transposition events caused 9-bp target site duplications. In a separate study, we demonstrate that the target site preferences are the same for the in vitro as for the in vivo transposition.4 Thus the hyperactive EK54/MA56/LP372 Tnp system is a reasonable facsimile of Tn5 transposition.
Macromolecules Involved in Tn5 Transposition, the 19-bp End Sequences and Tnp-- Transposition is a complex multistep process, and thus it is not surprising that many transposition systems are very elaborate involving a complex series of DNA sequences and several different polypeptides. In vivo studies suggested that this might not be the case for Tn5. If this were true, an in vitro Tn5 transposition system might offer a unique opportunity to study how a single polypeptide and single type of DNA sequence had evolved to perform multiple sequential steps. In vivo genetic studies have suggested that the only Tn5 related sequences required for transposition are two inverted 19-bp OE sequences (27-30). This situation is in clear distinction with that found for Mu (in which four 22-bp att sites and an internal enhancer sequence participate) (2) and Tn7 (in which approximately 150 bp are required on each end) (8). Even in the case of Tn10, an IHF binding sequence adjoining the 23-bp end sequence is intimately involved in the reaction resulting in the preferred end sequences being 70 bp long (4). The results reported in this communication are consistent with two inverted and appropriately spaced 19-bp OE sequences being the only specifically required cis-active sequence for Tn5 transposition.
The in vivo analyses of protein participants in Tn5 transposition are not as clear since several host mutants do affect the frequency of Tn5 transposition (reviewed in Refs. 3 and 15). The most obvious candidate for a required host protein is DnaA, because DnaA binds specifically to the OE sequence (34) and because the frequency of Tn5 transposition is reduced approximately 10-fold in a dnaA host (35). Our in vitro studies suggest that no host protein including DnaA is required for Tn5 transposition at least through the strand transfer step. We have used three different types of purified Tnp. The standard preparation is 86% pure, and two additional chromatographic steps result in 96% pure Tnp (see Fig. 4). Both preparations are active in mediating efficient in vitro transposition. Furthermore, a GST-Tnp fusion purified by affinity chromatography is also active in mediating efficient transposition (data not shown). It is possible, of course, that a required host protein has copurified with Tnp in all of these preparations. In fact topoisomerase I is known to be present in the 86% pure preparation (but not in the 96% pure transposase preparation), although topoisomerase 1 is not active in our standard transposition reaction conditions.5 However, it is highly unlikely that any contaminating protein is present in the stoichiometric amounts usually required for host proteins involved in transposition. There are two caveats to our proposal that Tnp is the only required protein for transposition. First, we have been using hyperactive mutant Tnp. It is possible that one or more of the mutant changes bypasses the need for a required host protein. A more interesting possibility is suggested by the results shown in Fig. 3C. Some protein(s) appears to be tightly associated with many of the Tn5 transposition products. Presumably this protein is Tnp. Thus release of Tnp may require a host factor. It is of interest that release of Mu Tnp from its products requires the presence of ClpX (6). We are currently testing whether DnaA is this required protein. We have also studied the Tnp concentration dependence for the reactions leading to DBB release (Fig. 5). These reactions demonstrate a modest sigmoidal dependence upon Tnp abundance suggesting that very few Tnp protomers are needed for this partial transposition process. Since Tnp has gel exclusion properties of a monomeric peptide,6 we propose that the reaction is initiated by the binding of single Tnp molecules to each of the two ends, and no other Tnp molecules are needed at least until the transposon is cleaved free of the DBB.Cut and Paste Mechanism-- It was first hypothesized 20 years ago that Tn5 transposes through a conservative cut and paste mechanism (36). This is in contrast to the replicative transposition mechanism used by Mu, Tn3, and related transposable elements (1). The data presented in this communication confirm this hypothesis. A cut and paste transposition mechanism is strongly supported by the time course analysis presented in Fig. 6, in which the excised transposon band is generated first and then is reduced in abundance in parallel with the appearance of final transposition products. It is also important to note that certain DNA structures that would be produced by a replicative process, in particular cointegrate type molecules that would travel more slowly than relaxed intramolecular products, are not observed in our experiments (see Figs. 3, 5, and 6). Finally, our analyses of the Tn5 donor backbone cleavage products strongly support the proposed cut and paste mechanism and specifically indicate that both stands are cleaved at the precise OE donor backbone boundary (Fig. 7).
Usefulness of the Tn5 System-- Other investigators studying transposition systems may find the isolation and use of hyperactive mutants a useful approach. In addition, the Tn5 in vitro system is defined, efficient, and simple to use, thus it should find applications in the in vitro manipulation of DNA.
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ACKNOWLEDGEMENTS |
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We thank Lisa Braam and Dona York for help in the preparation of Tnp; Maggie Zhou for providing the EK54 mutation; Warren Johnson for comments on the manuscript; Barb Shriver for preparing bacterial media and other solutions; and Michael Cox for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM50692.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Wisconsin, 420 Henry Mall, Madison, WI 53706.
1 The abbreviations used are: Tnp, transposase; OE, outside end; IE, inside end; bp, base pair(s); Inh, inhibitor; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate; PCR, polymerase chain reaction; DBB, donor backbone; Inter, intermolecular transposition product; ETF, excised transposon fragment.
2 L. Braam, personal communication.
3 I. Y. Goryshin, J. A. Miller, and W. S. Reznikoff, unpublished observations.
4 I. Y. Goryshin, J. A. Miller, Y. V. Kil, V. A. Lanzov, and W. S. Reznikoff, submitted for publication.
5 H. Yigit and W. S. Reznikoff, manuscript in preparation.
6 L. Braam and W. S. Reznikoff, unpublished observations.
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REFERENCES |
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Top
Abstract
Introduction
Procedures
Results
Discussion
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C.-D. Lu, Y. Itoh, Y. Nakada, and Y. Jiang Functional Analysis and Regulation of the Divergent spuABCDEFGH-spuI Operons for Polyamine Uptake and Utilization in Pseudomonas aeruginosa PAO1 J. Bacteriol., July 15, 2002; 184(14): 3765 - 3773. [Abstract] [Full Text] [PDF]
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Y. Shevchenko, G. G. Bouffard, Y. S. N. Butterfield, R. W. Blakesley, J. L. Hartley, A. C. Young, M. A. Marra, S. J. M. Jones, J. W. Touchman, and E. D. Green Systematic sequencing of cDNA clones using the transposon Tn5 Nucleic Acids Res., June 1, 2002; 30(11): 2469 - 2477. [Abstract] [Full Text] [PDF]
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T. A. Naumann and W. S. Reznikoff Tn5 Transposase Active Site Mutants J. Biol. Chem., May 10, 2002; 277(20): 17623 - 17629. [Abstract] [Full Text] [PDF]
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H. W. Mankouri, T. J. Craig, and A. Morgan SGS1 is a multicopy suppressor of srs2: functional overlap between DNA helicases Nucleic Acids Res., March 1, 2002; 30(5): 1103 - 1113. [Abstract] [Full Text] [PDF]
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A. Lamberg, S. Nieminen, M. Qiao, and H. Savilahti Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu Appl. Envir. Microbiol., February 1, 2002; 68(2): 705 - 712. [Abstract] [Full Text] [PDF]
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T. A. Naumann and W. S. Reznikoff Tn5 Transposase with an Altered Specificity for Transposon Ends J. Bacteriol., January 1, 2002; 184(1): 233 - 240. [Abstract] [Full Text] [PDF]
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Y. Nakada, Y. Jiang, T. Nishijyo, Y. Itoh, and C.-D. Lu Molecular Characterization and Regulation of the aguBA Operon, Responsible for Agmatine Utilization in Pseudomonas aeruginosa PAO1 J. Bacteriol., November 15, 2001; 183(22): 6517 - 6524. [Abstract] [Full Text] [PDF]
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L. Hamer, K. Adachi, M. V. Montenegro-Chamorro, M. M. Tanzer, S. K. Mahanty, C. Lo, R. W. Tarpey, A. R. Skalchunes, R. W. Heiniger, S. A. Frank, et al. Gene discovery and gene function assignment in filamentous fungi PNAS, April 5, 2001; (2001) 91094198. [Abstract] [Full Text]
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P. Guerry, C. P. Ewing, T. E. Hickey, M. M. Prendergast, and A. P. Moran Sialylation of Lipooligosaccharide Cores Affects Immunogenicity and Serum Resistance of Campylobacter jejuni Infect. Immun., December 1, 2000; 68(12): 6656 - 6662. [Abstract] [Full Text] [PDF]
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A. M. Gehring, J. R. Nodwell, S. M. Beverley, and R. Losick Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation PNAS, August 6, 2000; (2000) 170059797. [Abstract] [Full Text]
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T. A. Naumann and W. S. Reznikoff Trans catalysis in Tn5 transposition PNAS, July 19, 2000; (2000) 160107997. [Abstract] [Full Text]
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 D. R. Davies, I. Y. Goryshin, W. S. Reznikoff, and I. Rayment Three-Dimensional Structure of the Tn5 Synaptic Complex Transposition Intermediate Science, July 7, 2000; 289(5476): 77 - 85. [Abstract] [Full Text]
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L. R. O. Tosi and S. M. Beverley cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics Nucleic Acids Res., February 1, 2000; 28(3): 784 - 790. [Abstract] [Full Text] [PDF]
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A. Bhasin, I. Y. Goryshin, and W. S. Reznikoff Hairpin Formation in Tn5 Transposition J. Biol. Chem., December 24, 1999; 274(52): 37021 - 37029. [Abstract] [Full Text] [PDF]
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D. J. Lampe, B. J. Akerley, E. J. Rubin, J. J. Mekalanos, and H. M. Robertson Hyperactive transposase mutants of the Himar1 mariner transposon PNAS, September 28, 1999; 96(20): 11428 - 11433. [Abstract] [Full Text] [PDF]
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H. Yigit and W. S. Reznikoff Escherichia coli DNA Topoisomerase I Copurifies with Tn5 Transposase, and Tn5 Transposase Inhibits Topoisomerase I J. Bacteriol., May 15, 1999; 181(10): 3185 - 3192. [Abstract] [Full Text]
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D. R. Davies, L. M. Braam, W. S. Reznikoff, and I. Rayment The Three-dimensional Structure of a Tn5 Transposase-related Protein Determined to 2.9-A Resolution J. Biol. Chem., April 23, 1999; 274(17): 11904 - 11913. [Abstract] [Full Text] [PDF]
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S. Schmid, B. Berger, and D. Haas Target Joining of Duplicated Insertion Sequence IS21 Is Assisted by IstB Protein In Vitro J. Bacteriol., April 1, 1999; 181(7): 2286 - 2289. [Abstract] [Full Text]
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L. A. M. Braam, I. Y. Goryshin, and W. S. Reznikoff A Mechanism for Tn5 Inhibition. CARBOXYL-TERMINAL DIMERIZATION J. Biol. Chem., January 1, 1999; 274(1): 86 - 92. [Abstract] [Full Text] [PDF]
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I. Y. Goryshin, J. A. Miller, Y. V. Kil, V. A. Lanzov, and W. S. Reznikoff Tn5/IS50 target recognition PNAS, September 1, 1998; 95(18): 10716 - 10721. [Abstract] [Full Text] [PDF]
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A. E. Leschziner, T. J. Griffin IV, and N. D. F. Grindley Tn552 transposase catalyzes concerted strand transfer in vitro PNAS, June 23, 1998; 95(13): 7345 - 7350. [Abstract] [Full Text] [PDF]
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L. A. M. Braam and W. S. Reznikoff Functional Characterization of the Tn5 Transposase by Limited Proteolysis J. Biol. Chem., May 1, 1998; 273(18): 10908 - 10913. [Abstract] [Full Text] [PDF]
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M. Steiniger-White and W. S. Reznikoff The C-terminal alpha Helix of Tn5 Transposase Is Required for Synaptic Complex Formation J. Biol. Chem., July 21, 2000; 275(30): 23127 - 23133. [Abstract] [Full Text] [PDF]
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S. S. Twining, I. Y. Goryshin, A. Bhasin, and W. S. Reznikoff Functional Characterization of Arginine 30, Lysine 40, and Arginine 62 in Tn5 Transposase J. Biol. Chem., June 15, 2001; 276(25): 23135 - 23143. [Abstract] [Full Text] [PDF]
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L. Hamer, K. Adachi, M. V. Montenegro-Chamorro, M. M. Tanzer, S. K. Mahanty, C. Lo, R. W. Tarpey, A. R. Skalchunes, R. W. Heiniger, S. A. Frank, et al. Gene discovery and gene function assignment in filamentous fungi PNAS, April 24, 2001; 98(9): 5110 - 5115. [Abstract] [Full Text] [PDF]
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T. A. Naumann and W. S. Reznikoff Trans catalysis in Tn5 transposition PNAS, August 1, 2000; 97(16): 8944 - 8949. [Abstract] [Full Text] [PDF]
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A. M. Gehring, J. R. Nodwell, S. M. Beverley, and R. Losick Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation PNAS, August 15, 2000; 97(17): 9642 - 9647. [Abstract] [Full Text] [PDF]
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