From the Department of Biochemistry, University of
Wisconsin, Madison, Wisconsin 53705 and the § Department
of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin
53226-4801
Received for publication, November 29, 2000, and in revised form, March 26, 2001
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ABSTRACT |
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Three N-terminal basic residues of
Tn5 transposase, which are associated with
proteolytic cleavages by Escherichia coli proteinases, were
mutated to glutamine residues with the goal of producing more stable
transposase molecules. Mutation of either arginine 30 or arginine 62 to
glutamine produced transposase molecules that were more stable toward
E. coli proteinases than the parent hyperactive
Tn5 transposase, however, they were inactive in vivo. In vitro analysis revealed these mutants were inactive, because both Arg30 and Arg62 are required for
formation of the paired ends complexes when the transposon is attached
to the donor backbone. These results suggest Arg30 and
Arg62 play critical roles in DNA binding and/or synaptic
complex formation. Mutation of lysine 40 to glutamine did not increase
the overall stability of the transposase to E. coli
proteinases. This mutant transposase was only about 1% as active as
the parent hyperactive transposase in vivo; however, it
retained nearly full activity in vitro. These results
suggest that lysine 40 is important for a step in the transposition
mechanism that is bypassed in the in vitro assay system,
such as the removal of the transposase molecule from DNA following
strand transfer.
Transposases move transposons from one genomic location to another
by either a conservative (cut and paste) or a replicative mechanism.
Although Tn5 uses the simpler cut and paste mechanism of
transposition, there are still multiple steps involved (see Fig.
1 below). Genetic engineering of
Tn5 transposase to generate a hyperactive form
(EKLP)1 and the optimization
of the transposon end sequences allow many of the individual steps of
the Tn5 transposition mechanism to be studied in
vitro (1-6). The hyperactive EKLP form of Tn5
transposase contains the mutations E54K, M56A, and L372P (4). The
optimized mosaic transposon end sequence (ME) is a hybrid between the
bases found in the inside end (IE) and outside end (OE) sequences of the IS50 transposon (7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, model of Tn5 transposition
mechanism. The N-terminal domain plus several residues in the catalytic
domain of two Tn5 transposase molecules bind monomerically
to the transposon end sequences. The two bound transposase molecules
form synaptic complexes in which the C-terminal ends of the two
transposases dimerize (9). The catalytic domain of the transposase
molecule bound to the left end is positioned to cleave the transposon
end on the right end, and the transposase bound on the right end is
positioned to cleave the left transposon end (10, 21). In the presence
of Mg2+, the donor backbone DNA is cleaved via a mechanism
involving a hairpin intermediate (11). Target DNA is captured and the
transposon is inserted into the captured DNA. This step results in nine
unpaired bases at each end of the transposon (2). The transposase
molecules are removed, and the 9-bp gaps are repaired producing 9-bp
duplications. B, in vitro transposition assays
used to characterize Tn5 transposase mutants. Paired ends
complex formation examines the binding of transposases to transposon
end sequences present on short pieces of DNA with and without donor
backbone. The in vitro transposon excision assay tests these
steps plus the ability of the transposases to cut the transposon from a
plasmid. The in vitro transposition reaction using precut
transposons bypasses the transposon cleavage step to test the effect on
a mutation of target capture and strand transfer. The in
vitro transposition reaction using plasmids containing transposons
tests the transposition reaction through strand transfer complex
formation. For quantification of the in vitro transposition
reaction, the transposase molecules are removed with SDS or
chloroform-phenol treatment and the naked DNA is transformed into
E. coli. Repair of the 9-bp gaps occurs in vivo.
Acquisition of antibiotic resistance and presence of the transposon are
used for quantification of the reaction.
The first steps of the transposition mechanism are binding of the two end sequences of the transposon to two transposase molecules and dimerization of these transposase molecules to form a synaptic complex (see Fig. 1A below) (8, 9). Transposase cleaves the transposon from the donor backbone by a multistep reaction. This cleavage involves trans catalysis in which the transposon end held by one transposase molecule is cleaved by the second transposase molecule (10). In the first step of the cleavage reaction, the transposase in the presence of Mg2+ catalyzes a 3'-hydrolytic nick at position +1 of the transposon end that results in a free 3'-OH. This reactive group attacks the phosphodiester bond on the opposite strand to form a hairpin intermediate (11). The transposase then catalyzes the resolution of the hairpin by a second hydrolytic cleavage reaction. Cleavage of the two ends of the transposon is not a concerted process, because single end cleavage products have been observed (4).
Next, target DNA is captured by Tn5 transposase and the transposon is inserted into the target. Transposon insertion involves a catalyzed attack of the 3'-OH groups of the cleaved transposon on the phosphodiester bonds of the target DNA. This attack is staggered leading to 9-bp gaps. After repair, these are observed as duplications of the target sequence flanking the transposon. For this repair step to occur, the transposase, which binds to the DNA very tightly, must be removed. In the in vitro assays, the transposase is removed either by phenol-chloroform extraction or incubation with 0.5% SDS at 68 °C (4). The mechanism of this step in vivo is not known for Tn5 transposase. The IS903 and bacteriophage Mu systems are the only transposition systems in which transposase removal has been studied (12, 13). In both of these systems, a chaperone-mediated step is involved. In the Tn5 transposition system, the removal of the transposase probably requires proteins other than transposase (2).
In the Tn5 transposition mechanism, synaptic complex formation, transposon cleavage, target capture, and strand transfer into target DNA in vitro only require end sequence DNA, target DNA, the transposase, and Mg2+. In other transposition systems, additional transposon-encoded and/or host proteins are required for the transposition reaction such as the activator protein MuB for Mu transposition, the DNA integration host factor for Tn10 transposition, and TnsC for Tn7 transposition (14, 15).
Truncation of the N-terminal end of Tn5 transposase prevents
binding of the transposon end sequences and converts the transposase molecules into inhibitors of the transposase reaction (16). N-terminal
truncation products are generated by proteolysis or by the use of an
alternative promoter whose mRNA has a translation initiation site
corresponding to methionine 56 of the transposase molecule (16, 17). To
prevent synthesis directed by the alternative promoter, methionine 56 is mutated to an alanine to ensure full-length expression of the
transposase. This, however, does not prevent proteolysis of the
full-length transposase to form three stable N-terminal truncation
products (6). These act as transposase inhibitor molecules and react
with full-length transposase to form nonproductive complexes (18).
Three truncation products result from Escherichia coli
proteinase cleavage: 1 from cleavage of the
Arg30-Leu31 bond,
2 from cleavage of the
Lys40-Tyr41 bond and
from cleavage of the
Arg62-Phe63 bond of Tn5 transposase.
Arg30, Lys40, and Arg62 in the
N-terminal DNA binding domain were selected for mutation to genetically
engineer more stable forms of Tn5 transposase. The three
basic residues were individually mutated to glutamine, a residue that
potentially can interact with DNA directly through a hydrogen bond or
indirectly through interaction with a water molecule.
Here we show the stabilization of Tn5 transposase against
proteolytic degradation by E. coli enzymes by mutation of
Arg30 to Q and Arg62 to Q but not
Lys40 to Q. We identify two DNA binding residues
(Arg30 and Arg62) in Tn5 transposase
that are required to form stable complexes with transposon end
sequences attached to donor backbone. We also demonstrate that mutation
of another N-terminal basic residue, Lys40 to Q, does not
significantly alter synaptic complex formation, transposon excision,
target capture, or strand transfer using in vitro assays.
This mutation, however, affects a step in the transposition mechanism
that occurs in vivo not probed by the in vitro
assays such as the removal of the transposase from the DNA.
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EXPERIMENTAL PROCEDURES |
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Mutation of Tn5 Transposase-- The plasmid pRZPET2, which contains the hyperactive Tn5 transposase gene (EKLP), with the mutations E54K, M56A, and L372P (4), was used as a template to individually mutagenize residues Arg30, Lys40, and Arg62 in the N-terminal DNA binding region of the transposase to Q. The following PAGE purified oligonucleotide primers were obtained from Integrated DNA Technologies: R30Q FORWARD, TCGCCGTACTGCCCAATTGGTTAACGTCGCCGCCCAATT-3'; R30Q REVERSE, 5'-TTAACCAATTGGGCAGTACGGCGAGGATCACCCAGCGCC-3'; K40Q FORWARD, 5'-GCCCAATTGGCACAATATTCTGGTAAATCAATAACCATC-3'; K40Q REVERSE, 5'-ACCAGAATATTGTGCCAATTGGGCGGCGACGTTAACCAA-3'; R62Q FORWARD, 5'-GGCGCTTACCAATTTATCCGCAATCCCAACGTTTCTGCC-3'; PET21 TPASE FORWARD, 5'-CGACTCACTATAGGGGAATTGTGAGCGG-3'; and PET21 TPASE REVERSE 607, 5'-CTCATCATGCTGCCCATGCGTAACCGG-3'.
The R30Q (pRZPET2-R30Q) and the K40Q (pRZPET2-KQ40) mutants were
constructed by PCR using the template pRZPET2 and pfu DNA polymerase (Stratagene) to produce two overlapping products that contained the desired mutation and Xba and NotI
cleavage sites. For the R30Q mutant, the two overlapping PCR products
were obtained using the R30Q REVERSE primer and the PET21 TPASE FORWARD
primer for the first product and the R30Q FORWARD primer and the PET21 TPASE REVERSE 607 primer for the second product. For the K40Q mutant,
the two overlapping PCR products were obtained using the K40Q REVERSE
primer and the PET21 TPASE FORWARD primer for the first product and the
K40Q FORWARD primer and the PET21 TPASE REVERSE 607 primer for the
second product. The respective products for each mutation were purified
on agarose gels and extracted using the QIAquick PCR gel extraction kit
(Qiagen). The overlapping products were mixed with the PET21 TPASE
FORWARD and TPASE REVERSE 607 primers, and PCR was carried out. The
products were gel-purified and digested with Xba and
NotI (Promega), and the enzymes and small digestion products
were removed using a PCR purification kit (Qiagen). The plasmid pRZPET2
was digested with the same two enzymes, and the plasmid minus the
Xba-NotI fragment was gel-purified. The mutated
Xba-NotI fragments were ligated into the cut plasmids. These
plasmids were transformed into E. coli strain DH5, grown in Luria Bertani medium (LB) for 1 h, and plated on LB agar
containing ampicillin (100 µg/ml, Sigma Chemical Co.).
The R62Q (pRZPET2-R62Q) mutant was constructed using PCR to produce a
mega primer containing the mutation, and then this primer was used to
produce the mutated Xba-NotI fragment for insertion into the
digested pRZPET2. The first PCR reaction used the R62Q FORWARD primer
and the TPASE REVERSE 607 primer. After gel purification, this product
was used in a second PCR reaction as a mega primer with the PET21 TPASE
FORWARD primer to produce the full-mutated Xba-NotI product.
The resulting PCR product was purified and inserted into the digested
pRZPET2 plasmid as described above. All three mutant plasmids were
manually sequenced using Sequenase Version 2 (Amersham Pharmacia
Biotech) and [-32P]ATP from Amersham Pharmacia Biotech
to confirm the presence of the desired mutations and the absence of
unwanted mutations.
Papillation Assay--
The mutant plasmids and pRZPET2 were
isolated from DH5, transformed into E. coli strain
MDW320, and plated on
Trp
-X-gal-phenyl-
-D-galactoside
agar containing ampicillin (100 µg/ml) and tetracycline (15 µg/ml,
Sigma). The papillation assay was carried out as reported by Weinreich
et al. (16). This assay measures the rate of
Tn5lac transposition into an actively transcribed gene
through the appearance of blue papillae over time.
Mating Out Assay--
The mutant plasmids and pRZPET2 were
transformed into E. coli strain JCM101 containing pOX38-Gen,
an F' plasmid, and pFMA187OO, a plasmid containing a transposon with
two outside ends. The mating out assay was performed as previously
described using RZ224 as the recipient strain (18). To determine total
exconjugates formed in this assay, the bacteria were grown on LB agar
containing gentamicin (5 µg/ml, Sigma) and nalidixic acid (20 µg/ml, Sigma). To quantify exconjugates that contain the transposon
on the conjugated pOX38-Gen F-factor, the bacteria were grown on LB
agar containing gentamicin (5 µg/ml, Sigma), nalidixic acid (20 µg/ml, Sigma), and chloramphenicol (20 mg/ml, Sigma)
Expression of Tn5 Transposase Mutants-- The constructs for expressing mutated proteins were generated by swapping the Xba-NotI cleaved products containing the codon for glutamine at residues 30, 40, or 62 into the expression vector pGRTYB35 that encodes for the hyperactive EKLP Tn5 transposase fused at the C-terminal end to the intein-chitin binding domain (11). The plasmids, pRZPET2-R30Q, pRZPET2-K40Q, pRZPET2-R62Q, and pGRTYB35 were digested with the restriction enzymes Xba and NotI. The mutated Xba-NotI fragments and the cleaved pGRTYB35 plasmid were gel-purified, and then the mutated fragments were individually ligated into the cleaved expression vector. These vectors were transformed into E. coli strain BL21 DE3 plys. The hyperactive EKLP transposase and the mutated transposases were expressed and then purified on a chitin column (New England BioLabs) in the presence of a proteinase inhibitor mixture (Roche Molecular Biochemicals) as previously reported (11). 50 mM dithiothreitol (DTT) was used to induce intramolecular cleavage of the protein to release the free transposase molecules.
Proteolysis of Transposases by E. coli Proteinases--
The
mutant and hyperactive transposases were expressed in parallel 1-liter
cultures at 23 °C as described above. After 4 h of induction of
protein expression with
isopropyl-1-thio--D-galactopyranoside, all cultures were
normalized to an optical density of 0.6 with LB (11). The E. coli organisms in 1 liter were pelleted by centrifugation, resuspended, and then sonicated in the presence or absence of the
proteinase inhibitor mixture. The transposase molecules were isolated
as described above. The C-terminal portion of proteolysis products
bound to the chitin column through the C-terminal intein-chitin binding
domain. The transposase portion of the fusion protein was released upon
treatment with DTT. The N-terminal portions of proteolysis products
were lost in the purification process. The isolated molecules were
separated by SDS-PAGE, stained using Sypro orange (Molecular
Probes), and visualized on a FluoroImager (Molecular Dynamics).
Limited Proteolysis of Transposases by Thermolysin-- To remove the EDTA and the DTT present in the elution buffer from the chitin column, the isolated transposases were diluted three times in 50 mM Tris buffer, pH 7.0, containing 2 mM CaCl2 and then concentrated using a Centricon 30 (Millipore) at 4 °C. The mutant transposases and the hyperactive EKLP transposase (15 µg) were incubated with thermolysin (100 ng, Calbiochem) at 37 °C. Samples were taken at 0, 5, and 15 min and immediately placed into SDS-PAGE sample buffer containing DTT and boiled for 3 min. The cleaved samples were separated on 12% SDS-polyacrylamide gels and then stained with Coomassie Brilliant Blue.
Paired Ends Complex Formation--
Six PAGE-purified
oligonucleotides were obtained from Integrated DNA Technologies:
60-bp oligonucleotides containing the mosaic end sequence
(underlined) plus 21 bp of the transposon beyond the
end sequence and 20 bp of the donor backbone, Top
5'-CTCAGTTCGAGCTCCCAACACTGTCTCTTATACACATCTTGAGTGAGTGAGCATGCATGT-3' and Bottom
5'-ACATGCATGCTCACTCACTCAAGATGTGTATAAGAGACAGTGTTGGGAGCTCGAACTGAG-3'; 40-bp oligonucleotides containing the mosaic end sequence plus 21 bp of
the transposon beyond the end sequence, Top
5'-CTCAGTTCGAGCTCCCAACACTGTCTCTTATACACATCT-3' and Bottom
5'- AGATGTGTATAAGAGACAGTGTTGGGAGCTCGAACTGAG-3'; and 20-bp
oligonucleotides containing 20 bp of the donor backbone Top
5'-CTCAGTTCGAGCTCCCAACA-3' and Bottom 5'-TGTTGGGAGCTCGAACTGAG-3'. The 5'-ends of the oligonucleotides were end-labeled using T4 polynucleotide kinase (New England BioLabs) and
[-32P]ATP (Amersham Pharmacia Biotech,
Redivue). Unincorporated nucleotides were removed with a nucleotide
removal kit (Qiagen). The oligonucleotides were annealed in 20 mM Tris-HCl, pH 7.5, containing 50 mM NaCl by
heating at 80 °C for 2 min and then by slowly cooling to room temperature. Four different labeled transposon forms were constructed by annealing the following oligonucleotides: 60-bp DBB,
[5'-32P]ATP top and bottom 60-bp oligonucleotides; 40-bp
precut transposon, [5'-32P]ATP top and bottom 40-bp
oligonucleotides; 60-bp 3' Nick, [5'-32P]ATP top 60-bp
oligonucleotide plus bottom [5'-32P]ATP 40-bp
oligonucleotide and unlabeled bottom 20-bp oligonucleotide; and 60-bp
5' Nick, [5'-32P]ATP bottom 60-bp oligonucleotide plus
unlabeled top 40-bp oligonucleotide and top [5'-32P 20-bp oligonucleotide.
The 5'-32P-labeled mosaic end DNA fragments (28 nM) were mixed individually with the mutant or hyperactive EKLP transposases (400 nM) in 20 mM HEPES, 100 mM potassium glutamate buffer and incubated at 30 °C for 3 h. The reaction mixtures were immediately mixed with native gel loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol FF, 30% glycerol in H2O), and the products were separated on a 5% native polyacrylamide gel. The bands were visualized using a PhosphorImager (Molecular Dynamics).
In Vitro Transposon Excision--
pGRST2, a plasmid that
contains a 1.3-kb transposon with the kanamycin gene flanked by the
mosaic ends, was used for in vitro assays as previously
reported (4, 17). The hyperactive transposase and the three mutant
transposases (200 nM) were individually incubated with
pGRST2 (34 nM) in transposition buffer (0.1 M
potassium glutamate, 25 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, 50 µg/ml bovine serum albumin, 0.5 mM -mercaptoethanol, 2 mM spermidine, 100 µg/ml tRNA, 50 mM NaCl) at 37 °C for 1 h.
XmaI (Promega) was added, and the reactions were incubated
for an additional hour at 37 °C. The transposases and restriction
enzymes were removed by chloroform-phenol extraction followed by
ethanol precipitation. The products were resuspended in H2O
and analyzed on 1.5% agarose gels. The gels were stained with
SYBR green (Molecular Probes) and visualized using a
FluoroImager (Molecular Dynamics).
Precut Transposon Insertion into Target DNA--
pGRPK7876,
containing a 1.8-kb transposon, which includes the Tn5
mosaic end sequences and the R6Kori and a
kanR gene from Tn903, was cleaved
with PvuII to remove donor DNA (3). The resulting transposon
was gel-purified as given above. The precut transposons (60 nM) were incubated for 2 h with the mutant or the
hyperactive Tn5 transposases (400 nM) and the
target DNA (pUC19, 60 nM) in 20 µl of transposition
buffer. Following incubation, SDS was added to 0.5%, and the tubes
were further incubated at 68 °C for 15 min to dissociate the
transposases from the DNA. The reaction mixtures were placed on
0.05-µm VM nitrocellulose membranes (Millipore, Bedford, MA)
suspended on the surface of deionized water to remove the SDS
and protein dissociated from the DNA. After 2 h, 3 µl of the
dialyzed mixture was electroporated into DH5
cells using standard
procedures. KanR-AmpR
colonies containing pUC19 (AmpR) with the transposon
(KanR) were quantified, and the presence of the transposon
was confirmed by analysis of the size of the plasmids on agarose gels.
In Vitro Transposition Using the Intact Plasmid pGRPK7876-- This assay was the same as given above for the precut transposon assay with the exception that uncleaved pGRPK7876 was used. In this assay the transposase must cleave the transposon from the donor backbone prior to insertion into pUC19.
In Vitro Transposition Using Intact Plasmids with Outside and Mosaic End Sequences-- Two plasmids that differ only by the presence of the outside end sequences in pRZTL1 and the mosaic end sequences in pRZTL4 (4, 7) were used for in vitro transposition reactions in which the transposon ends are inserted within the transposon-forming nested deletions and inversions of the transposon (1). The transposon contains a promoterless tetracycline-resistant gene that can be expressed upon insertion of the gene in the correct orientation in a unit of transcription within the transposon.
Either pRZTL1 or pRZTL4 (20 nM) were incubated with the
hyperactive or mutant transposases (300 nM) in 10 µl of
transposition buffer at 37 °C for 2 h. The reaction mixture was
treated with 0.5% SDS at 68 °C for 15 min and dialyzed against
water, and 2 µl of the dialyzed preparation was electroporated into
DH5 cells as given above. The numbers of tetracycline-resistant
colonies from three independent experiments were averaged.
Proteolysis of the Transposase in the Strand Transfer
Complex--
Annealed 5'-32P-labeled 40-bp mosaic ends
containing oligonucleotides (2.8 nM) were incubated in the
presence of pUC19 (6 nM) and either the K40Q mutant
transposase or the hyperactive EKLP transposase (20 nM) in
10 µl of transposition buffer without bovine serum albumin for 2 h at 37 °C. Either N-tosyl-L-phenylalanine chloromethyl ketone trypsin (10-1000 nM, Sigma) or
proteinase K (6 µM, Sigma) were added to the samples and
further incubated for 15 min at 37 °C. The reaction was stopped by
the addition of native gel loading buffer containing
N-tosyl-L-lysine chloromethyl ketone (4 mM, Sigma), and the samples were immediately separated on a
1% agarose gel. The DNA bands were visualized using ethidium bromide.
The gel was carefully dried under vacuum onto nitrocellulose. The bands
on the dried gel were visualized using a PhosphorImager and quantified
using the ImageQuaNT program (Molecular Dynamics).
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RESULTS |
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Stability of Tn5 Transposase Mutants--
With the goal to produce
more stable forms of Tn5 transposase, three basic
Tn5 transposase residues (Arg30,
Lys40, and Arg62), present at the P1 positions
(N-terminal side) of the sites cleaved by E. coli
proteinases during purification (6), were individually mutated to
glutamine residues by site-directed mutagenesis. The hyperactive EKLP
Tn5 transposase was used as the parent form because its
in vitro activity allows dissection of the transposition reaction mechanism (4, 17). The R30Q and R62Q mutants were much more
stable than either the parent EKLP or the K40Q mutant transposases
toward the proteinases present in E. coli (Fig.
2). The
EKLP transposase preparation isolated in the absence of proteinase inhibitors contained two stable bands of proteolytic products in
addition to full-length transposase. Previous studies showed the band contains transposase molecules cleaved after Arg30
(
1) and Lys40 (
2), and the
band contained
transposase molecules cleaved after Arg62 (6). In contrast
to the R30Q and the R62Q mutants, the intact K40Q transposase mutant
was sensitive to proteolysis like the EKLP transposase (Fig. 2). It was
degraded to two stable products with more of the
band than was
observed for the EKLP transposase. This probably represents the
1
product, because the
2 cleavage site is mutated in the K40Q mutant.
The R30Q and R62Q mutants were very stable with small amounts of the
and
products detected. The
product in the R62Q preparation
is probably due to cleavage at Arg65, a nearby positively
charged amino acid. In addition, a small amount of a 35-kDa product was
observed for both the R30Q and R62Q mutant transposases. The identity
of this product is not known.
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The increased overall stability of the R30Q and R62Q transposase
mutants suggests not only that the peptide bond next to the mutated
residue is protected against cleavage but also that the N-terminal
region of these mutants may be less flexible and/or less exposed to
solvents. Either of these possibilities would lead to an overall
decrease in susceptibility to proteolysis by proteinases that degrade
at sites other than the mutated basic amino acids. To test this
hypothesis, the three mutants and the parent EKLP transposase were
subjected to limited proteolysis by thermolysin, a metalloproteinase
that cleaves preferentially at peptide bonds with the hydrophobic
residues, Leu, Phe, Ile, or Val, in the P1'-site (C-terminal side of
the cleavage site) but that can also accommodate Met, His, Tyr, Ala,
Asn, Ser, Thr, Gly, Lys, Glu, or Asp at the P1'-position (19). The R62Q
mutant was much more stable than the parent EKLP transposase (Fig.
3), however, similar products were
observed for both molecules, suggesting that this mutation alters the
flexibility or solvent accessibility of the N-terminal end of the
transposase but not the overall conformation. The R30Q transposase
mutant was slightly more stable over the first 5 min of degradation
with thermolysin than the parent EKLP transposase, but similar products
were formed for the two transposases. By 15 min, similar amounts of
both transposases were degraded. The K40Q mutant transposase was
degraded at a similar initial rate to that of the parent EKLP with
similar degradation products, indicating similar conformations and
flexibilities. These results suggest that the overall conformations of
the mutant transposases were similar but the accessibility of the
N-terminal region of R62Q mutant and, to some extent, the R30Q mutant
to the proteinases may be decreased.
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In Vivo Transposition-- Two in vivo transposition systems were used to test the activity of the three mutants relative to the parent hyperactive EKLP transposase. The papillation assay was used as a qualitative initial screen of activity. Multiple papillae per colony were observed for both the EKLP and K40Q transposases but not for the R30Q and R62Q mutants within 7 days. Although multiple papillae were observed, the initial papillae on the K40Q mutant colonies were observed 8-12 h after those for the EKLP colonies, and the numbers of papillae per colony were fewer for the K40Q mutants than for the EKLP colonies.
For a more quantitative assay, the trans-mating out assay was used to compare the in vivo transposition frequencies. No activity over background was noted for the R30Q and R62Q mutants (Table I, column 1), suggesting the mutations prevent one or more of the required steps of transposition. The K40Q mutant transposase was about 100× less active than the parent EKLP transposase, indicating the mutation alters but does not knock out transposition activity.
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In Vitro Paired Ends Complex Formation by K40Q Mutant Tn5
Transposase--
To explore the step or steps in the transposition
mechanism altered by the K40Q mutation, the activity of this mutant was compared with the hyperactive parent EKLP Tn5 transposase
using in vitro assays (Fig. 1B). The first assay
used an electrophoretic mobility shift assay to determine the ability
of the K40Q mutant transposase to form synaptic complexes (paired ends
complexes) with three forms of the transposon encountered by the
transposase at various steps of the transposition process and a control
form of the transposon not associated naturally with the transposon (Fig. 4A). The transposon
forms are 1) the normal substrate form composed of transposon end
sequences attached on one end (+19) to transposon sequences and on the
other end (+1) to donor backbone (60 bp); 2) the first intermediate in
the transposon cleavage step, a transposon with a 3'-nick in the
transferred strand between the +1 position of the transposon end
sequence and the donor backbone (60-bp 3'-nick); 3) the cleaved
transposon (40 bp); and 4) a control for the 3'-nicked transposon form,
a 5'-nicked transposon with a nick in the nontransferred strand between
the +1 position of the transposon end sequence and the donor backbone
(60 bp 5'-nick). This last form is used as a control to determine
whether any differences observed between the intact and nicked forms of
the transposon-containing donor backbone are due to differences in the
flexibility of the DNA or whether the differences are due to specific
interactions between the DNA and the transposases.
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The K40Q mutant was equally active as the EKLP mutant in the formation of paired ends complexes with DNA with donor backbone sequences (Fig. 4B, 60 bp, compare lanes 14 and 18) or without donor backbone sequences (Fig. 4B, 40 bp, compare lanes 11 and 15). In addition, both forms of the transposase formed paired ends complexes equally well with DNA containing nicks in the backbone either at the natural position for formation of the hairpin intermediate (Fig. 4B, 60-bp 3'-nick, compare lanes 12 and 16) or on the opposite strand (Fig. 4B, 60-bp 5'-nick, compare lanes 13 and 17). This suggests the K40Q mutation does not alter DNA binding nor synaptic complex formation.
In Vitro Excision of Transposons by K40Q Mutant Tn5
Transposase--
The next step in the transposition reaction, the
cleavage of the transposon from donor backbone, was compared between
the K40Q mutant and the parent EKLP transposases using pGRST2 that contains a 1.3-kb transposon with mosaic ends (Fig.
5A). To characterize the
excision step, the products were digested with XmnI that
asymmetrically cleaves the donor backbone. This allows visualization of
cleavage of the transposon at one end as well as cleavage at both ends. Analysis of the products following digestion revealed that both the
K40Q mutant and parent EKLP forms of the transposase produced the same
single left end cut product (3.2 kb) and right end cut product (2.0 kb)
with slightly higher amounts for the R40Q mutant than for EKLP
transposase (Fig. 5B). Both forms of the transposase produced the same double-transposon end cleavage product (1.3 kb) at
similar amounts, as well as the same excised donor backbone products
(right, 1.9 kb; left, 0.7 kb) with slightly higher amounts for the EKLP
transposase that for the R40Q mutant.
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In Vitro Transposition by K40Q Mutant Tn5 Transposase-- With the knowledge that the K40Q mutant can bind the transposon end sequences and cleave the transposon from donor backbone, two additional assays (Fig. 1B) were used to quantify the transposase activities of the K40Q mutant and the parent EKLP transposase. In these assays, the transposition reaction was carried out in vitro by the transposase, and then the transposase was dissociated by treatment of the DNA·protein complexes with SDS and heating. The protein-free DNA was transformed into E. coli for quantification of the transposition events as antibiotic resistant colonies. The number of transposition events per microgram of transposase was similar for both the R40Q mutant and the parent EKLP transposases when either the precut transposon (Table II, columns 3 and 4) or the plasmid pGRPK7876 was used as the source of the transposon for insertion into pUC19 (Table II, columns 1 and 2). These results demonstrate the K40Q mutant transposase is as active as the parent EKLP transposase in these in vitro assays.
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Because these in vitro assays use transposons with mosaic end sequences and the in vivo assays use transposons with outside end sequences, the 100× difference observed in the transposition frequencies of the K40Q mutant in vitro compared with in vivo could be due to the end sequences used. To test this, two transposon-containing plasmids were used that differed only by the presence of outside end sequences in pRZTL1 and mosaic end sequences in pRZTL4. No significant differences were noted in transposition frequencies between the K40Q mutant and the hyperactive EKLP parent transposase with either form of the transposons; however, the transposition frequency using mosaic ends was about 10× greater than using outside ends for both transposases (Table I, columns 3-6). Based on these data, the 100× difference in activity between these two transposase molecules observed in the in vivo mating out assay is due to a process that occurs in vivo not measured by the in vitro assay.
Proteolytic Cleavage of the K40Q Mutant Tn5 Transposase from the
DNA following Strand Transfer--
One step that must occur in the
in vivo transposition reaction and is bypassed in the
in vitro assays is the removal of the transposase molecule
from the transposon·target DNA complex. In the in vitro
assays, the transposases are removed by incubating with 0.5% SDS at
68 °C followed by dialysis. A possible mechanism for the removal of
transposase from DNA after the strand transfer reaction in
vivo is direct proteolysis. To test whether the strand transfer
complexes formed with the K40Q mutant transposase are more stable to
proteolysis than those formed with the parent EKLP transposase,
in vitro transposition was carried out using 40 bp of
precleaved DNA containing the mosaic end sequence and pUC19 as the
target DNA. The complexes formed were subjected to trypsin cleavage, a
proteinase that cleaves proteins at lysine and arginine residues, to
mimic the cleavage observed for the E. coli proteinase(s) (6). The K40Q mutant and the parent EKLP transposases were equally
susceptible to trypsin cleavage (Fig. 6).
This suggests that the decrease in transposition activity of the K40Q
mutant in vivo is not due to increased stability of the
transposase·DNA complex toward direct proteolysis at basic residues.
This, however, does not rule out a difference in other mechanisms of
removal of the transposase from DNA such as the interaction of the
transposase with a chaperone (13).
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In Vitro Analysis of R30Q and R62Q Mutant Transposases-- Unlike the K40Q mutant transposase, the R30Q and R62Q mutant transposases were inactive in vivo (Table I, columns 1 and 2). Because the thermolysin degradation products of these two mutants and the parent EKLP transposases were similar (Fig. 3), the overall conformation of the mutants probably is not changed. This would suggest the active site is intact but the two mutations, which are in the N-terminal domain associated with DNA binding (6), alter the ability of the mutant transposases to either bind DNA and/or form synaptic complexes. To test these possibilities, in vitro transposition assays (Fig. 1B) were used to compare the properties of the R30Q and R62Q mutant transposases to the parent EKLP transposase.
In Vitro Paired Ends Complex Formation by R30Q and R62Q MutantTn5 Transposases-- The ability of the R30Q and R62Q mutant transposases to form paired ends complexes with DNA was compared with that of the parent EKLP transposase (Fig. 4B). The two mutants did not form paired ends complexes with the 60-bp DNAs (Fig. 4B, lanes 6 and 10) that contained donor backbone plus the mosaic end sequence and a small amount the transposon. The mutants did form complexes with the 40-bp DNAs that do not contain donor backbone (Fig. 4B, lanes 3 and 7). Although the two mutants formed complexes, a substantially lower amount was formed than with the parent EKLP transposase (Fig. 4B, compare lanes 3 and 7 with lane 15), suggesting a difference in either the binding affinity for the DNA or the stability of the complexes.
To further characterize this binding, DNA was tested that mimics the natural nicked DNA intermediate formed in the first step of the transposon cleavage reaction (Fig. 4B, 60-bp 3'-nick). The R30Q and R62Q mutant transposases were able to assemble paired ends complexes with this 3'-nick form of the DNA (Fig. 4B, lanes 4 and 8) but not with DNA containing the nick in the nontransferred strand (Fig. 4B, 60-bp 5'-nick, lanes 5 and 9). The 3'-nicked form of the 60-bp DNA was utilized with a similar efficiency as the 40-bp precut transposon form. This demonstrates that the two mutations prevent the binding of transposons attached to donor backbone unless the natural 3'-nick is present. Furthermore, because complexes were not formed with the 60-bp DNA with a 5'-nick, the interaction between the mutant transposases and the 60-bp DNA with the 3'-nick is probably due to specific interactions and not to an increase in flexibility of the DNA.
In Vitro Transposition Reaction of R30Q and R62Q Mutant Tn5
Transposases--
Because the R30Q and R62Q mutant transposases are
predicted to have similar overall conformations and can form synaptic
complexes in the absence of donor backbone, the mutants would not be
expected to excise transposons from plasmids but would be expected to
insert precut transposons into donor DNA. Neither of these mutants
excised the transposon from pGRST2 (Fig. 5) nor produced
antibiotic-resistant colonies diagnostic for transposition using
plasmids containing transposons with either mosaic or outer end
sequences (Table I, columns 3-6 and Table II, columns
1 and 2). However, both mutants were able to insert
precut transposons into pUC19 in vitro, albeit with lower
efficiencies, 25% for R30Q and 1.4% for R62Q, than for the parent
EKLP transposase (Table II, columns 3 and 4).
These results suggest that target capture and strand transfer are not affected by the R30Q and R62Q mutations and the major effect of these
mutations is on the interaction of the transposases with the transposon
end sequences attached to donor DNA.
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DISCUSSION |
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R30Q and R62Q Tn5 Transposase Mutants Are More Stable Than EKLP Transposase but Are Defective in Synaptic Complex Formation-- Because N-terminal-truncated Tn5 transposase molecules form inactive heterodimers with full-length transposase and act as inhibitor molecules (17, 18), mutations of the transposase at known proteolytic cleavage sites (6) were expected to stabilize the protein toward proteolysis and increase in vivo transposition. Mutation of either Arg30 to Q or Arg62 to Q in Tn5 transposase increased the stability of the transposase toward E. coli proteinases but completely inhibited in vivo transposition. Limited proteolysis using thermolysin, a general proteinase that prefers hydrophobic amino acids at the P1'-site, generated similar proteolysis products with these two mutants and the parent EKLP transposase, suggesting the overall conformations of the three molecules are similar. The ability of the mutants to catalyze in vitro transposition using precut transposons further supports the similarity of the overall conformations of the two mutant and the EKLP transposases. The R62Q mutant was more stable to thermolysin digestion than the R30Q mutant or the parent EKLP transposases, suggesting the N-terminal portion of the molecule of the R62Q mutant may not be as flexible as the other two forms of the transposase or the N-terminal end of the molecule forms a tighter complex with the C-terminal end (16, 20). The difference in thermolysin susceptibility of the R62Q mutant from the R30Q mutant may be related to the 18× difference in the in vitro transposition activity between the R30Q and R62Q mutants when the precut transposon from pGR7876 was used.
Overall, mutations of Arg30 and Arg62 in the Tn5 transposase molecule affect the transposition mechanism in a similar manner. Both mutants form synaptic complexes with precut transposons and 3'-nicked transposons containing donor backbone in the paired ends complex assay but form fewer complexes than the parent EKLP transposase. Neither mutant forms synaptic complexes with transposons containing donor backbone or 5'-nicked transposons containing donor backbone. These similarities suggest both residues may be important for formation and/or stabilization of the DNA·protein complex. This conclusion is supported by previous experiments showing transposon end sequences protected Arg30-Leu31 and Arg62-Phe63 of the transposase from trypsin cleavage (6) and by the recently solved crystal structure of ELKP Tn5 transposase complexed with Tn5 transposon outside end sequences in the form of a paired ends complex (21).
Both Arg30 and Arg62 in the N-terminal domain
interact directly with DNA (Fig. 7).
Although Arg30 is found on the second turn of helix 2, and
Arg62 is near the C-terminal end of helix 4, these two
residues are close in space. In fact, these amino acids both interact
with guanidine 13 of the transferred strand. The NH1 of
Arg62 forms hydrogen bonds with N7 of guanidine 13 of the
transferred strand (Fig. 7A) and the NH2 and NH1 of
Arg30 bind directly or indirectly through a water molecule
to the phosphate of this residue (Fig. 7B).
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At first glance, the requirement of both Arg30 and Arg62 for efficient binding of the transposon to Tn5 transposase is surprising, considering the large number of contact residues between the transposon end sequences and the transposase (21). In addition to Arg30 and Arg62, six residues, Arg26, Arg27, Ser45, Lys54, Gln57, and Glu58, of the N-terminal domain of the transposase and three distant residues, Arg342, Glu344, and Asn348, directly bind to the transposon ends at positions +4 to +17 (21). In the synaptic complex, 15 additional residues, Thr99, Arg210, Tyr237, Gln243, Lys244, Arg253, Trp298, Tyr319, Arg322, Lys330, Lys333, Thr334, Ser438, Lys439, and Ser445, of the second transposon molecule also bind to the transposon end DNA at positions +1 to +7.
The importance of individual specific interactions between the transposase and the transposon has been demonstrated by missing nucleoside experiments (7, 8, 22). Removal of individual nucleosides at positions +4 to +19 of the top nontransferred strand or +5 to +19 of the bottom transferred strand of the mosaic end sequences results in dramatic decreases in synaptic complex formation of EKLP transposase to the DNA (8). A single amino acid mutation in a DNA binding residue of the transposase probably affects binding of the transposon at least to the same degree as a missing nucleoside because, in both cases, critical interactions between the transposon end sequences and the transposase are altered or missing.
Although the R30Q and R62Q mutations are not expected to alter the
formation of the respective -helixes, closer examination of the
interactions of Arg30 and Arg62 of the
transposase molecule with DNA in the synaptic complex (2) suggests how
mutation of either of these residues to glutamine results in the
dramatic effects on transposition. Arg62 forms four
hydrogen bonds with bases at positions 11, 12, and 13 in the major
groove of the outer end sequence (Fig. 7A). The observed
41° bend in the DNA between positions 11 and 12 of the transposon end
probably requires the positive charge on Arg62. Mutation of
Arg62 to the noncharged shorter glutamine residue not only
disrupts the bonds between residue 62 and the DNA but likely also
alters the multiple interactions between the DNA and the transposase that are dependent on the presence of the bend. The interactions most
affected probably are those between the DNA and the other binding
residues on helix 4, Lys54, Glu58, and
Gln57 (21).
Arg30 forms one hydrogen bond through a water molecule and three distinct ionic bonds with two of the backbone phosphates at positions 13 and 14 of the transferred strand (Fig. 7B). Substitution of glutamine for this arginine results in the loss of the positive charge and the ability of the amide group on the side chain to interact with the DNA backbone because of the shorter side chain. The absence of the four bonds between the transposase and the DNA may influence the binding of the additional residues Arg26 and Arg27 on helix 2 to the outer end sequence DNA. Arg27 forms an ionic bond with the phosphate backbone at the 5'-side of guanidine 15 of the transferred strand (21). Arg26 forms hydrogen bonds with the O2 of thymidine 16 of the transferred strand and the N3 of adenosine 17 of the nontransferred strand. It is possible that these three residues bind DNA in a synergistic manner. If true, the loss of the interaction of one of these amino acids with the DNA may alter the binding of the second two amino acids.
Transposase mutations at residues Arg30 and
Arg62 prevent synaptic complex formation when donor
backbone is attached at position +1 of the transferred strand but allow
complex formation, albeit at reduced levels, when DNA is precut or has
a 3'-nick. A possible explanation may be that a substantial DNA bend is
found at or near the 1/+1 cleavage site when the donor backbone is
present in the transposase·DNA complex (22, 23). Presumably, this bend is required for DNA binding when donor backbone is present. The
R30Q and R62Q mutations may decrease the affinity of the transposase for the DNA sufficiently so that it may not be able to grip the DNA
tightly enough to induce the bend, and thus binding cannot occur at
all. In the absence of donor backbone DNA, binding can occur in the
absence of this bend. The 3'-nick (which occurs as a natural
intermediate) may facilitate proper bend formation, whereas the 5'-nick
may not.
K40Q Tn5 Transposase Mutation Affects a Step in the Transposition Mechanism Not Measured by in Vitro Transposition Assays-- The third mutation, K40Q, affects in vivo transposition to a much greater extent than in vitro transposition. There are steps that may be affected by the mutation in vivo but are not tested in vitro. These include in vivo stability of the transposase, binding of potential accessory molecules to the Tn5 transposase·DNA complex not required in vitro, or the removal of the transposase following insertion of the transposon into target DNA. In vivo stability of the K40Q mutant transposase is probably similar to that of EKLP transposase, because similar amounts of the two intact transposases were recovered from the E. coli expression system.
The K40Q mutation may alter the interaction of the transposase·DNA complex with a binding molecule that is important for in vivo transposition but is not required in the in vitro transposition assays. This is supported by the surface location of R40 in the transposon end·transposase synaptic complex structure within a groove between the N-terminal DNA binding site and the C-terminal end of the transposase molecule (21). The mutation could alter the binding of molecules such as topoisomerase I or a chaperone molecule involved in the removal of transposase from the strand transfer complex. Topoisomerase I copurifies with Tn5 transposase, binds to the N-terminal end of the transposase, and can increase transposition rates (24). A more intriguing possibility is the interference by the K40Q mutation of the binding of a chaperone involved in removal of the transposase from the strand transfer complex. This step is bypassed in the in vitro system by the removal of the transposase by either heating in the presence of SDS or by phenol-chloroform extraction prior to transformation into E. coli for quantification of the transposition reaction.
Nothing is known about the mechanism by which the Tn5
transposase is removed from the strand transfer complex in
vivo; however, a chaperone and a protease may be required as shown
for the bacteriophage Mu and the IS903 transposition systems
(12, 13). Mu transposase (MuA) is removed and degraded by the
ClpX·ClpP chaperone·proteinase complex (13). The
IS903 transposase is degraded by Lon (12), a protein that
contains a chaperone domain at the N-terminal end, a proteinase domain
on the C-terminal end and is homologous to ClpX·ClpP (25). The
mutation of Lys40 to Q of Tn5 transposase may
alter the ability of the sensor, and substrate discrimination domains
of the Clp chaperones or protease Lon to recognize, bind, and remove
the transposase. Experiments are ongoing to test this hypothesis.
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ACKNOWLEDGEMENTS |
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We thank Ivan Rayment for help with the structural analysis in Fig. 7, Doug Davies, Lisa Mahnke, Todd Naumann and Mindy Steiniger-White for helpful conversation, and Barb Schriver for technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM50692 (to W. S. R.) and EY12931 (to S. S. T.).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-Madison, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-262-3608; Fax: 608-262-3453; E-mail: reznikoff@ biochem.wisc.edu.
Published, JBC Papers in Press, March 29, 2001, DOI 10.1074/jbc.M010748200
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ABBREVIATIONS |
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The abbreviations used are:
EKLP, hyperactive
form of Tn5 transposase with the mutations M56A, E54K, and
L372P;
bp, base pair(s);
DBB, donor backbone;
DTT, dithiothreitol;
IE, inner end sequence of Tn5 transposon;
ME, mosaic end
sequence of transposon;
OE, outer end sequence of Tn5
transposon;
P1, amino acid residue on the N-terminal side of a
proteolytic cleavage site;
P1', amino acid residue on the C-terminal
side of a proteolytic cleavage site;
PEC, paired ends complex;
PAGE, polyacrylamide gel electrophoresis;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
PCR, polymerase chain
reaction.
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