(Received for publication, March 16, 1995; and in revised form, September 6, 1995)
From the
Insertion sequence IS3 encodes two, out-of-phase, overlapping open reading frames, orfA and orfB. The OrfAB transframe protein that is IS3 transposase is produced by -1 translational frameshifting between orfA and orfB. Efficient production of the IS3 transposase in the cells harboring the IS3-carrying plasmid has been shown to generate miniplasmids as well as characteristic minicircles, called IS3 circles, consisting of the entire IS3 sequence and one of the 3-base pair sequences flanking IS3 in the parental plasmid. Here, we show that the IS3 transposase also generates the linear molecules of IS3 with 3-nucleotide overhangs at the 5`-ends. The nucleotide sequences of the overhangs are the same as those flanking IS3 in the parental plasmid, suggesting that the linear IS3 molecules are generated from the parental plasmid DNA by staggered double strand breaks at the end regions of IS3. The linear IS3 molecules are likely to be the early intermediates in the transposition reaction, which proceeds in a non-replicative manner.
Transposable elements are characterized by their ability to
transpose, and many of such elements have been identified so far in
plasmids and chromosomes of a wide variety of organisms. IS3 (1258 bp ()in length) is an insertion element present
in the Escherichia coli chromosome and in plasmid F (Malamy et al., 1972; Hu et al., 1975; Deonier et
al., 1979; Timmerman and Tu, 1985; Umeda and Ohtsubo, 1989).
IS3 does not mediate cointegration and is thus supposed to
transpose in a non-replicative manner (Sekine et al., 1994).
This element has imperfect inverted repeats (IRL and IRR) of 39 bp at
its terminal regions and encodes two open reading frames, orfA and orfB, which are in phase 0 and -1,
respectively, and overlap each other (Timmerman and Tu, 1985; Fayet et al., 1990; Sekine and Ohtsubo, 1991). In addition to the
OrfA and OrfB proteins, which are produced from each of the two orfs, a
transframe protein (the OrfAB protein) that is IS3 transposase
is produced by -1 translational frameshifting at the
A
G motif present in the overlapping region between the two
orfs (Sekine et al., 1994). An IS3 mutant with a
single guanine insertion in the A
G motif to give
A
G
produces the transposase protein without
frameshifting. This mutant causes a deletion of a sequence adjacent to
IS3 in a plasmid to produce miniplasmids and generation of
characteristic minicircles, called IS3 circles, which consist
of the entire IS3 sequence and a 3-bp sequence intervening
between the IS3 ends (Sekine et al., 1994). The orfB frame of IS3 codes for a polypeptide segment
showing homology with a conserved amino acid sequence motif found in
retrovirus and retrotransposon integrases (Fayet et al., 1990;
Khan et al., 1991), while the orfA frame codes for a
polypeptide segment with the
helix-turn-
helix motif, which
may be involved in recognition of the IS3 end regions
(Prère et al., 1990; Sekine and Ohtsubo,
1991). A group of IS elements that are structurally related to IS3 have been isolated from diverse bacterial genera. These elements,
called the IS3 family (Schwartz et al., 1988), code
for two orfs in phase 0 and -1, respectively (Fayet et
al., 1990; Sekine and Ohtsubo, 1991), and the predicted amino acid
sequences encoded by the downstream orf are similar to one another and
have homology with the motif in retrovirus/retrotransposon integrases
(Fayet et al., 1990; Khan et al., 1991). In addition
to IS3, two other members of the family produce transposases
by frameshifting between the two orfs (Vögele et al., 1991; Polard et al., 1992).
Here, we report that IS3 transposase generates linear IS3 molecules in addition to miniplasmids and IS3 circles. The molecules have 5`-overhangs, suggesting that they are generated from the parental plasmid DNA carrying IS3 directly by staggered breaks. We point out that the transposition reaction in IS3 is similar to the transposition reaction in other transposons, such as Tn10 and Tn7, which occurs by a non-replicative mechanism (Morisato and Kleckner, 1984; Benjamin and Kleckner, 1989; Bainton et al., 1991), and even more to the integration reaction in retroviruses, which generate linear DNA molecules as well as characteristic circles with long terminal repeat sequences.
Plasmids pSEK183 and
pSEK1831 used were pUC118 derivatives and carry wild type IS3 and an IS3 mutant, IS3-1, respectively (Sekine et al., 1994). IS3-1 contains a guanine insertion in
the AG motif at nt 328-332 by the coordinates given
to the IS3 sequence (Timmerman and Tu, 1985), leading to
in-frame alignment of orfA and orfB. Plasmid pSEK1832
belongs to type I miniplasmids derived from pSEK1831 and is deleted for
the region (1018 bp) extending from the end of IRR to a site within the
IG region in pSEK1831 (Sekine et al., 1994).
Reagents and chemicals used
were: [-
P]ATP (222 TBq/mmol) and
[
-
P]dNTP (110 TBq/mmol) (Amersham Corp.);
dNTPs and agarose (Takara); polyacrylamide (Wako).
Large scale preparation of the linear IS3 molecules was carried out as follows. Strain MV1184 harboring pSEK1831 or pSEK1832 was grown in 500 ml of L rich broth overnight at 37 °C. The cells were collected and lysed by the method of Clewell and Helinski(1970). The DNA was separated by CsCl/ethidium bromide equilibrium density centrifugation (Sambrook et al., 1989), and the upper band DNA containing linear DNA molecules and nicked circular plasmid DNA were collected. The DNA preparation was then separated by electrophoresis in a 1.2% agarose gel, and the band of the linear IS3 molecules was cut out and eluted.
The
3`-ends of the linear IS3 molecules were analyzed as follows.
The linear IS3 molecules were isolated from strain MV1184
harboring pSEK1831 as described above. In one reaction, the linear
IS3 molecules (0.01 pmol) were digested with BsmI or NcoI, treated with bacterial alkaline phosphatase, and labeled
with [-
P]ATP. In the other reaction, the
linear IS3 molecules were treated with bacterial alkaline
phosphatase, labeled with
P at their 5`-ends, and then
digested with BsmI or NcoI. The samples so prepared
were alkaline-denatured and electrophoresed in 8% polyacrylamide
sequencing gels containing 7 M urea. Size markers appearing as
sequence ladders were prepared using the pSEK183 DNA as template and
the
P-labeled BsmI or NcoI primer (see Table 1).
Figure 1:
Generation of the linear
IS3 molecules. A, ethidium bromide-strained 0.7%
agarose gels. Panel a shows DNA prepared from the YK1100 cells
harboring pSEK1831 (lanes 1 and 2), pSEK183 (lanes 3 and 4), and pSEK1832 (lanes 5 and 6). The DNA samples in lanes 1, 3, and 5 and
those in lanes 2, 4, and 6 were prepared under the
alkaline and neutral conditions, respectively. Panel b shows
DNA prepared from the YK1100 cells harboring pSEK1831 under neutral
conditions. The DNA samples shown in lanes 1-4 were
those treated with no enzyme, E. coli exonuclease III, phage
exonuclease, and MluI, respectively. Positions of the
monomer closed circular molecules of the parental plasmid and
miniplasmids, which are classified into six types (I-VI) are
indicated. Note that the linear IS3 molecules comigrate with
type V minicircular molecules and that IS3 circles correspond
to type VI molecules. There actually exist type VII molecules composed
of a portion of IS3 (Sekine et al., 1994), but they
are too small and too few to be visualized in the gel. Molecular sizes
of linear DNA molecules are shown in bp on the side of the gel. B, ethidium bromide-strained 3.5% polyacrylamide gels. Panel a shows DNA prepared from the YK1100 cells harboring
pSEK1831 (lanes 1 and 2) and pSEK183 (lanes 3 and 4) under alkaline conditions (lanes 1 and 3) or under neutral conditions (lanes 2 and 4). Panel b shows DNA prepared from the YK1100 cells
harboring pSEK1831 under neutral conditions. The DNA samples shown in lanes 1-4 were treated with no enzymes, E. coli exonuclease III, MluI, and both E. coli exonuclease III and MluI, respectively. Open
arrowheads indicate the fragments generated upon MluI
digestion of the linear IS3 molecules. Molecular sizes of
linear DNA fragments are shown in bp on the side of the
gel.
The linear molecules are approximately 1.3 kb in length, as estimated from their electrophoretic mobility. This size is almost the same as that of the IS3 sequence (1,258 bp). When the DNA sample prepared under the neutral condition was digested with MluI, which cleaves the IS3 sequence at one site, there appeared two bands of DNA fragments, about 0.8 and 0.5 kb in length (see the bands indicated by open arrowheads in Fig. 1Bb, lane 3). Such DNA fragments were not contained in the DNA sample treated with exonuclease III, followed by digestion with MluI (see Fig. 1Bb, lane 4). The sizes of the two fragments are the expected ones of the MluI-digested IS3 sequence, which are 0.78 and 0.48 kb in length, suggesting strongly that the molecules identified above are the linear IS3 molecules. Note here that MluI digestion resulted in a shift of the position of the band corresponding to IS3 circles (type VI molecules) to the position where the linear IS3 molecules were originally present, due to conversion from the circular form to the linear form of the IS3 circles (Fig. 1Ab, lane 4; Fig. 1Bb, lanes 3 and 4).
The linear IS3 molecules as well as small circular molecules including IS3 circles were not detected in the DNA sample prepared under either the alkaline or neutral condition from the cleared lysate of cells harboring pSEK183 carrying wild type IS3 (Fig. 1Aa, lanes 3 and 4; Fig. 1Ba, lanes 3 and 4), demonstrating that all these molecules are generated by the action of the IS3 transposase.
Figure 2:
Identification of the 5`-ends of the
linear IS3 molecules. A, strategy used for primer
extension experiments. The IS3 sequence and the overhanging
sequences at the 5`-ends of IS3 are indicated by shaded and open thick lines, respectively. Filled boxes indicate L and R primers (see Table 1), each labeled with P at the 5`-end. Striped arrows indicate the
direction of synthesis of DNA extended from each primer. B,
polyacrylamide gels (8%) showing the primer extension products. Lane P in panel a or b shows the products
extended from primer L or primer R, respectively. Lanes marked M are the sequence ladders used as size markers, which were
prepared using pSEK183 as template and the primer used in each primer
extension experiment. Nucleotide sequences of critical regions around
IRL and IRR in pSEK183 are indicated together with coordinates to
IS3(8) (Timmerman and Tu, 1985) on the side of the
gels.
Figure 3:
Identification of the 3`-ends of the
linear IS3 molecules. A, strategy used for
identification of the 3`-ends. The IS3 sequence and the
overhanging sequences at the 5`-ends of IS3 are indicated by shaded and open thick lines, respectively. Filled
boxes indicate the BsmI and NcoI primers (see Table 1) used to prepare sequence ladders using pSEK183 as
template. Striped arrows indicate the direction of synthesis
of DNA extended from each primer. Asterisks indicate the
5`-ends labeled with P. Sizes of the single-stranded DNA
fragments are indicated in nt. B, polyacrylamide gels showing
single-stranded DNA fragments generated upon denaturation of the linear
IS3 molecule. Panel a, an 8% polyacrylamide gel
showing DNA fragments generated upon BsmI digestion. Panel
b, an 8% polyacrylamide gel showing DNA fragments generated upon NcoI digestion. Lane 1, the DNA sample obtained by
restriction enzyme digestion followed by labeling with
P
as depicted in strategy I in A. Lane 2, the
DNA sample obtained by labeling with
P followed by
restriction enzyme digestion as depicted in strategy II in A. Lanes marked M are the sequence ladders
used as size markers, which were prepared using BsmI primer or NcoI primer. Sizes of the single-stranded DNA fragments are
indicated in nt. The sizes of the DNA fragments with the 3-nt sequence
attached to the 5`-end of IR are in parentheses, since they
are calculated based on the results shown in Fig. 2. Nucleotide
sequences of critical regions around IRL and IRR in pSEK183 are
indicated together with coordinates to IS3 on the side of the
gels.
To determine the 3`-ends at IRR of the linear IS3 molecules, the fragments containing IRR were isolated after
digestion of the molecules with NcoI, which cleaves IS3 at one site, and labeled with P at their 5`-ends (see Fig. 3A, strategy I). After denaturation of
the sample and electrophoresis of the fragments in a sequencing gel, we
detected two bands of single-stranded DNA fragments (Fig. 3Bb, lane 1). The large DNA fragment is
supposed to correspond to the fragment, whose 3`-end is the
IRR-proximal end of the linear IS3 molecules and whose 5`-end
is labeled with
P at the NcoI site. The small
fragment is supposed to be the fragment with a 3-nt sequence attached
to the 5`-end of IRR (Fig. 3A, strategy I). To
confirm this, the linear IS3 molecules were labeled with
P at their 5`-ends and then digested with NcoI (Fig. 3A, strategy II), and the DNA sample was
denatured and electrophoresed in a sequencing gel. The small fragment
was generated, but the large one was not (Fig. 3Bb, lane 2), confirming the assumption above. The large fragment
was determined precisely by size markers to be 171 nt long (Fig. 3Bb), indicating that the 3`-end at IRR of the
linear IS3 molecules is the 3`-end of the IS3 sequence. This and the result obtained above show that the linear
molecules have 3-nt overhangs at the 5`-ends of the IS3 sequence. Plasmid pSEK1831 used here carries IS3, which
is flanked by the same sequences, 5`-AGC-3`/3`-TCG-5`, that are the
target sequence duplicated upon IS3 insertion (Sekine et
al., 1994). It is possible that the 3-nt overhangs are the target
sequence.
To know whether the linear molecules observed
are of IS3 with 3-nt overhangs at the 5`-ends of IRR and IRL,
which are supposed to be 5`-TCC-3` and 5`-AGC-3`, respectively, we
carried out sequencing analysis as follows. To determine the nucleotide
sequence of the overhangs attached to the 5`-end of IRL, the linear
IS3 molecules were first incubated with a modified T7 DNA
polymerase (Sequenase), which lacks the 3` 5`-exonuclease
activity, in the presence of [
-
P]dNTP. The
DNA sample obtained was digested with BsmI, heat-denatured,
and electrophoresed in a sequencing gel. As shown in Fig. 4A, a single-stranded DNA fragment, 106 nt in
length, with an extension of 1 nt from the 3`-end of IRL was generated
in the presence of [
-
P]dGTP but was not in
the presence of either [
-
P]dATP,
[
-
P]dTTP, or
[
-
P]dCTP. This shows that only dGTP was
incorporated and thus that the nucleotide next to the 5`-end of IRL in
the 3-nt overhang is dC. Next, when the linear IS3 molecules
were incubated in the presence of [
-
P]dGTP
plus dATP or dTTP, the BsmI digestion of the sample did not
generate any fragments larger than the fragment (106 nt) that was
generated in the presence of [
-
P]dGTP alone (Fig. 4A). However, in this experiment, when dCTP was
added instead of dATP or dTTP, BsmI digestion generated the
107-nt fragment, 1 nt larger than the 106-nt fragment (Fig. 4A). This shows that dCTP is incorporated after
dGTP and thus that the nucleotide at the middle position in the 3-nt
overhang is dG. Finally, when the linear IS3 molecules were
incubated in the presence of dTTP in addition to
[
-
P]dGTP and dCTP, BsmI digestion
of the sample generated the 108-nt fragment, 1 nt larger than the
product (107 nt) that was generated in the presence of
[
-
P]dGTP and dCTP (Fig. 4A). However, in this experiment, when dATP was
added instead of dTTP, BsmI digestion did not generate such
extension products (Fig. 4A). This shows that dTTP was
incorporated after dGTP and dCTP and thus that the nucleotide at the
5`-end position in the 3-nt overhang is dA. All of these results show
that the nucleotide sequence of the overhang attached to the 5`-end of
IRL is 5`-AGC-3`. Note that this sequence is identical to the sequence
flanking IRL of IS3 in the parental plasmid pSEK1832.
Figure 4: Determination of the nucleotide sequence of the overhangs at the 5`-ends of the linear IS3 molecules. A, polyacrylamide gels (8%) showing the BsmI fragment having incorporated dNTP(s) into the 3`-ends at IRL of the linear IS3 molecules. B, polyacrylamide gels (8%) showing the NcoI fragment having incorporated dNTP(s) into the 3`-ends at IRR of the linear IS3 molecules. The DNA sample in each lane was obtained by incubation of modified T7 DNA polymerase (Sequenase) in the presence of dNTP(s) indicated. Sizes of the single-stranded DNA fragment in nt are indicated. Lanes marked M are the sequence ladders used as size markers, which were prepared using pSEK1832 as template and the BsmI primer (panel A) or NcoI primer (panel B). Nucleotide sequences of critical regions around IRL and IRR in pSEK1832 are indicated on the side of the gels together with coordinates to IS3.
To
determine the nucleotide sequence of the 3-nt overhang at the 5`-end of
IRR, the linear IS3 molecules isolated were incubated with
Sequenase in the presence of [-
P]dNTP and
digested with NcoI, which cleaves IS3 at one site. In
the presence of [
-
P]dGTP, NcoI
digestion generated an extension product, 173 nt in length, 2 nt longer
than that with the exact 3`-end of IRR (Fig. 4B). In
the presence of [
-
P]dCTP or
[
-
P]dTTP, NcoI digestion did not
generate such extension products, but in the presence of
[
-
P]dATP, NcoI digestion gave rise
to two faint bands of the fragments, 172 and 173 nt in length (Fig. 4B). These results show that the nucleotide(s)
next to the 5`-end of IRR in the linear IS3 molecules is dCC,
dTT, or dT and that the molecules with the overhanging sequence dCC are
major. When the linear IS3 fragments were incubated in the
presence of [
-
P]dGTP plus dATP, an
extension product, 174 nt in length, 1 bp longer than that generated in
the presence of [
-
P]dGTP alone, and in
addition, another extension product, 173 nt in length, forming a faint
band, were generated (Fig. 4B). In this experiment,
when dTTP or dCTP was added instead of dATP, the extension product of
173 nt long was generated, but the extension product of 174 nt long was
not (Fig. 4B). However, when the three nucleotides,
dCTP, dTTP, and dATP, were added in the presence of
[
-
P]dGTP, the extension product, 174 nt in
length, was generated (Fig. 4B). These results show
that the nucleotide sequence of the overhang at the 5`-end of IRR in
the major linear IS3 molecules is 5`-TCC-3` and that in the
minor ones is either 5`-VTT-3` (where V is A or G or C) or 5`-RCT-3`
(where R is A or G), etc. Note that the overhanging sequence 5`-TCC-3`
is identical to the sequence adjacent to IRR in the parental plasmid
pSEK1832. We will discuss later the reason the linear fragments with
overhanging sequences other than 5`-TCC-3` are generated.
We have shown in this paper that the efficient production of IS3 transposase results in generation of the linear IS3 molecules having 5`-ends with overhanging sequences of 3 nt. Nucleotide sequences of the 3-nt overhangs of the major linear IS3 molecules generated from pSEK1832 are identical to those flanking IS3 in the parental plasmid. This result indicates that the linear molecules are excised from the parental plasmid by staggered breaks at both end regions of IS3 by the action of the transposase. In transposons Tn10 and Tn7, which transpose in a non-replicative manner, double strand breaks occur at both end regions of the elements to excise the linear transposon fragments, which are subsequently inserted into a target site (Morisato and Kleckner, 1984; Benjamin and Kleckner, 1989; Bainton et al., 1991). Identification and characterization of the linear IS3 molecules further support the previous notion that IS3 transposes in a non-replicative manner (Sekine et al., 1994).
As described under ``Results,'' some of the linear IS3 molecules were found to have the 3-nt overhanging sequences different from those flanking IS3 in the parental plasmid. We have observed here and previously (Sekine et al., 1994) that IS3 mediates deletion frequently in the region adjacent to IRR of IS3 to produce miniplasmids, which now have IS3 flanked by different sequences, and that these miniplasmids still generate many kinds of smaller miniplasmids. It is therefore quite likely that the linear IS3 molecules with different overhanging sequences were generated by excision from the smaller miniplasmids.
It should be noted that unlike Tn10 and Tn7, IS3 generates IS3 circles consisting of the entire IS3 sequence and a 3-bp sequence intervening between the IS3 ends (Sekine et al., 1994) (Fig. 5). Most of the IS3 circles contain the intervening 3-bp sequence, which is identical to either one of the sequences flanking IS3 in the parental plasmid, but others contain the 3-bp sequence different from the original sequences flanking IS3. The latter IS3 circles are supposed to be produced from miniplasmids with a flanking sequence different from that in the parental plasmid. It is likely that these IS3 circles are derived by circularization of the linear IS3 molecules (Fig. 5), such that only one 3`-end of IS3 is joined to the 5`-end of the 3-nt overhanging sequence on the other side, and the resulting 3-nt gap on the opposite strand is subsequently converted to a homoduplex form through DNA repair. Alternatively, both 3`-ends of IS3 are joined with the 5`-ends of the 3-nt overhanging sequence on the other side to give circles with a 3-bp heteroduplex sequence intervening between IRL and IRR. (If the 3-bp sequences flanking IS3 are identical, the circles should have a homoduplex sequence intervening between IRs.) The IS3 circles with the sequence of one or the other strand could have been obtained by cloning. It is not clear at present whether the IS3 circles participate as substrates in transposition or not. Polard et al.(1992) have reported that an IS3 family element, IS911, also generates IS circles similar to the IS3 circles and that the IS911 circles are, however, not the obligatory transposition intermediates.
Figure 5: Proposed models for IS3 transposition and retrovirus integration. See details described in the text. LTR, long terminal repeat. An, a polyadenylate tail.
As described above, IS3 generates both circular and linear molecules, while Tn10 and Tn7 generate linear molecules but not circles. Retroviruses are, however, known to generate circular DNAs with two long terminal repeats in addition to double-stranded linear DNA molecules after reverse transcription from the viral RNA genome (Varmus and Brown, 1989) (Fig. 5). In this respect, IS3 resembles retroviruses. The linear molecules of retroviruses are considered to be the intermediates for their integration, in which 2 nt from each 3`-end of the linear viral DNA are removed by integrase to produce 5`-protruding ends (Craigie et al., 1990; Katz et al., 1990; Katzman et al., 1989; Sherman and Fyfe, 1990), and the 3`-ends of the linear molecules are subsequently joined to the 5`-ends generated at a target site (Fujiwara and Mizuuchi, 1988; Brown et al., 1989) (Fig. 5). Considering the conservation of the amino acid sequence motif in transposases of IS3 family elements and retroviral integrases, we assume that the linear IS3 molecules are the transposition intermediates and are inserted into a target site by a similar mechanism to that in the retroviral system. Probably, the 3`-OH of the linear IS3 molecule is joined to 5`-P of the target DNA, which is supposed to be generated by 3-bp staggered breaks (Fig. 5), since IS3 has been shown to give a 3-bp target duplication at its point of insertion (Sommer et al., 1979; Timmerman and Tu, 1985; Yoshioka et al., 1987; Spielmann-Ryser et al., 1991). The 3-nt gap on the opposite strand is subsequently repaired to convert the gap to a duplex form and to remove the 3-nt donor sequence attached to the 5`-end of the linear IS3 molecule (Fig. 5).
The
linear IS3 molecules are accumulated to a level that can be
readily detected in the DNA preparation from a small overnight culture
by gel electrophoresis and staining the gel with ethidium bromide. This
may imply that the ends of the linear IS3 molecules are
protected from the attack of cellular nucleases. In Tn10,
there exists such a protein-DNA complex, which is an active form of
transposition intermediate (Haniford et al., 1991). It is
likely that a gapped donor molecule, i.e. a donor backbone, is
produced when IS3 has been excised. We have, in fact, detected
such donor backbone molecules in the DNA sample in a smaller amount
than that of the linear IS3 molecules. ()This
suggests that the donor backbone molecules released do not form a
protein-DNA complex and are thus subjected to the attack of nucleases
unlike the linear IS3 molecules.