 |
INTRODUCTION |
Numerous site-specific DNA recombination systems and DNA
transposition systems have been characterized biochemically and have been found to follow two distinct chemical pathways for DNA cleavage and strand transfer in recombination (reviewed in Refs. 1-4). Site-specific recombination, mediated by the recombinases of the
-integrase and Hin/resolvase families, involves a two-step
transesterification reaction in which the intermediate is a covalent
recombinase-DNA linkage. This covalent attachment is the result of
nucleophilic attack on the DNA phosphodiester backbone by a hydroxyl
group of the conserved serine (Hin/resolvase), or tyrosine
(
-integrase), of the recombinase. In the second transesterification
reaction, the phosphodiester linkages of the exchanged DNA strands are
restored (reviewed in Refs. 2 and 3). In contrast, DNA transposition, mediated by transposases containing the catalytic DDE amino acid motif,
utilizes a hydrolysis reaction for cleavage at the ends of the
transposable element. This first cleavage leaves 3'-OH ends to act
directly as the attacking nucleophile in a one-step trans-esterification reaction resulting in strand exchange. Resolution of the transposition process involves DNA replication or DNA repair activity to fill in gaps left at the target site due to the staggered cut mediated by the transposase and the 3' hydroxyl groups at the
element ends (reviewed in Refs. 1 and 4).
These features of the recombination reactions mediated by site-specific
recombinases and transposases suggest that a group of related
recombinases would not mediate both site-specific recombination and
transposition. Therefore, it is surprising that the site-specific recombinase Piv, which directs site-specific DNA inversion in Moraxella lacunata and Moraxella bovis, exhibits
significant homology to the transposases of the
IS110/IS492 family of IS elements (approximately 25-35% amino acid identity and 45-55% similarity, 5). Furthermore, Piv and the IS110/IS492 transposases do not
appear to be related to the site-specific recombinases of the
-Int
or Hin/resolvase families or the transposases containing the DDE motif
(4). Therefore, Piv and the IS110/IS492
transposases may define a new family of DNA recombinases.
The homology between Piv and the IS110/IS492
transposases includes several highly conserved amino acid regions (5),
which, based on mutational analyses, contain functionally relevant
amino acid motifs.1 Although
there is no completely conserved DNA sequence among all the IS elements
and the Piv invertible DNA segment, there is a consensus sequence for
the ends of a subgroup of the IS elements, which is also found
overlapping the recombination sites of the Piv invertible
element.1 To determine if indeed Piv and the
IS110/IS492 transposases define a new family of
DNA recombinases that utilize a common mechanism for both site-specific
recombination and transposition, we must characterize the recombination
reactions mediated by Piv and transposases from the
IS110/IS492 family. As a first step in
understanding the mechanism for Piv-mediated assembly of a synaptic
nucleoprotein complex, DNA cleavage, and strand exchange, we have
characterized the interactions of Piv with the invertible DNA segment
of Moraxella lacunata.
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MATERIALS AND METHODS |
Reagents--
DNase I was obtained from Worthington Biochemical
Corp. Amylose resin, restriction enzymes, T4 DNA ligase, Klenow
(exo
), and T4 polynucleotide kinase were purchased from New England
Biolabs (NEB). Pfu DNA polymerase was obtained from
Stratagene. The His tag XPRESS purification system and the TA cloning
kit were purchased from Invitrogen. Radionucleotides were obtained from
NEN Life Science Products.
Isopropyl-1-
-D-galactopyranoside
(IPTG),2 dithiothreitol, and
EDTA were purchased from Sigma. Dimethyl sulfate was purchased from Aldrich.
Plasmids--
The pAG607 plasmid is composed of the pMal-C2
vector (NEB) containing the piv gene inserted into the
XmnI and BamHI sites such that it is in frame
directly downstream of a factor Xa protease site in the
malE gene. The piv gene was obtained by the
polymerase chain reaction (PCR) from the pMxL1 plasmid, subclone of
M. lacunata inversion region provided by C. Marrs (6), using
Pfu DNA polymerase and the primers:
5'-GCCAGCACGTGTCTAAAACTTACATTG-3' and 5'-CCTAAGCTTCTAGGATACCAATAAAT-3'. Similarly, the piv gene from pMxL1 was PCR-amplified with
the primers: 5'-CTCGTCTCGAGTTCATGAATGCGTTTGTCAAAAG-3' and
5'-CAGTTCACATATGTCTAAAACTTACATTGGGATT-3', cleaved with
XhoI and NdeI, and inserted into the same
restriction sites in pET21a (Novagen). This placed piv in
frame with a COOH-terminal His6 tag sequence, creating the
Piv-His6 expression vector, pAG1300.
A 245-bp fragment was generated from the pMxL1-dl24 plasmid (6) using
Pfu DNA polymerase and the primers
5'-CATAGGATCCAAAATTACCTGCCAGACATC-3' and
5'-CCGGAATTCGCTAACCTTACACTCATAC-3'. This 245-bp fragment, containing
the strong upstream binding site (sub1), was cloned into the
pCR2.1 vector (TA cloning kit, Invitrogen). The resulting plasmids,
pAG604 and pAG605, contain sub1 in opposite orientations. The inserted DNA was sequenced (T7 Sequenase version 2.0 DNA sequencing kit; Amersham Pharmacia Biotech) to check for any mutations due to the
PCR reaction.
To construct the DNA inversion test plasmid pAG862, a 5865-bp DNA
fragment containing the invertible segment was obtained by partial
digestion of pMxL1 with EcoRI. This fragment was
gel-purified and ligated into EcoRI-digested pACYC184 (NEB)
creating the pAG850 plasmid. To inactivate piv, the
fragment encoding the SpcR/SmR genes was
recombined into the piv gene of pAG850 by homologous recombination with piv::
on the pMxL5 plasmid (6) in the
Rec+ strain JM101 (NEB). The recombinant plasmid, pAG862,
was confirmed by restriction digestion and DNA sequencing.
Protein Purification--
To produce the Piv protein fused at
its amino terminus to maltose-binding protein (MBP-Piv), DH5
containing pAG607 was grown in Luria broth to OD600 = 0.5, induced with 0.5 mM IPTG, aerated at 37 °C for 2 h,
and lysed in a French pressure cell. Crude extract was immediately
loaded onto an amylose column, washed with column buffer (20 mM Tris-Cl, pH 7.4, 500 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol),
and then eluted with 10 mM maltose in column buffer (7, 8).
Fractions were collected and frozen in 20% or 30% glycerol at
20 °C. Protein concentration was quantitated using the Bio-Rad DC
protein assay kit.
To produce the His6-tagged Piv, pAG1300 was introduced into
BL21(DE3) (Novagen), grown to mid-log phase in Luria broth, then induced with 1 mM IPTG. After 1 h aeration at
37 °C, cells were collected by centrifugation, resuspended in 20 mM phosphate, 500 mM NaCl, pH 7.8, and lysed in
a French pressure cell at 4 °C. The lysate was centrifuged at
30,000 × g. The supernatant was loaded onto an
Invitrogen ProBond column (Ni2+ affinity column). Following
washes with 500 mM NaCl, 20 mM phosphate, pH
7.8 buffer, protein was eluted with an imidazole step gradient (50, 200, 350, and 500 mM). Piv-His6 eluted at 300 mM imidazole. The protein was dialyzed into 20 mM phosphate, 500 mM NaCl, pH 7.8, and 30%
glycerol at 4 °C and stored at
20 °C.
Antibody Production and Purification--
Antibody to the Piv
protein was produced in rabbits injected with purified MBP-Piv protein
(BAbCO; Berkeley Antibody Company). Nonspecific antibodies were removed
from the rabbit sera by absorption to whole cells of DH5
cultures,
followed by multiple passages over a Sepharose column bound with
Escherichia coli proteins and MBP. This affinity column was
made by lysing a culture of pMal-C2/DH5
that had been induced with
0.5 mM IPTG to express the MBP protein, then binding the
proteins from this lysate to cyanogen bromide-activated Sepharose resin
using the protocol of the resin manufacturer (Amersham Pharmacia
Biotech). The eluate was stored at 4 °C.
Western Blot and SDS-PAGE Analysis--
SDS-polyacrylamide gel
electrophoresis was performed using the protocol of Sambrook et
al. (9). Western blot analysis was accomplished following
the method of Ausubel et al. (10) with the following
modifications; polyvinylidene difluoride membrane (Bio-Rad) was used
for blotting, and the blot was probed with a 1:400 dilution of
partially purified Piv antibody. The protein markers were SDS-PAGE
standards, low range, or Kaleidoscope prestained standards (for Western
blots) from Bio-Rad.
Inversion Assay--
DH5
containing pAG862, pAG862 and pAG607
or containing pAG862 and pMal-C2 were grown to OD600 = 0.6, induced with 0.05 mM IPTG, and incubated for 2 h at
37 °C. Plasmids were extracted using an alkaline lysis protocol (9)
and digested with HindIII and KpnI. The
restriction digests were examined on a 0.8% SeaKem (FMC Bioproducts)
agarose gel. The DNA was visualized by ethidium bromide staining and
documented by digital imaging of the UV-illuminated gel.
Labeling of DNA Fragments Used in DNA Binding
Assays--
Double-stranded DNA oligonucleotides, INV and SUB (see
"Results"), were labeled with [
-32P]ATP using T4
polynucleotide kinase following the protocols of Sambrook et
al. (9). pAG604 and pAG605 were digested with SpeI and
EcoRV to obtain 298-bp fragments containing the
sub1 site to be used in DNase I protection assays. These
fragments were labeled at the SpeI site with
[
-32P]ATP using Klenow (exo
) DNA polymerase under
conditions recommended by the manufacturer with the modification of
incubating the reactions at 25 °C; the labeled fragments were
electrophoresed on a 5% polyacrylamide gel, electroeluted from the
gel, and stored at
20 °C. The pMxL1-dL24 plasmid, a deletion
derivative of pMxL1, provided by C. Marrs (6), and the pMxL1
invR fragment (obtained by PCR from pMxL1, using
Pfu DNA polymerase and the primers:
5'-GCCAGCACGTGTCTAAAACTTACATTG and 5'-CCATACACCATCAGCAGCACG) were
digested with various restriction enzymes as indicated under
"Results." The DNA fragments created were labeled with
[
-32P]dATP or dCTP using Klenow (exo
) polymerase,
gel-purified, and stored at
20 °C.
Gel Electrophoresis Retardation Assays--
Gel electrophoresis
retardation, or gel shift, assays were initiated by mixing MBP-Piv
protein with each radiolabeled DNA fragment (5 × 10
12 M) in 1× binding buffer: 80 mM KCl, 20 mM Tris-Cl, pH 7.6, 5 mM
CaCl2, 250 mg/ml poly(C), 1 mM dithiothreitol,
and 50 mg/ml bovine serum albumin (NEB). The reactions were incubated
for 20 min at room temperature, immediately loaded onto a nondenaturing polyacrylamide gel, and run at 4 °C in 0.5× TBE buffer. For the competition assays, increasing amounts of competitor DNA ranging from 1 to 500 molar excess was added to the reaction before addition of the
protein. The specific DNA competitors were the SUB and INV
double-stranded oligonucleotides and the nonspecific DNA competitor was a 39-bp oligonucleotide:
5'-TTAAGATCGATGACGTCAGATCTGAGCTCGATACTCGAG annealed to
5'-CTCGAGTATCGAGCTCAGATCTGACGTCATCGATC.
Nuclease and Chemical Cleavage Assays--
For the DNase I
protection assays performed at the sub1 site, the 298-bp
fragments from pAG604 and pAG605, labeled on the top strand and bottom
strand for sub1 (5 × 10
10
M), were incubated for 20 min at room temperature with or
without MBP-Piv (7 × 10
6 M) in 1×
binding buffer. DNase I protection assays were performed in binding
buffer plus 5 mM MgCl2 with 0.02 units of DNase
I as described previously (11). Chemical cleavage using the Maxam and
Gilbert sequencing reactions A/G and C/T were performed with the same
labeled fragments (12). The footprinting and sequencing reactions were
run on a 6% sequencing polyacrylamide gel.
At the site of inversion, dimethyl sulfate interference assays were
performed as described previously (11). Indirect DNase I and
1,10-phenanthroline-copper protection assays were also performed at the
site of inversion following the protocol described by Kuwabara and
Sigman (13) with the following modifications for the DNase I protection
assay: gel slices were treated with DNase I in 1× binding buffer plus
5 mM MgCl2 for 5-8 min before quenching the reaction with the addition of 20 mM EDTA.
Cooperativity Studies--
Gel electrophoresis retardation
assays were performed with labeled DNA fragments (5 × 10
12 M) and increasing amounts of MBP-Piv
protein ranging from 0.6 to 1582 nM for binding to SUB and
0.6 to 4560 nM for binding to INV. Binding of MBP-Piv (P)
to each DNA binding site (X) is represented by the equation:
K = [P]n[X]/[PnX],
which translates into:
ln([PnX]/[X]) = n
ln[P]
ln[K]. [P] is the concentration of MBP-Piv monomers, [Pn] is the concentration of active MBP-Piv
oligomers, n is the number of active monomers in the
oligomer, [PnX] is the concentration of bound
DNA, and [X] is the concentration of unbound DNA. A plot
of ln[P] versus
ln([PnX]/[X]) gives a line with
slope n (14). The amount of bound and unbound DNA was
quantitated using a PhosphorImager: 445SI with ImageQuant software (Molecular Dynamics).
Dissociation Rate Analysis--
Binding assays were performed in
1× binding buffer. Labeled, double-stranded oligonucleotide (5 × 10
12 M) was incubated with MBP-Piv protein
(6 × 10
7 M) as indicated for 20 min at
room temperature. Unlabeled, competitor oligonucleotide (2.5 × 10
8 M) was added at time zero. At the
indicated times, 20-µl aliquots were loaded onto a 10% nondenaturing
polyacrylamide gel running at 120 V. Following electrophoresis, the
gels were dried and imaged with a PhosphorImager: 445SI. ImageQuant
software was used to quantitate the label in the bands corresponding to
MBP-Piv-DNA complexes and unbound DNA. The fraction of the total DNA in
the reaction that was bound by MBP-Piv was calculated by dividing the
amount of labeled DNA present in the shifted complexes by the total
labeled DNA in the reaction (i.e. the combined complexed and
unbound DNA fragments quantitated for each reaction). The percent bound
DNA was plotted as a function of the time after addition of competitor
DNA. The half-life for each complex was the time required for a 50%
reduction in protein-DNA complex due to dissociation of the MBP-Piv DNA complex.
 |
RESULTS |
Purification of Piv Protein for in Vitro DNA Binding
Assays--
Based on the sequence of the piv gene, the Piv
protein is predicted to be a 322-amino acid polypeptide with a
Mr of 36,935 and an isoelectric point of 10.72. To facilitate purification of Piv, M. lacunata piv was
introduced into the expression vector pMal-C2, fusing malE
to the 5' end of piv (pAG607) to produce the fusion protein,
MBP-Piv. MBP-Piv (79 kDa) was soluble and expressed at high levels.
Affinity chromatography (amylose resin) was used to purify the MBP-Piv
fusion protein for use in DNA binding assays. Fig.
1A shows a Coomassie-stained
SDS-PAGE gel containing samples from a crude lysate used for
purification of MBP-Piv and fractions collected from the 10 mM maltose elution of protein bound to the amylose column.
ImageQuant analysis of a digital image of a Coomassie-stained,
SDS-polyacrylamide gel containing serial dilutions of the purified
protein indicated that the fractions used in all the following
experiments are approximately 85% full-length MBP-Piv protein (data
not shown).

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Fig. 1.
Expression of MBP-Piv. A,
SDS-polyacrylamide gel, stained with Coomassie Blue, showing 20 µl of
crude lysate from DH5 containing pAG607 after induction for 2 h
with 0.5 mM IPTG and 20 µl from 3 ml fractions (fraction
numbers 3-10) collected during elution with 10 mM maltose
(in column buffer) of the bound proteins on an amylose column loaded
with 50 ml of the crude lysate. B, Western blot analysis of
the proteins expressed from cultures, uninduced (U) or
induced (I) with 0.5 mM IPTG, containing pMal-C2
(MBP) or pAG607 (MBP-Piv), and loaded onto a 10% SDS-polyacrylamide
gel along with affinity purified MBP-Piv (4 µg of purified protein).
The bands corresponding to MBP-Piv and the location where MBP is seen
on the corresponding SDS-PAGE gel are indicated with arrows.
"Marker" designates the protein molecular weight
standards (Bio-Rad), and the molecular sizes are indicated (kDa). In
both the Coomassie-stained gel (A) and the Western blot
(B), there appears to be some degradation of MBP-Piv; the
purity of the protein preparations were based only on full-length
fusion protein.
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Fig. 1B shows Western blot analysis of expression of the
MBP-Piv fusion protein from pAG607 using polyclonal antisera raised against MBP-Piv and affinity-purified to obtain anti-Piv antisera as
described under "Experimental Procedures." MBP-Piv could be detected in the soluble fraction of a crude lysate prepared from DH5
cells containing pAG607 after induction with 0.5 mM IPTG (Fig. 1B, lanes 3 and 4).
The affinity-purified antibodies are specific for Piv as no
cross-reactivity with MBP alone was observed (Fig. 1B,
lane 2).
Cleavage of MBP-Piv with the protease factor Xa, which recognizes a
specific cleavage site between MBP and Piv in the fusion protein,
resulted in cleavage at the fusion junction sequence and,
unfortunately, also within Piv itself. Because this additional cleavage
site prevented separation of the full-length Piv protein from MBP by
Factor Xa treatment, we determined whether the MBP-Piv fusion protein
could carry out the functions of wild type Piv.
An in vivo inversion assay was used to qualitatively
estimate recombinase activity as an indication of functional
protein-DNA interactions (Fig. 2). DH5
was cotransformed with a DNA inversion test plasmid, pAG862, and
expression plasmids encoding either MBP alone (pMal-C2), or MBP-Piv
(pAG607). After overnight expression of MBP or MBP-Piv, recombination
of the invertible segment on pAG862 was assayed by restriction enzyme
digestion of isolated plasmid DNA. In the original orientation of the
test plasmid, digestion with HindIII and KpnI
results in two bands, of 5.7 and 1.0 kb. If there is MBP-Piv-mediated
inversion, the same digestion will yield two different unique bands, of
4.1 and 2.6 kb. With MBP alone, no inversion products are seen,
whereas, in the presence of MBP-Piv, approximately 50% of the test
plasmid is in the inverted orientation (Fig. 2). In a similar inversion
assay, wild type Piv also directed inversion to yield approximately
50% of either orientation, representing an equilibrium point in the
inversion reaction (data not shown). Thus, these results indicated that the MBP-Piv fusion is a functional recombinase; consequently, the
uncleaved MBP-Piv fusion protein was used in subsequent in vitro DNA binding studies.

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Fig. 2.
In vivo inversion activity of the
MBP-Piv fusion protein. An inversion assay was performed as
described under "Materials and Methods" using the inversion
substrate plasmid, pAG862, which is phase-locked in the tfpI
expressing orientation. The pMal-C2 and pAG607 plasmids, which express
MBP and MBP-Piv, respectively, were used to test the ability of MBP-Piv
to invert the Moraxella DNA segment (in
parentheses) in E. coli. Restriction digestion
with HindIII (H) and KpnI
(K) yields distinct DNA fragments indicating that inversion
has occurred, as represented in the diagram beside the EtBr-stained
agarose gel of these inversion products. These DNA fragments are
designated I and Q, denoting the orientation of the pilin genes,
tfpQ and tfpI, relative to their promoter in the
inversion substrate. The I bands are marked with *, and the Q bands are
indicated with arrows. The plasmids are not drawn to
scale.
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Piv Binding Sites within the Inversion Region of M. lacunata--
Based on the binding specificity of other site-specific
recombinases and transposases (reviewed in Refs. 1 and 2), we expected
that Piv would bind DNA sequences encoding the cross-over sites for
inversion (invL and invR) as well as possible
accessory sites involved in recombination or regulation of
piv expression. An assay for Piv DNA binding sites within
the inversion region was carried out using gel electrophoresis mobility
shift assays (EMSA; data not shown) with a series of restriction
fragments that spanned both the invertible segment and the adjacent
sequences required for inversion in E. coli (Fig.
3A). Several of the fragments were shifted in the presence of MBP-Piv (* in Fig. 3A). Two
fragments, 430 and 870 bp, were chosen for further analysis by DNase I
footprinting. The 430-bp fragment was selected because it spans the
left recombination site of the invertible element (invL).
The 870-bp fragment also contains the invL recombination
site, plus a potential accessory site (see below).

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Fig. 3.
Binding of MBP-Piv to the inversion
region. A, a diagram of the labeled DNA fragments from
the Moraxella inversion region used in gel electrophoretic
mobility shift assays is shown (not to scale). Restriction sites used
to create the DNA fragments are indicated (N is
NcoI, B is BstXI, Sp is
SpeI, R is EcoRV, and St is
StyI). The length of each restriction fragment is given in
base pairs. * marks the DNA fragments that EMSA indicated
were specifically bound by MBP-Piv. The location of the MBP-Piv binding
site is indicated by an X on the 850-bp fragment.
B, a summary of the results from the DNase I protection
assays with the 870-bp fragment (data not shown) and with the 298-bp
fragment shown in C. The symbol # indicates a DNase I
enhancement, and marks protected bases. The numbering of the
base pairs is relative to the first base pair of the invertible segment
(+1). C, DNase I protection assays were performed as
described under "Materials and Methods" on the top and bottom
strands of the 298-bp fragment. + and indicate the presence or
absence of MBP-Piv, respectively. A/G and C/T are
Maxam-Gilbert sequencing reactions. Arrows indicate sites of
enhanced cleavage, while bars represent areas of protection.
The positions of the first and last base of the DNase I protected
regions are indicated to the right of the gels.
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DNase I protection assays with MBP-Piv and the 870-bp fragment showed
protections and enhancements from about
285 to
265 bp upstream of
the invL cross-over site (data not shown). However, due to
the relatively large size of the fragment, the DNase I cleavage pattern
at the site was not easily resolved. Therefore, a smaller fragment of
298 bp, containing the protected DNA sequence, was examined by DNase I
protection assays (Fig. 3, B and C).
The DNase I cleavage pattern in the MBP-Piv binding region has long
tracts of no cleavage due to A-tract repeats that narrow the minor
groove, thus inhibiting DNase I binding and cleavage (15). These
A-tracts are also appropriately phased along the DNA helix to
contribute sequence-directed DNA bending to this region of the DNA
(16.). Protections and enhancements from
233 to
247 and from
266
to
285 (Fig. 3, B and C) could be resolved in
these DNase I protection assays. No other protected regions were
detected on the 298-bp fragment. This MBP-Piv binding site is probably
too distant from the site of DNA cleavage (+1) and strand exchange in
the inversion reaction to be the binding site for the Piv subunit that
actually catalyzes the recombination reaction. However, it is likely
that this Piv binding site is an accessory site utilized in the
assembly of an active synaptic complex for Piv-mediated DNA inversion
(see "Discussion").
The 430-bp fragment (Fig. 3A) contains the 26-bp site of
inversion (invL) that was defined based upon sequence
homology at the ends of the invertible segment (17). The initial EMSA
analysis of MBP-Piv binding to the 430-bp fragment indicated that
MBP-Piv does bind specifically, albeit weakly, to a sequence within
this fragment (data not shown). However, DNase I and
1,10-phenanthroline-copper protection assays with the 430-bp fragment
and MBP-Piv showed no evidence of protein-DNA interactions (data not
shown). Because MBP-Piv does shift the 430-bp fragment in EMSA gels,
indirect DNase I protection assays were performed with the 430-bp
fragment in which the electrophoretically separated protein-DNA complex and unbound DNA are treated with DNase I in the non-denaturing polyacrylamide gel and then isolated from gel slices for
electrophoresis on the DNA sequencing polyacrylamide gel (see
"Materials and Methods"). Even though this assay should have
enriched for DNA complexed with MBP-Piv, no protection of DNA sequence
by MBP-Piv was observed. These results suggested that the binding of
MBP-Piv to the 430-bp fragment was either unstable or not specific.
To determine if the poor interactions of MBP-Piv with invL
are due to the MBP protein at the amino terminus of Piv, Piv was tagged
at the COOH terminus with six histidines (see "Materials and
Methods"). The Piv-His6 fusion protein was partially
purified and used in EMSA and DNase I protection assays with the 430-bp fragment and also with the 298-bp fragment containing the accessory Piv
binding site (data not shown). All of the DNA binding results with
Piv-His6 corresponded exactly with those obtained with
MBP-Piv, indicating that the poor binding of MBP-Piv to invL
is not due to steric hindrance by MBP of Piv-specific binding.
Therefore, stable binding of Piv may require other factors such as
accessory DNA binding sites, accessory proteins, and/or supercoiled DNA substrate to facilitate binding to the recombination sites (see "Discussion").
Piv Binds Two Different Recognition Sequences--
To characterize
the interactions of MBP-Piv with the isolated invL and
upstream accessory site, double-stranded oligonucleotides corresponding
to each site were synthesized (designated INV and SUB, respectively;
Fig. 4A). The sequence of INV
included the 26-bp region of homology from either end of the invertible
element and 14 bp of sequence immediately upstream and downstream from the invL site. The DNA sequence of SUB (or
strong upstream binding site
oligonucleotide) was designed based on the DNase I footprint originally
obtained with the 870-bp DNA fragment (i.e. the protected region
269 to
284, but not the
233 to
245 region). The INV and
SUB DNA binding substrates were used in an EMSA to examine MBP-Piv
binding at each site (Fig. 4B).

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Fig. 4.
Comparison of MBP-Piv binding to the INV and
SUB oligonucleotides. A, the positions of the INV and
SUB oligonucleotides are indicated by bars above the diagram
of the Moraxella inversion region. The oligonucleotide
sequences are listed below this diagram. The possible 26-bp site of
inversion is boxed in the INV sequence. The DNase I
protected bases and enhanced cleavages within the SUB oligonucleotide
are indicated as described in Fig. 3. B, EMSA was performed
with labeled INV and SUB double-stranded oligonucleotides. The binding
reactions were done as described under "Materials and Methods" and
contained 32P-labeled INV and/or SUB and 63 nM
MBP-Piv protein. The complexes formed between MBP-Piv and SUB are
labeled S-1 and S-2. The complexes formed between
MBP-Piv and INV are labeled as I-1 and I-2. The
unbound INV and SUB substrates are labeled U. The
lane numbers are indicated below the gel.
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Two shifted complexes (S-1 and S-2,
lane 2, Fig. 4B) were formed with the
SUB oligonucleotide, indicating that at least two MBP-Piv proteins bind
SUB. Because the SUB oligonucleotide does not include the DNA sequence
from
233 to
247 bp (see Fig. 3), we have designated the MBP-Piv
binding site encoded within the SUB oligonucleotide,
sub1.
MBP-Piv binding to INV resulted in one primary complex (I-1) that
migrated at approximately the same rate as the slower migrating complex
formed with SUB (S-2). Because the difference in the molecular weights
of the INV and SUB oligonucleotides is nominal in comparison to the
MBP-Piv molecular weight, this result suggests that the I-1 and S-2
protein-DNA complexes contain the same number of MBP-Piv protomers. No
faster migrating protein-DNA complex was seen with INV when the
concentration of MBP-Piv was decreased (data not shown). The presence
of a slower complex (I-2) was detected with some variability in the
EMSAs. The formation of this complex suggests that a higher multimeric
MBP-Piv may bind INV or an additional INV oligonucleotide may bind the
I-1 complex.
Based upon the predicted molecular weights of INV, SUB, and MBP-Piv,
the electrophoretic mobilities of I-1, S-1, and S-2 indicate that INV
is bound by at least two MBP-Piv protomers while SUB is bound by either
single (S-1) or multiple (S-2) MBP-Piv protomers. Comparison of the Piv
DNA binding substrates, INV and SUB, revealed very little homology
(TATNC), suggesting that Piv has two different DNA recognition sites.
Specificity of MBP-Piv Binding to INV and SUB--
To further
examine the interactions of MBP-Piv with the apparently different
binding sites on INV and SUB, competition assays were performed using
each of the oligonucleotides as both binding substrate and competitor
DNA. In Fig. 5A, the SUB
oligonucleotide was labeled and used in DNA binding assays with MBP-Piv
and the unlabeled competitor DNAs: SUB, INV, and the nonspecific
oligonucleotide (see "Materials and Methods"). Although SUB
competed for both S-1 and S-2 complex formation at 100- and 500-fold
excess unlabeled oligonucleotide, INV competed effectively for only S-2
complex formation at the same molar excess of competitor DNA. This
result is consistent with MBP-Piv interacting with invL only
in a multimeric form (Fig. 4). The nonspecific DNA competitor also
competed (less effectively) with SUB for MBP-Piv binding to give S-2
complex. This competition for S-2 complex formation by the nonspecific competitor may indicate that multimeric MBP-Piv protein has a faster
off rate and so it is titrated out of the reaction by nonspecific protein-DNA interactions at 100-500-fold excess nonspecific competitor DNA. Fig. 5B shows that only the INV oligonucleotide
effectively competed for MBP-Piv binding to labeled INV; SUB did not
show significant competition even at 500-fold molar excess. These
results again suggest a different interaction between MBP-Piv and
invL as compared with binding of MBP-Piv to
sub1.

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Fig. 5.
Specificity of MBP-Piv binding to the INV and
SUB oligonucleotides. Competition DNA binding assays were
performed as described under "Materials and Methods" for both
labeled INV and SUB. A, an EMSA gel is shown on which
reactions containing 32P-labeled SUB oligonucleotide, 32 nM MBP-Piv protein, and cold competitor DNA were
electrophoresed. The -fold molar excess of competitor DNA is indicated
above each lane, and the lanes are grouped according to the competitor
DNA used: INV, SUB, or nonspecific cold competitor DNA. Probe indicates
the lane where no protein was added to the reaction. B,
competition assays are shown in which each DNA binding reaction
contained 32P-labeled INV oligonucleotide (see
"Materials and Methods"), 630 nmol of MBP-Piv protein, and cold
competitor DNA as indicated. The lanes are labeled as described in
A. S-1, S-2, and I-1
indicate the same complexes as in Fig. 4, and U is the
unbound DNA binding substrate. The nonspecific competitor
oligonucleotide has no homology to the INV or SUB
oligonucleotides.
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Dissociation Rate Analysis--
The results of the DNase I
footprinting, gel shift assays, and DNA binding competition analyses
could be interpreted as indicating that the interactions of MBP-Piv
with inv are less stable than with sub. The
inability to obtain more than 40% of the INV DNA bound with increasing
concentrations of MBP-Piv might be explained by a rapid dissociation
rate for MBP-Piv when bound to the site of inversion, such that no more
than 40% of the INV oligonucleotide complexed with MBP-Piv can be
detected by the DNA binding assays used. Dissociation rate experiments
with INV (Fig. 6A), using 5000-fold molar excess of competitor (unlabeled INV), showed that the
percent bound DNA in the I-1 complex decreased >50% after 15 s
of incubation with the competitor oligonucleotide. Surprisingly, even
though 5000-fold excess INV competitor DNA prevented any complex
formation when added before MBP-Piv (control lane
in Fig. 6A), there was still close to 10% I-1 complex after
a 1-h incubation of the preformed MBP-Piv-INV complex with 5000-fold
excess unlabeled INV. The dissociation rate experiments with SUB (Fig.
6B) gave a half-life for the S-2 complex of about 15 s,
similar to the I-1 complex. The S-1 complex, however, had a much higher
half-life of 12 min, indicating that MBP-Piv binding as a single
protomer in this complex is more stable. The apparent rapid
dissociation rate of I-1 and S-2 is also consistent with
multimerization of MBP-Piv producing an unstable protein complex.

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Fig. 6.
Dissociation rates of MBP-Piv complexed with
INV and SUB. Gel shift DNA binding assays are shown for
protein-DNA complexes that were challenged with 5000-fold molar excess
of competitor DNA. The percentage of bound DNA in each complex at each
time point is represented on a graph. A, a
dissociation rate assay performed with labeled INV produces a
graph showing that the MBP-Piv protein bound in the I-1
complex has a half-life of about 15 s. B, a
dissociation rate assay with SUB gives a result for both the S-1
(12-min half-life) and S-2 (15-s half-life) complexes as determined
from the graph below the gel. S1, S2, and
I-1 indicate the same complexes as in Fig. 4.
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Examination of the Cooperativity of Piv Binding--
Cooperative
interactions stimulate binding of multiple proteins to DNA sites.
Therefore, cooperative binding of MBP-Piv may facilitate complex
formation with the inv or sub sites where earlier results suggest that multiple protomers of MBP-Piv are binding. However, a multimeric form of MBP-Piv may bind noncooperatively if
assembled in solution before binding to the inv or
sub sites. To determine cooperativity, ln[P]
versus ln ([PX]/[X]) was plotted producing a slope n, where [P] is the concentration of
MBP-Piv protein added, and [PX] and [X] are
the concentration of the bound and unbound DNA, respectively. For
example, if MBP-Piv binds as a dimer to the DNA, n equal to
2 indicates that the binding of the two proteins is cooperative;
n equal to 1 indicates that the binding is not cooperative,
and therefore, the dimer is forming before binding to that DNA site
(14).
To determine the cooperativity of MBP-Piv binding in the S-1 and S-2
complexes, increasing amounts of MBP-Piv were incubated with the 298-bp
DNA fragment encoding the full sub site and subsequently analyzed by EMSA. Focusing on the MBP-Piv concentrations that gave the
S-1 complex, the ratio of bound to unbound DNA was determined and
compared with the concentration of MBP-Piv used. The ln[P] versus the ln([PX]/[X]) was
plotted for binding reactions with 0.63 to 31.6 nM MBP-Piv
(Fig. 7A). The slope of the
resulting graph is 1, showing noncooperative binding by MBP-Piv at
sub in the S-1 complex. Analysis of the S-2 complex revealed
that Piv is binding cooperatively. The ln [P] versus the
ln ([PX]/[X]) was plotted for binding
reactions with 47.4 to 1580 nM MBP-Piv (Fig.
7A), and the resulting graph has a slope of 2, indicating that the binding is cooperative. This result supports the conclusion that at least two MBP-Piv proteins are binding to the sub
site in the S-2 complex.

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Fig. 7.
Cooperativity of MBP-Piv binding to the
invL and sub sites.
A, an EMSA gel is shown in which the labeled 298-bp fragment
is incubated with increasing amounts of MBP-Piv. The results for the
S-1 and S-2 complexes (see Fig. 4) are plotted as ln[P]
versus ln([PX]/[X]), as described
under "Results," for the range of MBP-Piv concentration that gives
a single retarded complex and the range that gives two retarded
complexes, respectively. B, the binding of MBP-Piv to the
INV oligonucleotide is examined, and the results for the I-1 complex
are plotted as above.
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Cooperativity was also examined for Piv binding to the site of
inversion using the INV oligonucleotide (Fig. 7B). The I-1 complex accumulated with increasing amounts of Piv. Again, no intermediate protein-DNA complex was seen. The ln[P]
versus the ln[PX]/[X] was plotted
for each lane, and the slope of the resulting graph is 0.6; this result
indicates that noncooperative binding is occurring. Therefore,
multimeric MBP-Piv must assemble in solution or at another site before
binding to invL.
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DISCUSSION |
The site-specific recombinase Piv is a member of the novel family
of DNA recombinases that includes the IS110/IS492
transposases (5). To determine the mechanism for DNA cleavage and
strand exchange mediated by this unique DNA invertase, we must
understand how Piv interacts with the chromosomal DNA segment that it
inverts in M. lacunata. We have presented here DNA binding
studies with MBP-Piv and DNA sequences from the type IV pilin inversion
region of M. lacunata. The activity of the MBP-Piv fusion
protein in these gel electrophoresis DNA binding assays and
nuclease/chemical protection analyses is likely to correlate to
the activity of wild type Piv, as MBP-Piv is active in
vivo for DNA inversion (see Fig. 2).
Previous examination of the DNA sequence of the inversion region of
M. lacunata revealed 26-bp inverted repeats, invL
and invR, which defined the left and right ends of the
invertible element (17, 18). Piv-mediated DNA cleavage and strand
exchange must occur within these two 26-bp sequences for site-specific DNA inversion of the pilin segment. Therefore, based on other site-specific recombination and transposition systems (1, 2), it seemed
likely that Piv binds to a DNA sequence overlapping or adjacent to
invL and invR. The results of electrophoretic
mobility shift assays indicate that MBP-Piv does indeed interact with
DNA fragments containing both invL and invR, as
well as with a few DNA fragments from both outside and within the
invertible DNA segment of M. lacunata (see Fig.
3A). The binding of MBP-Piv to invL and one of
the additional sites located outside of the invertible segment was
further characterized.
MBP-Piv interactions with the invL site are quite weak, as
indicated by the gel shift DNA binding assays in which less than 40%
of the labeled DNA substrate (54-bp double-stranded INV oligonucleotide or 430-bp DNA fragment) could be specifically bound, no matter how high
the concentration of MBP-Piv protein (see Fig. 7B and data
not shown). In addition, MBP-Piv did not give a "footprint" at the
invL site in either nuclease or chemical protection assays. Measurements of the dissociation rate for the MBP-Piv bound to invL (on INV, see Fig. 6A; or the 430-bp
fragment, data not shown) show that the complex is unstable; the
half-life of the complex is less than 15 s. While this weak
binding of MBP-Piv to invL could have been due to the
presence of the maltose-binding protein at the amino terminus of Piv,
we found that Piv containing a small His6 tag at the COOH
terminus also bound invL very weakly.
Piv is certainly not the first site-specific recombinase to exhibit
poor affinity for the site where it mediates DNA cleavage and strand
exchange.
integrase (Int) binds very poorly to the "core" sites
where it mediates DNA cleavage and strand exchange at the
attP/attB or attL/attR sites. Cooperative
interactions with Int bound to the "arm-type" sites and DNA bending
mediated by IHF facilitate binding of Int to the core sites of
attP and attL (19, 20). Int binds with very low
affinity to the attB core sites (21); indeed, Int does not
give a DNase I footprint at the core site of attR, even in
the presence of the accessory proteins and arm-type sites (19).
Int is a bivalent protein, having two different, separable DNA
binding domains. The Int amino-terminal domain binds with high affinity
to the arm-type sites, while the carboxyl-terminal domain binds weakly
to the core sites and catalyzes DNA cleavage and strand exchange. Thus,
monomers of Int can bridge the arm and core sites within att
sites and bring att sites together in synapsis (20, 22).
Because Piv binds so poorly to the invL site, we did not
find it surprising that, like Int, Piv binds other DNA sequences that
may facilitate the formation of a synaptic complex. The nuclease
protection and gel shift DNA binding assays show that Piv binds with
higher affinity to a different DNA recognition sequence located
upstream of invL (Figs. 3, B and C,
4A, and 7, A and B). This strong
upstream binding site (sub) is bound cooperatively by at
least two protomers of MBP-Piv (see Figs. 4B and
7A). The dissociation rate assays (see Fig. 6B)
performed with the sub site suggest that the first MBP-Piv
protomer that binds to sub forms a stable complex (S-1) but
the multimeric complex (S-2) is less stable.
The interactions of MBP-Piv with invL appear to differ from
those with sub, not only in the affinity and stabililty, but
also in the form of the protein required for binding to the site. The cooperativity assays and competition analyses suggest that only the
multimeric form of MBP-Piv binds to invL, while single
protomers of MBP-Piv bind cooperatively to sub (see Figs. 5
and 7). Again, this is not unusual behavior for a bivalent protein that
mediates DNA recombination. The transposase of bacteriophage Mu, MuA,
binds as a monomer to sites within the transpositional enhancer
sequence which facilitates assembly of a synaptic structure (23, 24). But, MuA recognizes the recombination sites at the ends of the transposable element with a different domain of the protein, and then
assembles a tetramer that is bound to both ends of the element on a
supercoiled DNA substrate (25, 26). The interactions of MuA with the
end recombination sites actually promote tetramer formation, and it is
this complex that is active for recombination (27). In fact, under
altered reaction conditions MuA can form a tetramer upon binding a
single recombination site, but not on the enhancer sites (27).
Therefore, when we consider the nature of the MBP-Piv complexes with
sub and invL, it is possible that the
sub site is an assembly site for the multimeric form of Piv that binds to the recombination sites. The instability of the MBP-Piv
multimeric complex with invL may reflect the need for stabilization within a correct synaptic complex, like the binding of
integrase to the core sites, or, perhaps Piv transiently interacts
with the inv sites within an invertasome-like structure and
the multimeric complex rapidly falls apart following catalysis of the
recombination reaction. As has been suggested for a number of
transposition and site-specific recombination systems (Ref. 28; for
reviews see Refs. 1 and 2), it is undesirable for the recombinase to be
active at recombination sites until a synaptic structure is assembled;
this ensures that the correct strand exchange and religation occurs
after DNA cleavage.
If sub acts as an assembly site for Piv, it is likely to
stimulate the rate of inversion or to be required for Piv-mediated DNA
inversion. Previous deletion analyses of the M. lacunata
invertible segment and surrounding DNA sequences have shown that the
region encoding the sub site is not essential for inversion
of the Moraxella DNA segment in E. coli, although
it may have an affect on the rate of inversion (14). Like the P2 arm
site in
attR (22), the sub site may be
stimulatory only under certain conditions, such as when recombinase
levels are low or in the presence/absence of accessory factors.
Analysis of the nucleotide sequence of the inversion region using the
GCG Wisconsin Package Bestfit program with the sub site
sequence revealed two other possible sites within the 535-bp and 250-bp
DNA fragments that were bound by MBP-Piv in the gel shift DNA binding
assays (see Fig. 3A; data not shown). These other sites may
substitute for the sub site or coordinately regulate the
inversion reaction with sub. In order to define the roles of
Piv and its different binding sites in the inversion reaction, we are
currently using in vitro recombination assays to isolate
intermediates in the DNA inversion reaction.