(Received for publication, August 29, 1994; and in revised form, November 30, 1994)
From the
The recombinase, encoded by the Gram-positive bacterial
plasmid pSM19035, is unable to mediate DNA recombination in vitro unless a host factor is provided. The factor has now been
identified as the Bacillus subtilis Hbsu protein. Hbsu is a
nonspecific DNA-binding and DNA-bending protein. The
recombinase,
in the presence of highly purified Hbsu protein, is able to catalyze in vitro intramolecular recombination between two specific
recombination sites on a supercoiled DNA molecule. DNA resolution was
obtained when the two crossing over sites (six sites) were
directly oriented, whereas DNA inversion was the product when the six sites were in inverse orientation. The ability of the Escherichia coli chromatin-associated proteins HU, IHF, Fis,
and H-NS to substitute for Hbsu was investigated. HU efficiently
stimulated
-mediated recombination, while the effect of IHF was
partial and that of Fis and H-NS was undetectable. In addition, the
protein was able to mediate DNA recombination in both wild-type
and IHF-deficient E. coli cells, but failed to do so in an
HU-deficient strain. The data presented provide direct evidence that a
chromatin-associated protein is strictly required for
-mediated
recombination.
The Gram-positive broad host range plasmid pSM19035, originally
isolated from Streptococcus pyogenes, has extraordinarily long
inverted repeated sequences that comprise about 80% of the plasmid
molecule ( (1) and (2) and Fig. 1). The
functions required to ensure DNA replication and ordered partition at
cell division in Bacillus subtilis are located within the
inverted repeated segments and therefore are duplicated ((3, 4, 5, 6) , Fig. 1).
Genetic evidence suggests that the plasmid-encoded recombinase
maximizes pSM19035 partition in B. subtilis by catalyzing the
conversion of dimers or higher oligomeric forms into monomers (DNA
resolution) and that it also mediates a DNA inversion process within
the plasmid molecule(4) . Both recombination activities are
needed because the plasmid has two replication origins facing each
other (4) and uses a unidirectional
replication
mode(7) . Hence, activation of both origins should lead to
premature termination when the replication forks meet(8) . This
problem can be solved with a DNA recombination system analogous to that
used by the 2-µm plasmid to amplify its copy number; if a
recombination event inverts the orientation of one of the replication
forks, then both forks will move in the same direction. In this way, no
termination of replication will occur, and multimeric forms of the
plasmid will be generated. When one of the replication forks is
inverted again by the site-specific recombinase, replication is
terminated at the site where the two forks meet. Multimers of the
plasmid should then be converted into monomers (DNA resolution) by a
site-specific recombinase. In the case of the 2-µm plasmid, both
DNA inversion and resolution of multimers are catalyzed by the FLP
recombinase in the absence of accessory factors (for review, see (9) ). In contrast, the
recombinase from plasmid pSM19035
requires the help of a host-encoded accessory factor to catalyze both
DNA inversion and DNA resolution(6) .
Figure 1:
Physical map of the plasmid pSM19035. A, HindIII cleavage map of the plasmid (HindIII fragments A to I). The presence of
an apostrophe in a given HindIII DNA fragment
indicates that the segment occurs twice, once in each arm. Duplicated
sequences are indicated by a heavyline, and unique
sequences are indicated by a thinline. The arrowhead on the heavyline denotes the
polarity (arbitrary) of the inverted repeats. The grayboxes within the heavyline denote the
minimal replication region. The two openarrows indicate the direction of DNA replication. The protein
binding region is blown up in one of the repeated arms. The plasmid
replication origin (ori), and the site of crossing over (six), are indicated. The arrows indicate the open
reading frames in the region. The repS and
genes encode
for the initiation replication protein and the site-specific
recombinase, respectively, whereas orf
encodes an
uncharacterized product. B, nucleotide sequence of the six site, showing binding sites I and II for the
recombinase.
The regions protected by the
recombinase from the DNaseI attack
are shaded. Dyad axes of symmetry are indicated by convergentarrows.
There are two
well-characterized families of site-specific DNA recombinases, namely
the Tn3 family and the Integrase (Int) family (for review, see (9, 10, 11, 12) ). The enzymes of
the Tn3 family catalyze intramolecular recombination mediating
either DNA resolution or DNA inversion, although not both, and strictly
require supercoiled DNA substrates (for review, see (13) and (14) ). However, enzymes belonging to the Int family catalyze
inter- and intramolecular recombination with nearly equal frequencies,
they can promote both DNA resolution and DNA inversions, and in general
they do not require supercoiled DNA (for review, see (10, 11, 12, 13, 14, 15) ).
Although in terms of sequence homology the recombinase clearly
belongs to the Tn3 family, it shares properties with both
families of enzymes. It resembles the Int family in that it can promote
both DNA resolution and DNA inversions with comparable efficiencies,
but it differs in that the
recombinase does not catalyze
intermolecular recombination between sites located on separated
plasmids (see (6) ).
The enzymes of the Tn3 family
can be divided into three major groups: DNA resolvases, DNA invertases,
and resolvo-invertases. The recombinase is the only member,
described so far, that can be classified within the last group (see (6) and this work). DNA resolvases, which do not require any
other accessory component to promote recombination, bind to a DNA
segment termed res containing three adjacent binding sites (I,
II, and III). DNA resolution occurs between two directly oriented res regions at the center of site I. All three sites are
required for efficient recombination (for review, see (11) and (13) ). DNA invertases work through a different mechanism;
their target consists of a single site, and efficient inversion between
two inversely oriented sites requires the presence of a stimulating
sequence in cis (an enhancer) to which the Fis (
)protein binds(14, 15, 16) . In
the case of DNA resolvases and DNA invertases, there is a strong bias
in the efficiency of the reaction in favor of one specific orientation
(direct or inverse, respectively) of the recombination sites.
Conversely, the
protein, in the presence of a host factor,
catalyzes both DNA resolution and DNA inversion with comparable
efficiency(6) . The
protein should, therefore, conform a
group of its own (resolvo-invertases) within the Tn3 family of
recombinases. The
recombinase binding site has been localized
within an 85-bp region that can be divided into two adjacent sites,
named I and II ((6) , Fig. 1B). The site of
crossing over (six) for the
recombinase, although it
resembles that of DNA resolvases of the Tn3 family, differs in
that only two adjacent sites are found (I and II). The peculiar
architecture of the
protein target site (lack of site III) and
the need for a host factor are expected to have important consequences
in the formation of the synaptic complex.
In this report we show
that the B. subtilis accessory factor required for
protein-mediated DNA recombination is the Hbsu protein, a nonspecific
DNA-binding and DNA-bending protein belonging to the histone-like
family of proteins(16, 17) . The HU protein of Escherichia coli can efficiently substitute Hbsu, while IHF
does so very poorly. However, neither H-NS nor Fis will substitute
Hbsu.
The B.
subtilis host factor required for protein-mediated
recombination was purified as follows. B. subtilis DB104 (18) was grown at 37 °C in TY broth (19) to mid-log
phase (about 6
10
cells/ml) with agitation. Cells
(2 liters) were harvested by centrifugation (6000 rpm in a Sorvall GS3
rotor) and resuspended in 35 ml of buffer A (50 mM Tris-HCl
(pH 7.5), 2 mM MgCl
, 5% glycerol) containing 1 M NaCl. Lysozyme was added to a final concentration of 200
µg/ml, and the cells were incubated in water/ice for 15 min. The
cells (7 g) were lysed by sonication (15 pulses of 100 watts, 15 s long
each, using a MSE sonicator). The lysate was centrifuged for 15 min at
12,000 rpm in a Sorvall SS34 rotor (fraction I). Polyethylenimine (10%
(pH 7.5)) was slowly added to the supernatant under constant stirring
to a final concentration of 0.25% (A
120).
The DNA and co-precipitating proteins were pelleted by centrifugation
(15 min at 12,000 rpm in a Sorvall SS34), and the supernatant was
saved. The pellet was extracted a second time with 15 ml of buffer A
containing 1 M NaCl and centrifuged as described above. The
proteins remaining in the supernatant were precipitated by the addition
of solid ammonium sulfate to a final concentration of 45%. The ammonium
sulfate pellet was resuspended in 5 ml of buffer A containing 50 mM NaCl (Fuller's fraction II, Refs. 6, 21). In the presence of
20 µl of this cell extract, the
protein (168 nM)
promotes DNA resolution in pCB8 DNA. Fraction II was diluted 10 times
in buffer A containing 50 mM NaCl and loaded onto a 3
10-cm column of phosphocellulose equilibrated with buffer A containing
50 mM NaCl. The host factor was eluted with 300 ml of a linear
100-1000 mM NaCl gradient. The fractions centered at around
600 mM NaCl, which were able to stimulate
-mediated
recombination, were pooled (fraction III). A 1.5
8-cm column of
heparin-Sepharose CL-6B was equilibrated with buffer A containing 100
mM NaCl, and fraction III was dialyzed and applied to the
column. The host factor was eluted with 200 ml of a linear 600-1000
mM NaCl gradient. The fractions containing the factor able to
stimulate
-mediated recombination (10 ng of the protein gave a
detectable activity), which eluted at about 700 mM NaCl, were
pooled (fraction IV). This fraction contained a protein with an
estimated molecular mass of 9.0 kDa that was more than 90% pure. The
highly enriched 9.0-kDa protein was further applied to a 0.8
4-cm DNA-cellulose column. The column was eluted with a 100-800
mM NaCl linear gradient. The host factor was able to stimulate
-mediated recombination eluted at around 420 mM NaCl. The
fractions containing it were pooled (fraction V); about 5 ng of
proteins of this fraction sufficed to show a detectable activity.
Fraction V was concentrated by ammonium sulfate precipitation and
stored at -20 °C in the presence of 50% glycerol. Fraction V
contained the 9.0-kDa protein with a purity higher than 98%.
Hbsu
concentration was estimated from the absorption at 258 nm based on an
absorption coeficient of 7.6 10
M
cm
according to Groch et al.(22) .
The sequence of the first 17 amino-terminal residues of the purified protein are identical to the amino acid sequence deduced from the nucleotide sequence of the B. subtilis hbs gene(23) . The hbs gene codes for a homodimeric type II DNA-binding protein named Hbsu. The Hbsu is a small, basic, and heat-stable protein that belongs to the family of the histone-like or chromatin-associated proteins. The Hbsu protein is 92 amino acid residues long and has a predicted molecular mass of 9.8 kDa(23, 24) .
The identity of the isolated protein
with Hbsu was further substantiated by using a homogeneously pure Hbsu
protein in the recombination assay. In the presence of authentic Hbsu
protein, the purified protein did mediate intramolecular
recombination in vitro, both DNA resolution (deletions) and
DNA inversion (see below). As previously reported, DNA resolution was
2-4-fold more efficient than DNA inversion(6) . Reactions
without Hbsu failed to yield recombinant products, which confirms that
the host factor is an essential component for
-mediated DNA
recombination ( (6) and this work). The results are shown in Fig. 2; digestion of plasmids pCB8 (six sites in direct
orientation, see Fig. 2A) or pCB12 (six sites
in inverse orientation) with endonucleases PstI and SalI render one fragment of about 4.8 and two fragments of
about 0.5-kb each for pCB8 (lane1) or one fragment
of about 3.9 and two fragments of about 0.5-kb each for pCB12 (lane6). In the presence of 840 nM protein
(lanes2 and 7) or 2.2 µM Hbsu (lanes3 and 8), recombination does not take
place (see Fig. 2B). However, if both components are
simultaneously added (168 nM of
protein and 400 nM of Hbsu), digestion of the plasmid DNAs with PstI and SalI enzymes shows the appearance of two new restriction
fragments, of about 2.7 and 2.1 kb in the case of pCB8 (lanes4 and 5) and of about 3.0 and 0.9 kb in the case
of pCB12 (lanes9 and 10). These fragments
correspond to the expected recombination products between the six sites. In the case of pCB8, the recombination products correspond
to a DNA resolution (deletion) event, whereas in the case of pCB12, the
recombination products indicate that a DNA inversion process between
the six sites had occurred. Indeed, when the recombinant
products were digested with a restriction enzyme that cuts the DNA once (PstI), linear and circular species appeared, as in the case
of pCB8; whereas only a linear species was detected in the case of
pCB12 (data not shown).
Figure 2:
In vitro mediated site-specific
recombination requires both and Hbsu proteins. A, map of
the plasmids used as substrates in the recombination assay (pCB8 and
pCB12). The dottedline denotes the vector DNA, and
the continuousline denotes the cloned segments. The
location of the six site (corresponding to the 447-bp AseI-BbrPI segment of pSM19035) is denoted by a filledbar, and its orientation is indicated by an arrow. The relevant restriction sites are also shown (P, PstI; S, SalI). The plasmid
size, in kilobases, and the distance between the indicated restriction
sites, are denoted. B, electrophoretic analysis of the
products generated by
protein-mediated recombination. The DNA
substrates pCB8 (10.6 nM) or pCB12 (6.3 nM) were
incubated with the protein combinations indicated below for 30 min at
room temperature and then digested with endonucleases PstI and SalI. The DNA fragments generated were separated in an 0.8%
agarose gel. The proteins added to the recombination reactions were as
follows: lanes1 and 6, no protein added; lanes2 and 7,
protein (840
nM); lanes3 and 8, Hbsu protein (2
µM); lanes4 and 9,
protein (168 nM) and B. subtilis Hbsu protein (400
nM); lanes5 and 10, same as above
but in the absence of 10 mM MgCl
, which was
present in all other reaction mixtures. The parenthesis in the band of
0.5 kb denotes that it is a double band.
As previously reported, we failed to detect
any intermolecular recombination between separated supercoiled plasmids
containing a single six site(6) , ()indicating that the
protein cannot function as an
integrase (for review, see (12) ). We also note that when Hbsu
was included in the reaction, the
recombinase could mediate DNA
rearrangements both in the presence (lanes4 and 9) or absence (lanes5 and 10) of
Mg
(6) .
The requirement of DNA supercoiling for the reaction was investigated using closed circular (a self-ligated DNA) or linear pCB8 DNA as substrates. No recombination product was formed when closed circular or linear DNA was used (Fig. 3). It is likely, therefore, that supercoiling of the substrate DNA is a strict topological requirement for the reaction to occur.
Figure 3:
protein-mediated recombination
requires supercoiled DNA. Reaction mixtures contained the
recombinase (168 nM), the Hbsu protein (400 nM), and
pCB8 DNA (10.6 nM). The substrates used for the reaction were
as follows: pCB8 HindIII-digested (lanes1 and 1`), self-ligated HindIII-cleaved pCB8 DNA (lanes2, 2`, and 2"), and pCB8
supercoiled DNA (lanes3 and 3`). In lanes 1`, 2`, 3`, and 2", the DNA
substrates were incubated with the
and Hbsu proteins for 30 min
at room temperature prior to digestion with either SalI (lanes 1` and 2`) or with the SalI + PstI (lanes 3` and 2") enzymes. The ligation
of pCB8 molecules in a head-to-head configuration could render the high
molecular weight DNA band that disappeared upon digestion with SalI + PstI (see lane2"). The parentheses in the band of 0.5 kb denotes that is is a double
band.
Figure 4:
Stoichiometry of the recombination
reaction promoted by protein and its accessory Hbsu protein.
Electrophoretic analysis of products produced by in vitro
protein-mediated recombination. A, the pCB8 DNA
substrate (10.6 nM) was incubated with
protein (400
nM), Hbsu (1 µM), or with a constant amount of
protein (80 nM) and an increasing concentration of Hbsu
protein (5, 10, 21, 32, 42, 64, 85, 128, 170, 260, 340, 680, 1000, and
2000 nM). B, pCB8 was allowed to react either with
the
protein (400 nM), with the Hbsu protein (1
µM), or with an increasing concentration of
protein
(20, 30, 40, 60, 80, 100, and 160 nM) and a constant amount of
Hbsu protein (200 nM). The DNA substrate was incubated, as
indicated, for 30 min at room temperature prior to digestion with PstI and SalI enzymes. The generated DNA fragments
were separated in an 0.8% agarose gel. The parentheses in the
band of 0.5 kb denotes that it is a double
band.
The
Hbsu binding affinity for pCB8 DNA (or for the protein pCB8
complex), measured as its ability to facilitate
-mediated
recombination, is at least 500-fold higher than its affinity for
double-stranded linear DNA (see (22) ). However, when DNase I
footprinting experiments were performed with
DNA complexes
in the presence or absence of Hbsu, no specific protection or
hypersensitivity could be attributed to the Hbsu protein. (
)
As revealed in Fig. 4B, in the presence
of pCB8 (10.6 nM) and an excess of Hbsu (200 nM), the
DNA recombination reaction reached a maximum when the concentration of
protein reached 64 nM. Under these experimental
conditions, about 6 dimers of protein
/DNA molecule are sufficient
to saturate the recombination reaction. Identical results were obtained
when two different
protein preparations were used (data not
shown).
The Hbsu and Hbst proteins
occur as homotypic dimer, whereas native E. coli HU (also
known as NS) protein is predominantly a heterotypic dimer,
although HU preparations also contain small amounts of
and
homodimers (for review, see (25) ).
The Hbsu protein shares a 57 and 52% identity with E. coli HU
and HU
protein, respectively (for review, see (29) ).
To investigate whether the E. coli HU,
HU, or HU
proteins could substitute for B. subtilis Hbsu, we tested their ability to stimulate
-mediated DNA
resolution. At a pCB8 concentration of 10.6 nM, a significant
stimulation of
-mediated recombination was observed after addition
of 100 nM of either HU or HU
protein dimers. A similar
extent of stimulation was observed when 10-20 nM Hbsu
dimers were added to the recombination reaction (see Fig. 4A and 5A). Hence, although the HU and the HU
proteins
can substitute for Hbsu in
protein-mediated recombination, they
are 5-10-fold less efficient than Hbsu. The purified HU
homodimer was as good a stimulator as the HU heterodimer, whereas the
HU
homodimer was about 3-fold less efficient (Fig. 5A). No direct correlation between the extent of
stimulation of
-mediated recombination and the amount of homology
between the B. subtilis and a given subunit of the E. coli HU protein was observed (see above).
Figure 5:
Stimulation of -promoted DNA
resolution by different histone-like proteins. Electrophoretic analysis
of products of in vitro
protein-mediated recombination
in the presence of increasing concentrations of a given histone-like
protein of either B. subtilis or E. coli origin. A, the reaction contained pCB8 DNA substrate (10.6
nM) and either
protein (800 nM), about 2
µM given histone-like protein, or a constant amount of
protein (80 nM) and increasing concentrations (50, 100,
and 200 nM) of either Hbsu, reconstituted HU heterodimer,
HU
homodimer, or HU
homodimer. B, pCB8 (10.6
nM) was incubated either with
protein (800 nM),
about 2 µM of a given Histone-like protein, or with a
constant amount of
protein (80 nM) and increasing
concentrations (180, 360, and 720 nM) of either Fis, IHF, H-NS
proteins or with
protein (80 nM) and reconstituted HU
heterodimer (100 nM). The DNA substrate was incubated, as
indicated, for 30 min at room temperature prior to digestion with the PstI-SalI enzymes. The generated DNA fragments were
separated in a 0.8% agarose gel. The parentheses in the band
of 0.5 kb denotes that it is a double band.
To determine whether the
stimulatory effect of HU on -mediated recombination is specific,
we investigated whether other E. coli histone-like proteins
such as Fis, IHF, or H-NS (27) could act in a similar fashion.
Fis and IHF were originally discovered as host factors required for in vitro site-specific recombination events and show
site-specific DNA binding (for review, see (13, 14, 15) ). The H-NS protein (30, 31) plays a role in the compaction of the
bacterial chromosome and binds tightly and nonspecifically to duplex
DNA (for review, see (27) ).
In the presence of pCB8 DNA
(10.6 nM) and the protein (200 nM), the Fis and
H-NS proteins showed no stimulatory activity on
-mediated DNA
resolution at protein concentrations ranging from 4 nM to 1.6
µM (Fig. 5B).
On the contrary,
IHF, which shares a significant homology (about 35%) with both HU and B. subtilis Hbsu (for review, see (25, 26, 27) and 29), was able to stimulate
the
-mediated recombination reaction, although it was about 6-fold
less efficient than HU. A partial stimulation of
-mediated-recombination was observed after the addition of 364
nM IHF protein and 10.6 nM pCB8 DNA (see Fig. 5B).
By gel
retardation assays we determined that Hbsu does not significantly
increase the binding efficiency of the protein to the six site.
As an alternative possibility, we investigated
whether Hbsu could work by facilitating the interaction of the
dimers bound to the six sites in a DNA-independent way,
therefore contributing to enhance the formation of the synaptic
complex. To test this hypothesis, we added to a recombination reaction
lacking Hbsu (200 mM of
protein and 10.6 nM of
pCB8 DNA) increasing concentrations (final concentration, 0.1-5%)
of the inert volume-occupying agent polyethylene glycol. The
macromolecular crowding promoted by polyethylene glycol did not
substitute for Hbsu in the
protein-mediated recombination,
although it does not affect the recombination reaction in the presence
of Hbsu (data not shown).
The E. coli HU protein is
required for the assembly of the active tetramer of the MuA transposase
under normal reaction conditions (37, 38) . It has
recently been shown that in the presence of 15% MeSO, a
single plasmid-borne end-type MuA transposase binding site is
sufficient to promote tetramer assembly(39) . To test whether
Me
SO could stimulate
-mediated recombination in the
absence of Hbsu protein, we incubated pCB8 (10.6 nM) DNA with
the
protein (200 nM) in the presence of increasing
concentrations of Me
SO (ranging from 1 to 35% final
concentration). Me
SO was unable to stimulate the
recombination reaction in the absence of the Hbsu protein (data not
shown).
Figure 6:
Scheme of the in vivo assay for
-mediated site-specific recombination. The
protein-mediated
recombination process was monitored using E. coli cells
harboring plasmid pCB17 (six sites in direct orientation) (A) or pCB18 (six sites in inverse orientation) (B) and plasmid pBT434, from which the
recombinase can
be expressed in an inducible manner. The six sites are
indicated as open and shadedboxes (the
distance between them is not drawn to scale); the arrow shows
the orientation of the site. The xylE reporter gene, which is
expressed from a vector promoter, is represented with an outerarrow that denotes its orientation. The vector promoter
is denoted with a bentwavyarrow.
Transcription of this gene can be monitored by spraying the colonies
with pyrocatechol, which is transformed by the xylE gene
product into a yellow compound. The plasmid replication origin (ori) is shown. Recombination (step a) on pCB17 leads
to a deletion of the reporter gene (step b), which is lost
from the cell population since it lacks a replication origin. In the
case of pCB18, the inversion process (a) renders a plasmid in
which the reporter gene is no longer expressed because its orientation
relative to the vector promoter has been inverted (step b).
Since the resulting plasmid can go through successive rounds of
recombination (step b`), it is expected that, on equilibrium,
the cells harbor a mixed plasmid population in which the xylE
gene can be expressed in only 50% of the plasmid molecules. To
distinguish both plasmid forms, the plasmid DNA was extracted from the
pool of cells and transformed into an E. coli strain lacking
the
recombinase. The expression of the reporter gene was then
assayed.
We have devised a recombination
assay in which the protein is provided by plasmid pBT434 (its
production can be induced by
isopropyl-1-thio-
-D-galactopyranoside) and in which
plasmids pCB17 or pCB18 (compatible with pBT434) are used as substrates
(see Fig. 6). Plasmid pCB17 contains two directly oriented six sites separated by a 2.2-kb DNA segment bearing the
promoterless reporter gene xylE (Fig. 6A).
Plasmid pCB18 contains two copies of the six site in inverted
orientation, separated by a 1.3-kb segment bearing the promoterless
reporter xylE gene (Fig. 6B). In both vectors,
the xylE gene is transcribed from a vector promoter.
Expression of the reporter gene renders yellow colonies after spraying
the plates with 0.5 M pyrocatechol(40) . The general
scheme is depicted in Fig. 6. In short, recombination between
two direct repeated six sites results in the deletion of the
intervening segment (containing the xylE reporter gene), which
is lost from the cell population. Recombination between two inverted
repeated six sites results in the inversion of the DNA
segment. This substrate, however, could go through a second or further
rounds of recombination. Hence, if both events occur at the same
frequency, we will expect to find only a half of the pCB18 substrate
molecules with an inversion between the six sites.
The E. coli strains XL-1-Blue (HU IHF
), WM2014 (HU
), and WM2017
(IHF
) were transformed with plasmids pBT434 and
pCB17, with pBT434 and pCB18, or with the recombination substrates
pCB17 or pCB18. Except for pBT434 + pCB17 in strain WM2017, we
obtained the desired transformants. Recombination does not occur in the
absence of the
protein. The synthesis of
recombinase from
the pBT434-borne
gene was induced in the transformants for about
30 min by the addition of 5 mM isopropyl-1-thio-
-D-galactopyranoside, and the cells
were plated in the absence of inducer. In case of cells containing
pCB17, about 34% of the wild-type colonies remained colorless after
spraying with pyrocatechol, indicating that resolution had taken place.
In the case of cells containing pCB18, about 12% of both wild-type and
IHF
colonies remained colorless, indicating
inversion. These values are corrected for the number of viable cells
since induction of
-recombinase reduces this titre. Nevertheless,
the same assay performed with HU
cells rendered only
3% of white colonies, indicating that HU protein plays an essential
role in the process. The substrate plasmids were purified from
colorless colonies, and restriction analysis with PstI and SalI resulted in DNA fragments of the size expected for
recombinant products (data not shown).
In a previous study we have shown that the recombinase
is unable to mediate DNA recombination in vitro unless a host
factor is provided(6) . The host factor has now been identified
as a nonspecific DNA-binding and DNA-bending protein, termed Hbsu in B. subtilis. This was confirmed by four independent
observations: (i) the purified host factor was found to have the same
amino-terminal sequence as the Hbsu protein; (ii) a homogeneously pure
and independently obtained Hbsu protein did promote
-mediated
recombination in vitro; (iii) purified E. coli HU
protein, which is the counterpart of B. subtilis Hbsu protein,
was shown to be able to substitute in vitro for Hbsu although
with a 5-10-fold reduced efficiency; and (iv)
protein-mediated DNA recombination was shown to occur in vivo in E. coli cells wild-type for HU protein and to be
10-fold less efficient when the strain used was deficient in the HU
protein (E. coli hupA hupB strain). Hence, it is
unlikely that any trace of a contaminant protein in our preparations
can account for the stimulatory effect in the recombination reaction.
Furthermore, our preliminary results have revealed that the mammalian
HMG-1 protein, which has no known sequence or structure homology to
Hbsu(41, 42, 43) , can substitute for Hbsu in
-mediated recombination. (
)Since Hbsu, HU, and the
HMG-1, which are chromatin-associated proteins that bind DNA and bend
it in a sequence-independent
manner(25, 27, 44) , stimulate
-mediated
recombination, it is likely that protein-protein interactions do not
mediate Hbsu stimulation of
-mediated recombination. Furthermore,
this provides an explanation for the broad host range of pSM19035, as a
chromatin-associated protein could stimulate
-mediated
recombination in each host species.
In the presence of purified Hbsu
protein, the recombinase was able to activate DNA resolution
between two directly oriented six sites and, with a
2-4-fold lower efficiency, DNA inversions between two inversely
oriented six sites.
About one to two Hbsu dimers are
required to stimulate -mediated DNA recombination. The reaction is
saturated after the addition of about 5 ± 1
dimers, which
agrees with one dimer bound at each site (sites I and II, see (6) ). It is curious that the in vitro reactions of
Hin recombination, the assembly of DnaA at oriC, or of MuA
transposase at the transpososome core also require a few HU dimers/DNA
molecule(33, 35, 45) . It is likely,
therefore, that HU (or Hbsu) has a more precise mode of action than a
general coating of the DNA (see (32) ).
The recombination process leading to DNA resolution is well studied in the case of the Tn3 family of resolvases(11, 13, 14) . Here, binding of the resolvase to an isolated res site (composed of sites I, II, and III) forms a resolvosome, while interaction between two res sites in a supercoiled molecule leads to the formation of a higher order structure named synaptosome, within which recombination takes place(46, 47, 48) . It is thought that the role of sites II and III is to facilitate the formation of a precisely organized complex in which the two sites I adopt the proper relative orientation for the reaction to occur; in this way, and since the crossing over takes place at site I, sites II and III would be acting as a kind of enhancer (for review, see Refs. 11, 13, and 14). No host factors are required by the Tn3 family of resolvases to function in vivo or in vitro.
The
recombinase binds to only two sites, within the six region,
and requires an accessory factor (Hbsu) for its activity. There are
several possible ways in which Hbsu could work. This nonspecific
DNA-binding and DNA-bending protein could help the
recombinase to
form a nucleoprotein complex with its target site or could assist in
the formation of the adequate architecture at the synaptic complex for
the reaction to occur. Despite the virtual lack of sequence
specificity, Hbsu (HU) protein is thought to bind preferentially to DNA
regions having a sequence-directed curvature or showing an anisotropic
flexibility (49) and to bend the DNA upon binding to
it(17, 50) . The role of HU protein in stabilizing
several nucleoprotein complexes at least transiently, and at low or
moderate HU/substrate DNA ratios (25, 26, 27, 34) , suggests that it
could have certain binding preferences, probably stabilizing curved DNA
conformations. This property could be the key role of Hbsu in the
protein-mediated recombination reaction, because the stabilization of
curved DNA conformation could be needed to facilitate interactions
between the
protomers in the synaptic complex. For example, HU is
believed to stimulate the Hin-mediated DNA inversion reaction under
certain conditions by providing sufficient bending of the DNA to enable
the physical association of one of the hix subsites with the
enhancer, to which Fis binds(35) .
The fact that the related
though not identical chromatin-associated proteins HU and IHF can
partially substitute for Hbsu in the recombination reaction suggests
that it is their common property of binding to DNA with low sequence
specificity, and stabilizing bent DNA conformations, that is important
in the activation of the recombination process. A role of Hbsu in
facilitating the binding of the protein to the six site
is unlikely because when the rate of
-DNA complex formation was
measured using linear DNA, the same results were obtained whether Hbsu
was present or not (not shown). Therefore, the role of Hbsu should most
likely be to assist in the assembly of the synaptic complex. Our
inability to identify chemical compounds that, being able to induce or
stabilize DNA curvatures, can replace Hbsu in
-mediated
recombination probably means that the repertoire of conformations they
induce does not satisfy the requirements for synaptic complex
formation.
It is not surprising that the E. coli proteins
HU and IHF can partially substitute for Hbsu in -mediated
recombination, while H-NS and Fis do not. The lower efficiency of IHF
probably derives from the fact that HU binds to DNA
nonspecifically(25, 26, 27) , while IHF
recognizes a partially specific DNA sequence (27, 29) that is context-dependent. The six site for
recombinase may not bind IHF efficiently, or IHF
may not properly fit in the synaptic complex not achieving, therefore,
the optimum architectural conformation. Fis and H-NS do not show
sequence homology with HU or Hbsu. Although both proteins bind
preferentially to curved DNA
sequences(17, 32, 52, 53, 54) ,
they should be expected to have a different structure and to form
nonequivalent protein-DNA complexes. Since the synaptic complex is a
highly ordered structure, not every protein stabilizing bent DNA
conformations should be expected to fit properly in it.
In summary,
all DNA resolvases of the Tn3 family except for the
recombinase bind to three contiguous sites and do not require accessory
factors(11, 12, 13) , while the
protein
binds to only two sites (I and II) and requires Hbsu. Therefore, we
propose that the role of Hbsu (or the DNA structure promoted by it) in
-mediated DNA resolution could be to substitute for the missing
site III in the assembly of the synaptic complex. The
-recombinase
also catalyzes DNA inversion between two inversely oriented six sites. DNA invertases of the Tn3 family require a 26-bp
binding site (equivalent to site I) and an enhancer sequence to which
the Fis protein binds specifically. Our preliminary results suggested
that an enhancer sequence and the Fis protein are not required for
-mediated DNA inversion. (
)This suggests that subsite
II and the Hbsu protein substitute for the Fis protein and the cis-acting enhancer sequence in
-mediated DNA inversion.