ABSTRACT
The
[beta]
recombinase from the broad host range Gram-positive plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented
recombination sites in the presence of a chromatin-associated protein (Hbsu). The recombination site had been localized to a
447 bp DNA segment from pSM19035. This segment includes a 90 bp region that contains two adjacent binding sites (I and II) for
[beta]
protein dimers. Using
in vitro
recombination assays, we show that this 90 bp region is necessary and
sufficient for
[beta]
protein-mediated recombination; this defines the
six
site as the region required for
[beta]
protein binding. The point of crossing over has been localized to the center of
site I. Hbsu has a strong binding affinity for an unknown site located within
the 447 bp segment containing the
six
site. We discuss the possibility that Hbsu recognizes an altered DNA structure,
rather than a specific sequence, generated in the synaptic complex.
The [beta] recombinase, encoded by the Gram-positive broad host range plasmid pSM19035, mediates the conversion
of plasmid multimers into monomers (DNA resolution) to maximize stable plasmid
inheritance and is also involved in an inversion process probably required for
correct plasmid replication (
1
-
3
). In the presence of the
Bacillus subtilis
chromatin-associated protein Hbsu, the purified [beta] recombinase is able to catalyze both deletions (resolution) and
inversions between two specific recombination sites, depending on their
relative orientation (
2
,
3
). The role of Hbsu protein is to facilitate the formation of the synaptic
complex, since in the absence of Hbsu, synaptic complexes are not formed (
4
). The chromatin-associated proteins HU from
Escherichia coli
or HMG-1 from mammals can substitute for Hbsu in the recombination reaction (
4
). HMG-1 shares neither sequence nor structural homology with Hbsu. Therefore, it is likely that these chromatin-associated proteins work by recognizing and stabilizing a bent or
altered DNA structure at the recombination site, rather than acting as a bridge
between [beta] recombinase dimers through protein-protein interactions (
4
).
Sequence homology comparisons allow the inclusion of the [beta] protein into the Tn
3
family of DNA recombinases (
2
,
5
), which includes three subfamilies: DNA resolvases, DNA invertases and
resolvase-invertases. The [beta] recombinase and the highly related Res[beta] protein of plasmid pAM[beta]1 are the only members of the last group that have been
analyzed
in vitro
(
2
,
3
,
6
). DNA resolvases are highly specialized in catalyzing deletions between two directly oriented
recombination sites, named
res
sites (reviewed in
7
,
8
). DNA invertases mediate inversions between two inversely oriented target sites, but deletions between two directly oriented sites occur
at a very low frequency (reviewed in
9
,
10
). The [beta] recombinase does not have such bias and can catalyze both deletions
(resolution) and inversions between two specific recombination sites with
comparable efficiency, depending on their orientation (
2
,
3
).
DNA resolvases bind to a 120 bp DNA segment termed
res
containing three adjacent binding sites with dyad axis symmetry (I, II and III;
11
). DNA resolution occurs between two directly oriented
res
sites, at the center of site I. All three sites are required for efficient
recombination, but no additional factors are needed (
12
; reviewed in
7
,
8
). DNA invertases bind to a 26 bp site, within which recombination occurs, and
require a 60 bp recombination enhancer sequence
in
cis
, to which the Fis protein binds (reviewed in
9
,
10
). The recombination site (
six
site) for [beta] recombinase has been localized to a 447 bp DNA sequence immediately downstream of the plasmid replication origin (
2
; see Fig.
1
). The [beta] protein, which is a dimer in solution, binds cooperatively to two
adjacent sites, named I and II, located within the
six
site (
2
; see Fig.
1
). This binding does not require Hbsu or any other factor (
2
). By using different footprinting techniques, the sequences required for [beta] protein binding have been shown to include a 10 bp inverted repeat at
site I, with a 14 bp spacer and a non-symmetrical 32 bp long sequence at site II (Fig.
1
;
13
). We have now investigated whether the recombination determinants are just the
sequences required for [beta] protein binding or if additional DNA sequences are required. This
information could be particularly relevant in unravelling the way in which Hbsu
helps to form the synaptic complex. Hbsu protein, which is needed in
stoichiometric amounts, can be competed out from the [beta]-mediated synaptic complex by an excess of a supercoiled DNA
containing the
six
site, but not by a DNA lacking the
six
site (
4
). This suggests that Hbsu has a marked binding preference for a site located
within the 447 bp DNA region from pSM19035 that contains the
six
site. Determination of the minimum sequences required for recombination could
unravel whether Hbsu binds to a specific sequence. The results presented show
that [beta]-mediated DNA resolution requires two 90 bp recombination sites in
direct orientation that contain sites I and II for [beta] protein binding. This would be the smallest recombination site described
so far for a DNA resolvase of the Tn
3
family. Furthermore, this suggests that no specific sequences are needed for
Hbsu binding and that Hbsu would perform its task by recognizing or stabilizing
an altered DNA structure at the recombination site. We have also investigated
where the recombination reaction occurs; we found that it takes place at the
center of site I.
The
Escherichia coli
strain XL-1 Blue was used (
15
). Plasmids pCB8 and pUC18[Omega] have been described previously (
2
). Plasmid pBT316A contains two directly oriented
six
sites, one from plasmid pSM19035 and the other from the highly related plasmid
pAM[beta]1, separated by a marker gene coding for resistance to phleomycin.
Plasmids pCB61, pCB62, pCB63, pCB64, pCB66 and pCB67 were obtained from pCB8 by
making nested deletions with the exoIII enzyme in one of the two 447 bp DNA
segments that include the
six
site (deletions were performed from the
Pst
I site; see Fig.
2
). The deletion end points were determined by nucleotide sequencing. Deletions
of 4 (pCB61), 27 (pCB62), 110 (pCB63), 121 (pCB64), 178 (pCB66) or 195 bp
(pCB67) were obtained; numbering of positions considers the cutting site for
Ase
I as position 1 (see Figs
1
and
2
B).
Protein [beta] was purified and its concentration determined as previously described (
2
,
16
). Purified Hbsu protein was a gift from Prof. U. Heinemann (Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany). Since both proteins are
dimers in solution, their concentration is expressed as mol of protein dimers.
Reaction mixtures to assay site-specific recombination contained 10.6 nM substrate plasmid, 20-335 nM purified [beta] protein, 100 nM purified Hbsu protein, 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl
2
, in a total volume of 25 [mu]l. Reactions were initiated by the addition of [beta] protein and incubated at 30oC for 30 min. To analyze the recombination products, the DNA was
digested with two restriction enzymes, one cutting at the multicloning site of
the vector (
Pst
I or
Hin
dIII) and the other one cutting at the region lying between the two
six
sites (
Sal
I,
Eco
RV or
Hpa
I). The DNA fragments generated were analyzed by agarose gel electrophoresis. The relative amounts of DNA present in any particular band were determined by
laser scanning densitometry of photographic negatives; negatives corresponding
to different exposure times were used to ensure linearity of the response with
respect to DNA concentration.
We have previously shown that two copies of the 447 bp
Ase
I-
Bbr
PI DNA segment from plasmid pSM19035, containing a binding region for the [beta] recombinase, are necessary and sufficient for [beta] protein-mediated DNA recombination; the 447 bp segment was termed the
six
site (
2
; see Fig.
1
). Within this DNA fragment, the sequences specifically recognized by the [beta] recombinase were localized to a 90 bp region (
2
,
13
; Fig.
1
). It was unknown, however, whether the 90 bp target segment is sufficient to
support the recombination reaction or if the rest of the sequence of the 447 bp
DNA fragment is also required. Plasmid pCB8 contains two of the 447 bp
six
sites in direct orientation, separated by a DNA region ~2.2 kb in length (Fig.
2
A), and is an efficient substrate for [beta] protein-mediated recombination (
2
). To determine which are the minimal sequences required for recombination, one
of the above-mentioned 447 bp DNA segments from plasmid pCB8 was sequentially reduced upstream of site I and downstream of site II.
Sequences upstream of site I were progressively eroded with exoIII on the 447 bp
six
site located on the left in Figure
2
A (marked L), leaving the other 447 bp
six
site unaltered (marked R in Fig.
2
A). The ability of the resulting plasmids to serve as substrates for [beta] protein-mediated recombination was analyzed
in vitro
(see Fig.
2
). In short, the deletions obtained upstream of site I, which eliminated up to
121 bp (plasmid pCB64), had no apparent effect on reaction efficiency. Deletion
of site I, however, totally abolished recombination (see plasmids pCB66 and
pCB67). The effect of sequences downstream of site II was analyzed by
generating a synthetic 91 bp segment lacking all sequences downstream of site
II (positions 234-447) and upstream of site I (positions 1-141) but containing sites I and II for [beta] protein binding (positions 142-233). In addition, this synthetic site contained two
point mutations in the spacer region between sites I and II that generate a
Sca
I restriction site (see Fig.
2
C). This 91 bp DNA fragment was used to replace the left 447 bp
six
site of plasmid pCB8, generating plasmid pCB105. This plasmid was as good a substrate for [beta]-mediated recombination as the parental pCB8; no difference in recombination efficiency
could be detected under the conditions used (not shown). Complete deletion of
site II, leaving an intact site I (plasmid pCB51), totally abolished [beta]-mediated recombination (Fig.
2
C). Substitution of the two 447 bp
six
sites of pCB8 by the 91 bp DNA segment that contains just the [beta] recombinase binding site (positions 142-233, plasmid pCB106) also gave an active recombination substrate
(Fig.
2
D). Therefore, binding sites I and II are strictly required for efficient
recombination, but the sequences upstream of site I or downstream of site II
are not and can be substituted by unrelated sequences with no apparent effect
on
in vitro
reaction efficiency. These results allow the reduction of the
six
site to the 91 bp segment comprising positions 142-233. Since positions 231-233 are not conserved in the equivalent region of the related
plasmid pAM[beta]1 (Fig.
3
), which can also serve as substrate for [beta]-mediated recombination (see below), we can reduce the minimal
six
site to the 89-90 bp segment required for the binding of [beta] protein to DNA.
The precise position within the 447 bp recombination region at which the
crossing over reaction takes place was unknown. To localize it more precisely,
we constructed plasmid pBT316A, which contains two similar, but not identical,
six
sites in direct orientation, one of them derived from plasmid pSM19035 and the
other from the related plasmid pAM[beta]1. Plasmids of the
inc
18 incompatibility group (pSM19035, pAM[beta]1 and pIP501) code for a recombinase (
2
,
17
) and recombination regions (
13
,
17
,
18
) that are very homologous (~92%; see Fig
3
). The [beta] protein from pSM19035 can bind to the recombination site of plasmid pAM[beta]1 (
2
), which suggests that it can recognize it as a recombination substrate. As in
pSM19035, the recombination site of plasmid pAM[beta]1 is composed of two binding sites for the recombinase, named R1-R2 and R3 (
6
). Site R1-R2 is equivalent to site I of pSM19035, while R3 corresponds to site II
for the [beta] protein of pSM19035 (
2
,
6
,
13
). Although sites I and R1-R2 are very similar, some differences are present in the sequences located immediately upstream and the homology decreases considerably downstream of these sites
(Fig.
3
;
16
). On the basis of these differences, we expected that the sequence of a
recombination product between the two recombination sites of pBT316A would
allow a more precise delimitation of the region of crossing over.
An
in vitro
recombination reaction was carried out with plasmid pBT316A, [beta] recombinase and Hbsu under standard conditions and the reaction products
were transformed into
E.coli
. Since recombination should eliminate a DNA region lying between the two
recombination sites that includes a marker gene conferring resistance to
phleomycin, recombinant plasmids were easily discriminated from parental
plasmids on the basis of sensitivity to phleomycin (the plasmid vector confers
resistance to ampicillin). Two such plasmids were purified and the nucleotide
sequence of their unique
six
site was determined and compared with that of the
six
sites of plasmids pAM[beta]1 and pSM19035 (Fig.
4
A). Sequences immediately upstream of the left arm of the inverted repeat of
site I belonged to pSM19035, while the last base of the right arm and those
lying downstream of it corresponded to pAM[beta]1 (Fig.
4
A). This indicates that recombination had taken place somewhere in site I. Since
pSM19035 site I is almost identical to pAM[beta]1 site R1-R2, the assay cannot indicate the precise crossing over point within site I. The sequences upstream of the left arm of the site I inverted
repeat are not required for recombination (see above). In addition, the central
4 nt of the pSM19035 site I are identical to the equivalent regions of site I
for the [gamma][delta] and Tn
3
resolvases. These two enzymes are known to produce staggered cuts at this
position (see Fig.
4
B). It is likely, therefore, that the [beta] protein produces the strand exchange somewhere in the spacer regions
between the two arms of the site I inverted repeat. We further analyzed this
possibility.
The above results suggest that the point of crossing over should lie within the
short spacer sequence that separates the two half-sites recognized by the [beta] protein. In the case of the [gamma][delta] and Tn
3
resolvases, the two central nucleotides of the corresponding site I, at which
the staggered cuts are introduced, are critical for the recombination reaction
and their mutation drastically reduces the formation of recombination products (
19
). Therefore, determination of the nucleotides essential for [beta] protein-mediated recombination should help to locate the point of crossing
over. To this end, every single nucleotide in the center of the spacer separating the two arms of the [beta] protein recognition target in site I was modified by site-directed mutagenesis to cytosine, except at the position where a
cytosine was already present, which was modified to guanine (see Fig.
5
). In each case, a plasmid was constructed containing two
six
sites in direct orientation, one of them with wild-type sequences and the other with a mutated position (plasmids pCB81-pCB90). The absence of unexpected mutations at the
six
sites of the resulting plasmids was confirmed in all cases by nucleotide
sequencing. The efficiency of each of these plasmids as a substrate for [beta]-mediated recombination was analyzed
in vitro
under standard conditions. As shown in Figure
5
, all except the two central positions could be modified without impairing the
recombination reaction; reaction efficiency was not altered either (data not
shown). Modification of the central 5'-AT-3' nucleotides gave substrates that were totally
inactive for recombination (see Fig.
5
).
Previous studies had indicated that [beta] protein-mediated recombination requires a 447 bp DNA segment from plasmid pSM19035, a segment that
contains two adjacent binding sites for the recombinase (
2
). Chemical footprinting assays delimited the target sequences for the [beta] protein to a 90 bp segment (coordinates 140-229) located within the 447 bp DNA fragment (
13
). The results presented in this work indicate that the sequences that are necessary and sufficient for the recombination reaction lie in a 89 bp DNA segment (coordinates 142-230) that coincides with the [beta] recombinase binding site. Sequences upstream of binding site I or
downstream of binding site II could be modified without significantly affecting
reaction efficiency. The first two bases of site I (coordinates 140 and 141) were dispensable for recombination. Though these positions are contacted by the [beta] protein, they are not necessary for protein binding (see
13
). Both binding sites I and II were essential for the recombination reaction:
elimination of either of them from just one of the two recombination sequences of the substrate plasmid abolished DNA recombination. Therefore, we can define the minimum
six
site as that containing the target sequences for the [beta] recombinase, i.e. the DNA segment including sites I and II. This is
consistent with the
six
site suggested for the Res[beta] recombinase of the pAM[beta]1 plasmid (
6
).
These results are particularly interesting in understanding how the accessory
protein Hbsu participates in assembly of the recombination complex. About 1-2 Hbsu dimers/DNA molecule are enough to activate the recombination
reaction (
3
), which suggests that it is binding with high affinity to a particular region
of the DNA. Since in the presence of the [beta] protein, binding of Hbsu can be competed out by a supercoiled DNA
containing the 447 bp DNA segment that includes the
six
site, but not by a DNA lacking these DNA regions, it was hypothesized that Hbsu
should bind with high affinity to a site located in this DNA segment (
4
). A similar assay with a supercoiled DNA containing just the minimal 91 bp
recombination region including sites I and II gave the same result (not shown).
Nevertheless, no binding was observed by footprinting techniques when using a
linear DNA fragment spanning positions 87-259, irrespective of the absence or presence of the [beta] protein (
13
). Our results showing that the DNA sequences upstream of site I and downstream
of site II can be substituted by unrelated DNA make it very unlikely that Hbsu
is recognizing a specific sequence outside the
six
site. Enzymatic (
2
) and chemical (
13
) footprinting analyses suggest that between [beta] protein binding sites I and II there is a sequence, 24 bp in length, that
is not contacted by the two [beta] protein dimers. It is also unlikely that Hbsu recognizes a specific
sequence in this region, for the following reasons: (i) 16 of the 24 bp are not
conserved in plasmid pAM[beta]1 (Fig.
3
), whose recombination
site is very homologous to that of pSM19035 and supports recombination with the
pSM19035 [beta] protein in the presence of Hbsu; (ii) we have shown here that we can modify two of the conserved positions in this 24 bp region (T189 and A190) without affecting [beta] protein-mediated recombination. Therefore, up to 18 of the 24 bp can
be modified with no detectable effect on recombination. This means that it is
very unlikely that Hbsu can recognize a specific sequence in this DNA region, unless recognition relies more on DNA deformability or structure than on the precise identity of the nucleotide sequence. In
agreement with this idea, although the HU and Hbsu proteins are known to bind
DNA with little or no sequence specificity, they are known to have a clear
binding preference for bent, kinked or distorted DNA sequences, irrespective of
their precise nucleotide composition (
21
,
22
). The mammalian HMG-1 protein, sharing neither sequence nor structural homology with Hbsu, can
substitute for Hbsu in the recombination reaction (
4
), which argues against a model in which Hbsu is acting as a bridge between [beta] protein dimers bound at the recombination sites. Therefore, our results
suggest that Hbsu may bind and stabilize an altered DNA structure that is
formed at the recombination site upon assembly of the synaptic complex. In
principle, this structure could arise either upstream of site I, downstream of site II or, perhaps, at the small region that lies between both sites. Hbsu would bind
efficiently to this target, but recognizing a DNA structure rather than a
specific sequence. Indeed, the HU protein has been shown to help to stabilize
higher order protein-DNA complexes in a number of cases (
23
-
27
) and it has been proposed to do it through its ability to stabilize altered DNA
structures.
On the other hand, we present evidence indicating that the strand exchange takes
place at the center of site I. Mutation of the two central nucleotides of site
I in one of the two directly oriented recombination sequences of the substrate
plasmid gave DNA molecules that were unable to generate recombination products.
These nucleotides are located at the center of the spacer of the inverted
repeat that forms the [beta] protein recognition sequences. Mutations at other positions in this
spacer had no effect on recombination. It is likely, therefore, that the two
central nucleotides are directly involved in the strand exchange reaction and
that the recombination reaction occurs by a mechanism that is probably similar
to that described for other recombinases of the Tn
3
family. The mechanism has been most extensively studied for the [gamma][delta] and Tn
3
DNA resolvases. As stated in the Introduction, both resolvases binds to three
adjacent sites (I, II and III) and crossing over takes place at the center of
site I (
20
). Mutations introduced at the central positions of [gamma][delta] or Tn
3
site I in one of the two directly oriented target sites of a DNA substrate for
the resolvases drastically reduced the generation of recombination products (
19
). It was interpreted that a mismatch at the positions of the two recombination
sequences that should hybridize after the strand exchange seriously affected
the outcome of the reaction. Therefore, it is very likely that DNA resolution
by [beta] recombinase occurs by a mechanism similar to that of [gamma][delta] and Tn
3
resolvases and, by inference, that the strand exchange reaction occurs at the
center of site I. The way of achieving the proper orientation of the two sites
I at the synaptic complex would nevertheless differ for the two recombinases, given the differences in their binding sites (three for [gamma][delta] and two for [beta] recombinase) and the need for Hbsu in the case of [beta] protein.
This work was supported by grant PB93-0116 from the DGICYT to JCA. We are grateful to Udo Heinemann for his
generous gift of Hbsu protein and to L.Yuste for excellent technical
assistance.
REFERENCES
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