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INTRODUCTION |
Site-specific recombinases of the Tn3/
family can be divided
into three major subfamilies: DNA resolvases, DNA invertases, and DNA
resolvase-invertases (reviewed in Refs. 1-3). The members of the three
subfamilies normally require supercoiled DNA substrates containing two
recombination sites (reviewed in Refs. 4-6). While DNA resolvases and
DNA invertases are highly specialized in catalyzing resolution or
inversion, respectively, members of the resolvase-invertase subfamily
do not have this bias and catalyze both kinds of reactions efficiently
(6, 7). The major difference between the enzymes of these three
subfamilies lies in the DNA site required for recombination. The
res site of DNA resolvases contains a crossover site
(subsite I) and two essential accessory sites (subsites II and III) to which the recombinase binds (reviewed in Refs. 1 and 2). In the case of
DNA invertases, the crossover site is analogous to the subsite I of DNA
resolvases. The accessory site (sis or enhancer), which is
not targeted by the recombinase, is located at some distance from the
recombination site and is targeted by the sequence-specific DNA-binding
and DNA-bending protein FIS (reviewed in Refs. 2, 8, and 9). The
recombination site (six site) of resolvase-invertases
includes a crossover site (subsite I) and an essential accessory site
(subsite II). The accessory site(s) to which a sequence-independent
chromatin-associated protein binds is not well defined (3, 11). The
recombinase is a well characterized member of the latter subfamily.
Both in vitro and in vivo, the activity of the
recombinase depends upon the presence of a chromatin-associated
protein such as Bacillus subtilis Hbsu, the
Escherichia coli HU, or eukaryotic HMG1, both when the
protein mediates deletions between two directly oriented six
sites, or inversions between inversely oriented six sites
(7, 10-12). We have shown that, for DNA resolution, the role of the
chromatin-associated protein is to facilitate the formation of the
recombination complex (11). Our current model holds that the
chromatin-associated protein works by recognizing and stabilizing a DNA
structure at the six site. Our previous work had indicated
that resolution occurred when the two six sites for the
recombinase are directly oriented and two or more Hbsu dimers per DNA
molecule are present. The inversion reaction, which is less defined,
takes place in the presence of 40 Hbsu dimers per DNA molecule and
requires a substrate with two inversely oriented six sites
(7, 12). With the aim of characterizing in detail the inversion
reaction, we have studied the effect of the
recombinase on
substrates containing two inversely oriented six sites. We have found that when such a substrate is supercoiled, both DNA inversion and DNA resolution can occur. The Hbsu concentration could
determine the final direction of the reaction on a supercoiled DNA
substrate. Relaxation of the DNA substrate totally inhibited the
resolution activity of the
recombinase but did not affect its
ability to catalyze inversion reactions. DNA inversion was observed
even on linear DNA substrates. Therefore, the combination of the
recombinase six site and Hbsu allows an unprecedented flexibility of the DNA substrate, making it competent for the assembly
of productive recombination complexes of different geometries.
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MATERIALS AND METHODS |
Bacterial Strains and Plasmids--
The Escherichia
coli strain XL-1 Blue (13) was used as host for DNA manipulations.
Plasmids pCB8 and pCB12 (6) were used as substrates for site-specific
recombination.
Proteins and Reagents--
The
protein was overexpressed and
purified as described previously (6, 14). Purified Hbsu protein was a
gift from Prof. U. Heinemann (Max-Delbrück-Centrum für
Molekulare Medizin, Berlin). Since both wild-type
(14) and Hbsu (6)
proteins are dimers in solution, their concentration is expressed as
mol of protein dimers.
Preparation of Plasmids with Different Superhelical
Densities--
Plasmids pCB8 and pCB12 were relaxed with topoisomerase
I in the presence of ethidium bromide concentrations ranging from 0 to
20 µM, essentially as described (15). The mean
superhelical densities of the samples obtained were calculated, as
described (16), from the linking number difference between the center of the topoisomer distribution of a given sample and the center of the
topoisomer distribution corresponding to relaxed DNA. The amount of DNA
is expressed as mol of plasmid molecules.
In Vitro Assays for Site-specific Recombination--
Plasmids
pCB8, which contains two directly oriented six sites for the
recombinase separated by about 2.3 kb,1 and pCB12, in which the
two six sites are inversely oriented and separated by about
1.4 kb, were used as substrates for site-specific recombination.
Reaction mixtures contained, in a total volume of 25 µl, 10 nM substrate plasmid, in 10 mM bis-Tris
propane-HCl, pH 7.0, 10 mM MgCl2, 10 mM NaCl, 1 mM DTT and proteins Hbsu and
recombinase at the concentrations indicated in the Fig. legends. The
protein-catalyzed resolution was saturated after 30 min of
incubation, whereas saturation of the inversion reaction was achieved
after 3 h. Hence, all reactions were incubated 3 h at 30 °C. To visualize the reaction products by agarose gel
electrophoresis, the DNA was digested with the KpnI and
EcoRV endonucleases. When working at higher ionic strength,
reactions contained 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl2, and the Hbsu
and
recombinase concentrations indicated in the Fig. legends
(6).
Electron Microscopy--
A sample containing supercoiled or
linear DNA (10 nM) was incubated for 30 min at 30 °C, in
the absence or presence of
protein (500 nM) and/or Hbsu
(800 nM), in a solution containing 10 mM triethanolamine-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT in a total volume of 25 µl. Glutaraldehyde was
then added to a final concentration of 0.1%, and allowed to react for
2 h at 30 °C. Samples were diluted with a buffer containing 10 mM triethanolamine-HCl, pH 7.5, 2 mM
MgCl2 to a final concentration of 2 µg/ml of DNA. Samples
were directly adsorbed to freshly cleaved mica, stained with 2% uranil acetate for 2 min and washed in double distilled water. After floating
the samples in water, these were deposited onto copper grids covered
with toluol. After being rotary shadowed with platinum at
10
5 torr, a carbon film was deposited. Finally, replicas
were obtained as described previously (17). Electron micrographs were
taken at 80 kV, routinely at a magnification of × 50,000. Contour
length measurements were carried out on photographic prints using a
Summagraphic-type digitizer tablet.
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RESULTS |
Recombinase Can Catalyze Both DNA Resolution and DNA Inversion
on Supercoiled DNA Substrates with Two Inversely Oriented six
Sites--
Previously, we have demonstrated that
recombinase, in
the presence of Hbsu at concentrations corresponding to about 40 Hbsu dimers per DNA molecule, catalyzes a DNA inversion reaction on a
supercoiled DNA substrate (plasmid pCB12) with two inversely oriented
six sites (6, 7, 18). In this study we found that both DNA
resolution and DNA inversion can occur on supercoiled DNA substrates
with two inversely oriented six sites and that the Hbsu
concentration present in the reaction mixture influenced whether
resolution or inversion products were formed preferentially. In the
presence of 10 nM supercoiled DNA substrate containing two
six sites in an inverse orientation (plasmid pCB12) and an excess of
recombinase (670 nM), the addition of 3-13
Hbsu dimers (30-130 nM) per DNA molecule (1 Hbsu dimer per
1600 to 370 base pairs) led to the appearance of recombination products
corresponding preferentially to a resolution process rather than to the
expected inversion reaction. Nevertheless, at higher Hbsu
concentrations (>150 nM, corresponding to about 15-200
Hbsu dimers per DNA molecule), the amount of resolution products
decreased while inversion products became predominant (Fig.
1, A and B). About
42 and 30% of the total DNA substrate was converted to resolution and
inversion products, respectively, in 3 h at 30 °C.

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Fig. 1.
recombinase can catalyze both DNA
resolution and DNA inversion on supercoiled DNA substrates with two
inverted six sites. A, recombination
reactions were carried out in 10 mM bis-Tris propane-HCl,
pH 7.0, 10 mM MgCl2, 10 mM NaCl, 1 mM DTT, in the presence of 10 nM pCB12, 670 nM recombinase, and increasing concentrations of Hbsu
(16, 34, 67, 135, 270, 540, 1080, and 2160 nM). The
products were digested with endonucleases KpnI and
EcoRV and analyzed by agarose-gel electrophoresis. Upon
inversion of the DNA segment between the two six sites,
digestion of the resulting recombinant molecules should give DNA
fragments 4.1 and 0.8 kb in length, whereas the products from a
resolution reaction between the same sites should yield DNA fragments
of 3.0 and 1.9 kb. Lanes 1 and 2,
correspond to undigested pCB12 (10 nM) incubated in the
absence or presence of 2 µM Hbsu protein. The
non-recombinant plasmid (lane 3; no recombinase
added) generates two bands of 3.5 and 1.4 kb. Except for the unexpected
2.6-kb segment (see text), the DNA fragments corresponding to inversion
or resolution products are indicated. B, the amounts of the
DNA fragments corresponding to inverted or resolved molecules in the
assay shown in panel A were quantitated by
laser-scanning densitometry. The graph shows the reaction efficiency
plotted versus the Hbsu concentration. Reaction efficiency
was calculated relative to the maximum recombination value observed in
each case.
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An additional unexpected DNA fragment of about 2.6 kb, apparently
associated to the resolution process, was also detected, although its
nature is at present unknown. The amount of
recombinase present
(from 30 nM to 2 µM) did not seem to bias the
reaction in either direction (resolution or inversion) (data not
shown).
The addition of 2 µM Hbsu (200 Hbsu dimers per DNA
molecule, or 1 dimer per 25 nucleotides) neither nicked nor unwound
pCB12 DNA (Fig. 1A, lanes 1 and 2, and
data not shown), ruling out the possibility that the decrease in
resolution efficiency at high Hbsu concentrations could be due to
nicking or unwinding of the substrate DNA (see below). It is likely,
therefore, that the effect of
recombinase on supercoiled DNA
substrates containing two inversely oriented six sites is
different from that of the enzymes of the other subfamilies (see the
Introduction) that are very selective for the orientation of the
recombination sites and generate only one product from a particular
substrate; DNA invertases catalyze only inversions and DNA resolvases
are highly specialized in resolution.
The Role of Hbsu in
-Mediated DNA Inversion Is to Facilitate the
Assembly of a Synaptic Complex--
When the substrate of
recombinase is a supercoiled plasmid containing two directly oriented
six sites (pCB8 plasmid DNA), the role of Hbsu is to
facilitate the joining of distant recombination sites to form a
recombination complex (11). To investigate whether Hbsu has the same
function in the inversion reaction, recombination complexes formed with
plasmid pCB12 (10 nM) under conditions which favor DNA
inversion (an excess of Hbsu) were analyzed by electron microscopy
after fixation of the protein-DNA complexes with glutaraldehyde. When
the DNA was incubated separately with
recombinase or Hbsu, neither
recombination complexes nor protein bound to DNA were observed (Fig.
2, A-C). Furthermore, the
addition of 50 dimers of
recombinase (500 nM), or 80 dimers of the Hbsu (800 nM) protein per DNA molecule, did
not seem to affect the level of supercoiling or to relax pCB12 DNA
(Fig. 2, A-C). When both Hbsu and
recombinase were
present in the reaction mixture, about 30 to 40% of the DNA molecules
showed a dot-like complex that held together two DNA segments of the
molecule, dividing the plasmid into two discrete domains. From the
length of the DNA segments in each domain, we infer that the dot-like
structures is located at the position expected for a recombination
complex (Fig. 2, D-F). The proportion of DNA molecules
containing a dot-like structure correlates with the amount of
recombinant products observed by gel electrophoresis.

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Fig. 2.
Role of Hbsu in the recombination reactions
catalyzed by the protein on supercoiled DNA with two inversely
oriented six sites. Supercoiled pCB12 DNA (10 nM) was incubated in the absence or presence of protein
(500 nM) and/or Hbsu (800 nM), and after fixing
with glutaraldehyde, the molecules were adsorbed to mica and visualized
under the electron microscope. A, no protein added;
B, only recombinase; C, only Hbsu;
D-F, both recombinase and Hbsu were present. The
putative synaptic complexes are indicated by
arrowheads.
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Although these assays do not allow us to discriminate whether the
complexes observed correspond to resolution or inversion reactions,
they indicate that Hbsu is essential for the joining of two distant
inversely oriented six sites into a recombination complex,
as was previously shown to be the case when the two six sites are in direct orientation (11).
The Supercoiling Density of the DNA Substrate Dramatically Affects
the Resolution Reaction but Not the Inversion
Reaction--
Supercoiling of the substrate DNA is essential for
site-specific recombinases of the Tn3/
family
(reviewed in Refs. 1 and 2). In the case of
recombinase, resolution
with a plasmid containing two directly oriented six sites
(plasmid pCB8) occurs efficiently if the molecule is supercoiled but is
undetectable if the substrate is relaxed (7). To determine whether
differences exist in the supercoiling requirements of the inversion and
resolution reactions, we relaxed plasmid pCB12 with topoisomerase I and
tested whether the relaxed DNA is a substrate for the inversion or
resolution reactions. Fig. 3 shows that
the relaxed pCB12 did not support the resolution reaction but was as
good a substrate for the inversion reaction as the supercoiled
form.

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Fig. 3.
Resolution and inversion reactions on relaxed
DNA substrates. pCB12 DNA (10 nM) was relaxed by
incubation with topoisomerase I (lanes 1 and
4). Relaxed (R) or supercoiled (SC)
DNA was incubated in the absence (lanes 1-3) or
presence (lanes 4 and 5) of 250 nM Hbsu and 500 nM protein. The products
were analyzed by digestion with endonucleases KpnI and
EcoRV. The lane labeled M corresponds
to a DNA size marker. The DNA fragments corresponding to inversion or
resolution products are indicated.
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To analyze this aspect in more detail, we prepared plasmid populations
with different supercoiling densities from plasmids pCB8 and pCB12 and
scored for DNA recombination. Under standard conditions (10 nM substrate DNA, 500 nM
protein, 300 nM Hbsu), resolution with pCB8 was efficient only for
plasmids having superhelical densities between -0.03 and -0.07; below
and above these values, the reaction efficiency decreased sharply (Fig.
4). Resolution was the only recombination
product observed with pCB8 DNA (Ref. 7; data not shown). Plasmid pCB12
showed that the resolution reaction between two inversely oriented
six sites has a DNA supercoiling requirement similar to that
of a substrate with two directly oriented six sites.
Nevertheless, efficient inversion was observed at all supercoiling
densities tested (Fig. 4). It seems, therefore, that
recombinase
does not strictly require DNA supercoiling for the inversion
reaction.

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Fig. 4.
Effect of the DNA substrate supercoiling
density on the resolution and inversion reactions. Recombination
reactions were carried out with plasmid preparations having different
superhelical densities (see "Materials and Methods"). Plasmids pCB8
(two directly oriented six sites) and pCB12 (two inversely
oriented six sites) were used. Reactions contained 10 nM substrate DNA, 300 nM Hbsu, and 500 nM protein. Samples were digested with endonucleases
KpnI and EcoRV, and the fragments generated were
resolved by agarose-gel electrophoresis. The amounts of the DNA
fragments corresponding to inverted or resolved molecules were
quantitated by laser-scanning densitometry. The graph shows
the reaction efficiency plotted versus the superhelical
density of the plasmid preparation (circles correspond to
resolution on pCB8, squares to resolution on plasmid pCB12,
and triangles to inversion on pCB12). Reaction efficiency
was calculated relative to the maximum recombination value observed in
each case; similar amounts of unreacted material were present in all
cases.
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Recombination was not detected between two negatively supercoiled or
linear plasmid substrates, each containing a single six site
(intermolecular recombination; data not shown).
The
Recombinase Catalyzes Inversion on a Linear DNA with
Inversely Oriented six Sites--
Since, as shown above, the
recombinase efficiently catalyzed DNA inversion on a relaxed substrate
containing two inversely oriented six sites, we asked
whether this reaction could also take place when the substrate was in
linear form. Plasmid pCB12 was linearized with KpnI and used
as substrate for recombination. As revealed in Fig.
5, under standard conditions inversion
products were readily observed. The reaction efficiency was similar, or even higher, than that obtained with a supercoiled pCB12 DNA. DNA
recombination between two inversely oriented six sites on linear DNA strictly required the presence of the accessory protein Hbsu
(data not shown).

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Fig. 5.
A linear DNA with inversely oriented
six sites is a substrate for recombinase. Plasmid
pCB12 was linearized with endonuclease KpnI and used as
substrate for recombination in the absence (-) or presence (+) of
proteins Hbsu and recombinase, as indicated. Samples were
subsequently digested with endonuclease EcoRV, and the
fragments generated were resolved by agarose-gel electrophoresis. The
DNA fragments corresponding to nonrecombinant plasmid (3.4 and 1.4, marked as nr), or to inversion products (4.1 and 0.8, marked
as inv), are indicated. Hbsu was present at 500 nM, pCB12 at 10 nM, and the recombinase at
248, 496, 992, or 1984 nM. The lane labeled as
M corresponds to a DNA size marker.
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To investigate the recombination products formed with linear pCB12
substrates, the protein-DNA complexes were fixed with glutaraldehyde and analyzed by electron microscopy. When the linear DNA was incubated separately with either
recombinase (500 nM) or Hbsu
(800 nM), neither recombination complexes nor protein dots
sitting on DNA were observed (data not shown). In the presence of both
(500 nM) and Hbsu (800 nM), about 30 to
40% of total DNA molecules were complexed with proteins. The gallery
presented in Fig. 6, which is
representative of the results obtained, was assembled with those DNA
molecules in which a dot-like structure was located at the position
expected for a recombination complex (77% of complexed molecules,
n = 90). The length of the DNA segments at each side of
the dot-like structure was measured in 39 molecules. The length of the
long arm was about 2821 ± 216 bp, that of the loop was 1,889 ± 197 bp, while the short arm was 167 bp ± 67 in length; the
length measured for the protein-free plasmid was 4,984 ± 55 bp.
It is likely, therefore, that the dot-like structures represent a
synaptic complex formed by the joining of the two distant inversely oriented six sites present in the linear molecule.

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Fig. 6.
Recombination complexes formed by recombinase on relaxed DNA substrates. KpnI-linearized pCB12
DNA was incubated in the absence or presence of recombinase and/or
Hbsu, and after fixing with glutaraldehyde, the molecules were adsorbed
to mica and visualized under the electron microscope. Panels
A to J show different examples of molecules
containing a nucleoprotein complex that organized the DNA into a short
arm, a loop, and a long arm whose estimated lengths corresponded to
those expected for the joining of the two six sites into a
synaptic complex (a scheme is shown on the
bottom). These molecules represented about 77% of the
dot-containing samples. Panel K shows the linear
DNA molecules used as a substrate in the absence of Hbsu and protein.
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In three cases (3.3%), the protein dot was bound to one DNA strand,
whereas in the remaining 20% of the cases (n = 18),
two or more individual DNA molecules bound to a protein dot were
observed (data not shown). These complexes, which are probably
preparation artifacts, were not further analyzed.
From these results, we conclude that the role of Hbsu with linear
substrates is to facilitate the formation of a recombination complex,
as it does with supercoiled plasmids (see Fig. 2).
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DISCUSSION |
Recombinase Does Not Show a Directionality Bias on a
Supercoiled Substrate with Two Inversely Oriented six Sites--
The
control of the reaction directionality of DNA resolvases and DNA
invertases results from the formation of higher order protein-DNA
complexes which control the DNA geometry within the synaptic complex.
This discrimination is further enhanced by the free energy associated
with DNA supercoiling (reviewed in Refs. 1 and 2). Our results show
that
recombinase can catalyze both DNA resolution and DNA inversion
on negatively supercoiled DNA substrates containing two inversely
oriented six sites, and that the type of reaction
(resolution or inversion) is determined to a significant extent by the
amount of Hbsu available relative to DNA substrate. The effect of Hbsu
in the directionality of the reaction is not the consequence of a
nicking of the DNA by contaminant nucleases since addition of an excess
of Hbsu did not nick the DNA. Previously, it has been shown that 10-13
dimers of HU (E. coli counterpart of Hbsu, see the
Introduction) per DNA molecule are required to generate a superhelical
turn on a relaxed DNA molecule (reviewed in Ref. 19). The results
reported in this work show that the efficiency of
-mediated DNA
resolution on a substrate with two inversely oriented six
sites decreases in the presence of an excess of Hbsu (Fig. 1), or at
superhelical densities above or below the optimal values (between
-0.04 and -0.07; Fig. 3). It is likely, therefore, that the decrease
in resolution efficiency observed at high Hbsu concentrations occurs, at least in part, because Hbsu increases the DNA supercoiling above the
optimum limits for resolution.
Unlike DNA resolution, DNA inversion was not sensitive to the level of
negative supercoiling of the substrate DNA, which suggests that Hbsu
can probably favor the formation of recombination complexes with two
different geometries, one of them leading to resolution and the other
one to inversion products. If this hypothesis is correct, whether
recombinase will catalyze resolution or inversion on these substrates
will depend on the DNA geometry within the synaptic complex rather than
on DNA site orientation discrimination. It should be taken into account
that this model would hold only when the substrate has two inversely
oriented six sites since, when the two six sites
are directly oriented, only DNA resolution occurs, independently of the
Hbsu concentration (6, 7).
Analysis of recombination complexes by electron microscopy showed that,
both in the inversion and resolution reactions, the role of Hbsu is to
stabilize the formation of the recombination complex (Ref. 11; this
work). Since
protein catalyzes DNA inversion between two inversely
oriented sites on linear DNA, we can rule out that the role of Hbsu is
solely to increase the supercoiling of the DNA substrate. The
stabilization of the recombination complex requires overcoming an
energy barrier to stabilize a DNA conformation that allows the proper
alignment of the two inversely oriented six sites in a
productive recombination complex which, after strand exchange,
generates recombination products. The results reported show that the
presence of 3 to 13 Hbsu protein dimers per supercoiled DNA molecule
helps to stabilize preferentially a complex with a geometry leading to
resolution products. When 15-200 Hbsu dimers per supercoiled molecule
are present, a recombination complex with a different geometry is
stabilized which, after strand exchange, yields inversion products.
We have previously shown that a mammalian HMG1 protein, which shares
neither sequence nor structural homology with Hbsu, or even plastid
HlpA, can stimulate
-mediated recombination (11, 20). These
chromatin-associated proteins bind preferentially to DNA sequences
showing bent or altered conformations (21-23). It is likely,
therefore, that the way through which the chromatin-associated protein
participates in the reaction is by stabilizing one or more bent DNA
conformations that arise upon the assembly of a recombination complex.
This complex should be stable enough to allow the recombinase molecules
to assemble and undergo the conformational changes necessary to adopt a
catalytically active state. A role for Hbsu in binding bent or altered
DNA structures would allow a high flexibility for its positioning in
the recombination complex: it may bind to more than one position,
provided that the DNA can be distorted, and that the protein can
physically fit into the complex. We believe that it is this
characteristic which confers less selectivity to the
recombinase
with respect to the final direction of the recombination reaction
(resolution or inversion) when the two
protein six sites
are in an inverted orientation.
Formation of Synapsis Depends on Conformational Effects--
As
discussed above, the
recombinase requires negative supercoiling of
the DNA substrate for the resolution reaction but not for the inversion
reaction. Productive recombination complexes were formed even on linear
DNA molecules containing inversely oriented six sites. This
behavior is different from that of the DNA resolvases or DNA
invertases. The analysis of the role of DNA supercoiling on the
recombination reactions catalyzed by DNA resolvases and DNA invertases
has led to the conclusion that the critical mechanistic assistance
provided by supercoiling leading to the formation of a synapsis depends
on conformational effects while the transition to postsynapsis and
strand exchange relies on torsional stress; the latter process requires
a higher superhelical density than synapsis (24, 25). DNA invertases
require an accessory protein, FIS, to mediate inversion and reaction
selectivity (26). FIS-independent mutant derivatives of the Gin
invertase do not require supercoiled DNA and are even active on linear
DNA substrates, albeit with a low efficiency. Those mutations lie in
the dimer interface and may render a constitutively active recombinase
(27, 28). FIS-independent Gin mutants have also lost the selectivity
for inversion. Furthermore, the requirement for supercoiling by DNA
resolvases can be circumvented by using special "permissive"
reaction conditions (5, 29). The
recombinase resembles in many
respects the FIS-independent Gin invertase mutants or DNA resolvases
assayed under special "permissive" reaction conditions (5, 28, 29).
An essential difference between the
and Gin systems is that FIS
binds to a specific site providing a directionality to the reaction,
while Hbsu could in principle bind to any region of the DNA in the
recombination complex that has an appropriately bent structure. This
characteristic is most likely what allows the
protein to form
recombination complexes of different geometries. It is worth mentioning
that, like other DNA resolvases of this family (24, 25), the
protein can form synaptic complexes with a relaxed DNA substrate
containing two directly oriented six sites, as judged by
electron microscopy (data not shown), although these complexes are not
productive (Figs. 3 and 4).
Finally, our results may shed some light on how the recombination sites
find each other to form the recombination complex. Several models have
been proposed to account for this process. Recombination sites could
initially juxtapose either by random collision (30, 31) or by
slithering in a plectonemically interwound molecule (32). When the
substrate DNA is circular, it is proposed that a productive
interwrapping of the sites and cognate proteins can only be achieved
when the recombination sites exist in the substrate DNA molecule in a
particular geometry that would impose a "topological filter" on the
reaction (31). When the sites are not in the appropriate orientation,
the energetic cost of distorting the DNA to achieve productive synapsis
is probably too high and recombination is precluded (25, 30, 31). We suggest that the
recombinase, in the presence of Hbsu, probably forms the recombination complex by random collision because inversion occurs efficiently on linear substrates. In linear DNA, encountering of
the recombination sites by slithering would only be successful when the
DNA is moving in one direction relative to the first target site for
the recombinase since the slithering in the opposite direction would
reach the end of the molecule before the second target is found. This
would considerably diminish recombination efficiency, and the
protein seems to be equally efficient with linear and supercoiled
molecules. In a supercoiled substrate stabilization of a distorted DNA
by Hbsu, binding could overcome the energetic cost of reorienting the
inappropriate orientation of the inverted six sites to
achieve productive synapsis; in the presence of an excess of Hbsu, the
constraints on the DNA impose a barrier for such a reorientation and
the juxtaposed, properly oriented sites form a synaptic complex.
We are very grateful to U. Heinemann for the
gift of purified Hbsu protein, to Martin Boocock and Marshall Stark for
useful comments on the manuscript, and to T. A. Trautner for
continuous interest in the project.