From the Department of Molecular and Medical Genetics, University
of Toronto, Toronto, Ontario M5S 1A8, Canada
The Flp recognition target site contains two
inverted 13-base pair (bp) Flp binding sequences that surround an 8-bp
core region. Flp recombinase has been shown to carry out strand
ligation independently of its ability to execute strand cleavage. Using
a synthetic activated DNA substrate bearing a 3'-phosphotyrosine group,
we have developed an assay to measure strand exchange by Flp proteins.
We have shown that wild-type Flp protein was able to catalyze strand
exchange using DNA substrates containing 8-bp duplex core sequences.
Mutant Flp proteins that are defective in either DNA bending or DNA
cleavage were also impaired in their abilities to carry out strand
exchange. The inability of these mutant proteins to execute strand
exchange could be overcome by providing a DNA substrate containing a
single-stranded core sequence. This single-stranded core sequence could
be as small as 3 nucleotides. Full activity of mutant Flp proteins in strand exchange was observed when both partner DNAs contained an
8-nucleotide single-stranded core region. Using suicide substrates, we
showed that single-stranded DNA is also important for strand exchange
reactions where Flp-mediated strand cleavage is required. These results
suggest that the ability of Flp to induce DNA bending and strand
cleavage may be crucial for strand exchange. We propose that both DNA
bending and strand cleavage may be required to separate the strands of
the core region and that single-stranded DNA in the core region might
be an intermediate in Flp-mediated DNA recombination.
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INTRODUCTION |
The Flp gene of the 2-µm circle plasmid of Saccharomyces
cerevisiae encodes a conservative site-specific recombinase (Flp) that is involved in amplification of the plasmid (1-3). The Flp protein is a member of the integrase family of recombinases whose members share a tetrad of conserved residues (arginine 191, histidine 305, arginine 308, tyrosine 343; numbers represent amino acid numbers of Flp; see Refs. 4 and 5). The mechanism of Flp action is
similar in many respects to that of the bacteriophage
integrase and
the Cre recombinase of bacteriophage P1 (6-8).
Flp mediates a recombination event between two
599-bp1 inverted repeats.
Embedded in each repeat are two Flp recognition target (FRT) sites that
are the targets of action of Flp (Fig.
1). The FRT site contains two inverted
13-bp symmetry elements (a and b) which surround
an 8-bp core region (open box). A third 13-bp symmetry
element (c) is in direct orientation with element
b but is dispensable for recombination both in
vivo and in vitro (9-11). Flp binds to each of the
symmetry elements in an ordered fashion; binding to the b
element occurs first, then binding to a, and finally binding
to c (12, 13). Binding of an Flp molecule to a single symmetry element results in a bend of approximately 60°, whereas binding of Flp molecules to two symmetry elements (a and b) flanking the core region results in a severe bend of greater than 144° (referred to as a type II bend; see Refs. 14 and
15). Intermolecular protein-protein interactions between Flp molecules
bound to FRT sites bring two partner FRT sites together to form a
synaptic complex (16). Nucleophilic attack on the DNA phosphodiester
bond at the margins of the core region by tyrosine 343 of Flp results
in strand cleavage, generating a free 5'-OH and a phosphotyrosine bond
between the 3'-phosphoryl end of the nicked DNA and the protein (17,
18). Strand exchange and ligation take place when the free 5'-OH of the
nick from one of the partner DNAs acts as a nucleophile to attack the
phosphotyrosine bond of the other partner and generate a new
phosphodiester bond. As a result of this event, a Holliday junction is
formed (19-22). This junction is resolved to yield recombinant
products by a second set of Flp-mediated strand cleavages and ligations
(23, 24). The active site of Flp is constituted from two molecules of
Flp (25). Cleavage by Flp takes place in trans-horizontal
fashion: an Flp molecule activates the scissile phosphodiester bond for a nucleophilic attack by tyrosine 343 of the Flp molecule that is bound
across the core from the bond to be cleaved (26, 27). However, ligation
occurs in cis, i.e. the monomer bound adjacent to
the site of cleavage executes ligation at that site (28). Flp can carry
out strand ligation independently of its ability to cleave and attach
covalently to the DNA (29).

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Fig. 1.
Diagram of the Flp recognition target site
(FRT site). The FRT site is characterized by three 13-bp symmetry
elements (a, b, and c,
horizontal arrows). Elements a and b
are in inverted orientation, flanking an 8-bp core region (open
box). Element c is in direct orientation with element
b and is dispensable for DNA recombination both in
vivo and in vitro. Flp-mediated cleavage sites are
indicated by two vertical arrows.
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Although we have learned a considerable amount about the mechanisms of
Flp-mediated DNA binding, bending, strand cleavage, ligation, and
resolution (6, 7, 30), little is known about the mechanism of DNA
strand exchange, the process by which two strands are exchanged and
ligated to the partner DNAs in the synaptic complex. One possible
mechanism that may contribute to this process could be the separation
of DNA strands of the 8-bp core regions of the FRT sites and
reestablishment of base pairings in the newly made hybrid core regions.
It has been suggested that Flp-mediated DNA bending and cleavage may
facilitate the process of separation of DNA strands in the core region
(31).
In this study we report the development of an assay to measure
Flp-mediated strand exchange that occurs independently of DNA cleavage.
We have shown that a single-stranded core region in the FRT site can
overcome the defect in strand exchange of certain mutant Flp proteins
that are deficient in either bending or strand cleavage. These results
suggest that both DNA bending and strand cleavage may be required to
separate the strands in the core region and that single-stranded DNA in
the core region may play a role in the process of strand exchange by
Flp.
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MATERIALS AND METHODS |
Flp Preparations--
Flp proteins were purified as described
previously by Pan et al. (32). The purity of wild-type Flp
was >90%, and mutant Flp proteins were greater than 60% pure. Mutant
proteins with purity of greater than 60% exhibit the same activity as
those purified to >95% when assayed for various steps of
recombination such as DNA binding, strand cleavage, and covalent
attachment, strand exchange, and ligation. The concentration of Flp was
estimated by densitometric comparison with highly purified Flp
standards on a Coomassie Blue-stained SDS-polyacrylamide gel. The
Bradford (33) assay was used to determine the concentration of the
homogeneous Flp standards. Flp plasmids encoding Flp proteins Y343F and
Y343S were obtained from M. Jayaram (Ref. 41; Y343F means that the tyrosine normally present at position 343 has been replaced by a
phenylalanine). The Flp gene bearing a 4-amino acid insertion at
position 115 (Ins115) was isolated in our laboratory (34).
Synthetic Substrates--
The full-FRT and half-FRT sites were
assembled by annealing complementary synthetic oligonucleotides in 5 mM MgCl2, 100 mM NaCl as described
previously (27, 35). The oligonucleotide containing
3'-phosphoryltyrosine was synthesized by R. Brousseau and C. Juby at
the Biotechnology Research Institute, Montreal, Quebec, Canada, as
described previously (36). All other oligonucleotides were synthesized
by the Carbohydrate Research Center, Faculty of Medicine, University of
Toronto. The sequences of the oligonucleotides forming half-FRT sites
and full-FRT sites are shown in Table I, and the substrates are illustrated in Table
II.
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Table I
Synthetic oligonucleotides used in this study
The underlined sequence represents the FRT site. The sequence of the
symmetry elements is shown in italics, and the core sequence is in bold. Where appropriate, oligonucleotides 2, 5, 11 were phosphorylated at their 5'-ends using T4 polynucleotide kinase (New England Biolabs) and cold ATP to give oligonucleotides 2', 5', and
11'.
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In Vitro Strand Exchange Assays--
Strand exchange assays by
Flp were done essentially as described by Zhu and Sadowski (27). DNA
substrates (0.02 pmol) were incubated with Flp proteins (~2.54 pmol)
in 30 µl of binding buffer containing 50 mM Tris-HCl (pH
7.5), 33 mM NaCl, 1 mM EDTA, and 2 µg of
sonicated and denatured calf thymus DNA at room temperature for 60 min
by which time the ligation products had reached their maximal levels. A
further 1-h incubation did not change the level of the reaction
products. The reaction was stopped by the addition of 3 µl of 10%
SDS and 10 µg of proteinase K, followed by incubation at 37 °C for
30 min. DNA was then extracted twice with phenol/chloroform, precipitated with ethanol, and redissolved in 10 mM
Tris-HCl buffer (pH 7.5) containing 1 mM EDTA. The strand
exchange products were analyzed on an 8% denaturing polyacrylamide gel
and quantitated using a Molecular Dynamics PhosphorImager. Each
experiment was repeated two to four times with similar results.
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RESULTS |
Mutant Flp Proteins Are Defective in Strand Exchange--
To
examine whether the abilities of Flp proteins to induce DNA type II
bending and to execute DNA cleavage were correlated with their
abilities to catalyze strand exchange, we carried out strand exchange
assays with wild-type Flp and mutant Flp proteins that are defective in
DNA type II bending and strand cleavage. As diagrammed in Fig.
2a, assays were carried out
between two full-FRT sites, one being the DNA acceptor containing a
3'-phosphotyrosine and the other being the DNA donor. The two
substrates were designed such that they both contained a nick at the
Flp cleavage site adjacent to symmetry element a. The nick
of the DNA acceptor contained the 3'-phosphotyrosine that was competent
for ligation and a 5'-PO4 group that would inhibit
intramolecular ligation on the same strand, thus allowing only
intermolecular strand exchange and ligation to occur. The nick of the
DNA donor bore a 3'-OH and a 5'-OH. The 5'-OH at the nick served as a
nucleophile for intermolecular strand exchange ligation, but
intramolecular ligation of the donor DNA could not occur because there
was no leaving group on the 3'-OH. Therefore, these DNA substrates
allowed us to measure specifically strand exchange by Flp proteins. If
Flp proteins were capable of carrying out strand exchange between the DNA acceptor and the DNA donor, the 32P-labeled strand
exchange products that were 40 nucleotides in length would be detected
on a denaturing polyacrylamide gel.

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Fig. 2.
Strand exchange assays between two nicked
full-FRT sites, the acceptor DNA (A) and the donor DNA
(D). Panel a, diagram of substrates and products
of strand exchange assays. Both the DNA acceptor and the donor contain
two Flp-binding symmetry elements a and b
flanking a duplex core region as well as a nick at the cleavage site
adjacent to element a. The nick of the DNA acceptor bears a
3'-phosphotyrosine group and a 5'-PO4 group. The
5'-PO4 group at the nick prevents ligation on the same DNA
strand, whereas the 3'-phosphotyrosine group is available for ligation
of an incoming strand. The DNA donor contains both 5'- and 3'-OH groups
at its nick. The 5'-OH at the nick serves as a nucleophile for
intermolecular ligation. The phosphotyrosine-containing strand of the
DNA acceptor was 5'-end labeled by 32P
(asterisk). If Flp-mediated strand exchange takes place, a
labeled DNA product 40 nucleotides (nt) long will be formed
and detected on an 8% denaturing polyacrylamide gel. In this and
subsequent figures, horizontal arrows represent Flp-binding
elements, and the open boxes refer to 8-bp duplex core
regions. Panel b, analysis of strand exchange by wild-type
Flp and mutant Flp proteins Ins115, Y343F, and Y343S. The reactions
shown here and in subsequent figures were done as described under
"Materials and Methods." Flp proteins were incubated with acceptor
and donor DNA substrates at room temperature for 60 min. Strand
exchange products were analyzed on an 8% denaturing polyacrylamide
gel. The contents of each reaction are shown above the
lanes. The small amount of product in lanes 3,
5, 7, and 9 provides a measure of the
amount of the oligonucleotide that escaped phosphorylation by T4
polynucleotide kinase. In this and subsequent figures, SEP
represents strand exchange products, and S refers to the
32P-labeled oligonucleotides.
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As shown in Fig. 2b, assays were carried out with wild-type
Flp as well as three mutant Flp proteins, Flp Ins115, Y343F, and Y343S
(Table III). Flp Ins115 is competent for
strand cleavage but is deficient in DNA type II bending (34), whereas
Flp Y343F is deficient in strand cleavage but competent for DNA type II bending (28). Flp Y343S is deficient in both strand cleavage and DNA
type II bending (14, 15). All proteins were competent for
intramolecular strand ligation as assayed by hairpin substrates and
linear nicked substrates containing a 5'-OH and a 3'-phosphotyrosyl group at the nick (28).2 As
shown in Fig. 2b, wild-type Flp was able to promote strand exchange, converting about 19.0% of substrate to strand exchange products (lane 4 and Table
IV), whereas both Flp Ins115 and Flp Y343F exhibited reduced strand exchange activity, converting less than
3% of substrate to products (lanes 6 and 8 and
Table II). No strand exchange product was detected with Flp Y343S
(lane 10 and Table II). These results suggest that the
abilities of Flp proteins to induce DNA type II bending and to carry
out strand cleavage are crucial for strand exchange. Alternatively, it
is possible that these mutant proteins are defective in strand exchange independent of their defects in bending and cleavage.
A Single-stranded Core Region in the DNA Acceptor Substrates
Mitigates the Defects of Mutant Flp Proteins in Strand
Exchange--
If DNA type II bending and strand cleavage play a role
in separation of DNA strands of the core region and facilitate strand exchange, then the defects in strand exchange resulting from the inabilities of mutant Flp proteins either to induce DNA bending (Ins115) or to execute strand cleavage and covalent attachment (Y343F)
or both (Y343S) might be suppressed by providing substrates containing
single-stranded core regions. To test this hypothesis, we carried out
strand exchange assays between the DNA acceptor (A1) containing a
single-stranded core region and the DNA donor (D) containing a duplex
core region as illustrated in Fig.
3a. DNA donor D was the same
as that diagrammed in Fig. 2. However, DNA acceptor A1 differed from
the DNA acceptor A shown in Fig. 2 in that A1 had an 8-nucleotide
single-stranded core region, whereas acceptor A had an 8-bp duplex core
region.

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Fig. 3.
Panel a, schematic diagram of strand
exchange between the nicked DNA donor and the DNA acceptor containing a
single-stranded core region. Both donor and acceptor DNA contained two
Flp-binding elements (a and b). The nicked DNA
donor D was described in Fig. 2. Unlike the DNA acceptor A in Fig. 2,
which had an 8-bp duplex core, acceptor A1 contained an 8-nucleotide
(nt) single-stranded core region. Panel b,
analysis of strand exchange by Flp proteins. AB represents
aberrant products resulting from ligation of the top strand of the DNA
acceptor to the phosphotyrosine-containing oligonucleotide by Flp
proteins. HP represents hairpin products resulting from
ligation of the top cleaved strand of the DNA acceptor to the
phosphotyrosine-containing oligonucleotide by Flp Ins115.
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As shown in Fig. 3b, when the DNA acceptor in the reaction
contained an 8-nucleotide single-stranded core region, the level of
strand exchange products made by wild-type Flp was increased by about
2-fold compared with that when the DNA acceptor containing a duplex
core region was used (Fig. 3b, lane 3 versus Fig.
2b, lane 4); approximately 37.1% of the
substrate was converted to products (Table IV). More importantly, the
single-stranded core region of acceptor A1 stimulated strand exchange
reactions by Flp Ins115 and Flp Y343F by more than 10-fold (Fig.
3b, lanes 4 and 5 versus Fig.
2b, lanes 6 and 8). As summarized in
Table IV, approximately 23.5% of the substrate was converted to
products by Flp Ins115, whereas about 15.0% of the substrate was
converted to products by Flp Y343F. Furthermore, when the core region
of the acceptor was single-stranded, Flp Y343S was also able to convert about 4.9% of the substrate into strand exchange products (Fig. 3b, lane 6 and Table IV). These results show that
defects of mutant Flp proteins in strand exchange accompanied by their
inabilities either to form DNA type II bend or to execute strand
cleavage, or both, can be overcome by the presence of the
single-stranded core region of the DNA acceptor. This suggests that a
single-stranded core region of the DNA acceptor might be involved in
Flp-mediated DNA recombination.
A Single-stranded Core Region in the DNA Donor Also Overcomes the
Defects of Mutant Flp Proteins in Strand Exchange--
Because a
single-stranded core region of the acceptor facilitated strand exchange
by Flp proteins, it was of interest to investigate whether a
single-stranded core region of the DNA donor had a similar effect on
strand exchange. To answer this question, we carried out strand
exchange assays between an acceptor DNA containing a full-FRT site and
a donor DNA containing a half-FRT site (Fig. 4a). The latter substrate
enabled us to make single-stranded DNA in the 5'-OH terminus of the
core region independent of cleavage of the donor DNA. As shown in Fig.
4a, DNA donors D1 and D5 differed in that D1 contained an
8-nucleotide protrusion of the core region with a 5'-OH, whereas donor
D5 contained an 8-bp duplex core region with a 5'-OH. The DNA acceptor
was the same as that described in Fig. 2, bearing a nick at the
cleavage site adjacent to element a. The nick contained a
3'-phosphotyrosine and a 5'-PO4. As shown in Fig.
4b, when the half-FRT site donor DNA with an 8-nucleotide
protrusion was used in the reaction, wild-type Flp and mutant Flp
proteins produced similar levels of strand exchange products,
converting about 26.0% (wild-type Flp), 22.1% (Ins115), 18.2%
(Y343F), and 18.1% (Y343S) of the substrates to products (Fig.
4b, lanes 3, 6, 9 and,
12, respectively, and Table IV). However, when the core
region of donor DNA was rendered double-stranded, the level of strand
exchange products decreased by approximately 3.6-fold for wild-type
Flp, 5.2-fold for Ins115, 8.5-fold for Y343F, and 36-fold for Y343S
(Fig. 4b, lanes 4, 7, 10,
and 13, respectively, and Table IV). These results
demonstrate that a single-stranded core region of donor DNA had an
effect on strand exchange similar to that of a single-stranded core of
acceptor DNA, overcoming the defects of mutant Flp proteins in strand
exchange.

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Fig. 4.
Panel a, schematic diagram of strand
exchange between the nicked DNA acceptor and half-FRT site donors. The
two half-FRT site donors contained only element b and
differed from each other in that one had an 8-nucleotide
(nt) protrusion (D1), and the other had an 8-bp duplex core
(D5). The 5'-OH groups of the bottom strands of the DNA donors served
as nucleophiles for ligation after strand exchange. DNA acceptor A was
described in Fig. 2. Panel b, analysis of strand exchange by
Flp proteins. The small amount of product in lanes 2,
5, 8, and 11 provides a measure of the
amount of the oligonucleotide that escaped phosphorylation by T4
polynucleotide kinase.
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Defects of Mutant Flp Proteins in Strand Exchange Can Be Overcome
by the Core Region Containing a 3-Nucleotide Protrusion--
To
investigate the minimum degree of single-stranded DNA in the core
region required for strand exchange, assays were first carried out
between a full-FRT site acceptor DNA and a half-FRT site donor DNA
containing various degrees of single-stranded core region as diagrammed
in Fig. 5a. Although a
complete 8-nucleotide single-stranded donor core permitted strand
exchange by mutant Flp proteins, it was not essential. As shown in Fig.
5b, a donor core containing only a 3-nucleotide protrusion
was also able to stimulate strand exchange by mutant Flp proteins to a
level similar to that achieved with the 8-nucleotide donor core
(lanes 6, 11, and 17 versus lanes 3,
8, and 14). Similar results were also obtained by
wild-type Flp (data not shown).

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Fig. 5.
Schematic representation of strand exchange
between the nicked full-FRT acceptor and a series of half-FRT donor
DNAs containing various amounts of single-stranded DNA in the core
region. DNA acceptor A and DNA donors D1 and D5 were described in
Fig. 4. The DNA donors (D2-D4) are half-FRT sites containing only
element b and the core sequence. D2, D3, and D4 had 5, 4, and 3 unpaired nucleotides (nt) in the bottom strand of the
core region, respectively. Short vertical lines in the core
region represent unpaired nucleotides; long vertical lines
in the core represent base-paired nucleotides. Panel b,
analysis of strand exchange products made by mutant Flp proteins. The
small amount of product in lanes 2, 7, and
13 is an indication of incomplete phosphorylation by T4
polynucleotide kinase.
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Unlike mutant proteins Ins115 and Y343F, mutant protein Flp Y343S
seemed to use the donor DNA (D4) containing a 3-nucleotide single-stranded protrusion more efficiently than the donor DNA (D3)
containing a 4-nucleotide single-stranded protrusion (Fig. 5b, lane 17 versus lane 16). It is not clear why
Flp Y343S behaves differently from mutant proteins Ins115 and
Y343F.
Similar experiments were also carried out to examine the requirement of
single-stranded DNA in the core region of the acceptor to allow strand
exchange by mutant Flp proteins. As shown in Fig. 6, the acceptor core containing
3-nucleotide single-stranded DNA also overcame defects of mutant Flp
proteins in strand exchange. These results suggest that DNA strands of
the core region might be at least partially separated during strand
exchange.

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Fig. 6.
Panel a, representation of strand
exchange between the nicked DNA donor and the DNA acceptor containing a
3-nucleotide single-stranded region in the core adjacent to the
cleavage site in the core. Both acceptor and donor DNA contained two
Flp-binding elements flanking the core sequence. DNA donor D and
acceptor A were described in Fig. 2. Short vertical lines in
the core region represent the unpaired oligonucleotides
(nt); long vertical lines in the core represent
base-paired nucleotides. DNA acceptors A2 and A were 5'-end labeled
with 32P on the bottom strand as indicated by an
asterisk. Panel b, analysis of strand exchange
products made by mutant Flp proteins. The small amount of product in
lanes 1, 4, and 7 is an indication of
incomplete phosphorylation by T4 polynucleotide kinase.
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Strand Exchange Is Stimulated Greatly When Both Donor and Acceptor
Contain a Single-stranded Core Region--
Because a single-stranded
core region in either the donor or the acceptor DNA was able to
stimulate strand exchange by Flp proteins, it was of interest to know
whether the presence of single-stranded core regions in both the donor
and the acceptor DNA would maximize strand exchange. We found that
strand exchange reactions by Flp proteins were stimulated greatly when
both donor and acceptor DNA contained a single-stranded core (Fig.
7). Wild-type Flp and mutant Flp proteins
were able to convert approximately 70% of substrates to products
(Table IV). This exceeded the sum of levels of products when either the
donor or the acceptor core was single-stranded. These results strongly
suggest that the single-stranded core region plays an important role in
strand exchange. Furthermore, the fact that mutant Flp proteins that
are defective in either cleavage or type II bending or both can carry
out efficient strand exchange in the presence of the
single-stranded core region in both the donor and the acceptor DNA
indicates that Flp-mediated type II bending and cleavage activities may
play an important role in separating the core sequence of DNA before
strand exchange.

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Fig. 7.
Panel a, representation of strand
exchange between a full-FRT DNA acceptor and the half-FRT DNA donor.
DNA acceptor A1 and donor D1 were described in Figs. 3 and 4,
respectively, both containing an 8-nucleotide (nt)
single-stranded core sequence. Panel b, analysis of strand
exchange by Flp proteins. The substrates and protein are indicated
above each lane.
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These results are not simply attributable to intermolecular strand
ligation facilitated by base pairing between the single-stranded core
regions of donor and acceptor DNA because the levels of strand exchange
exceed those obtained between acceptor A1 and a single-stranded donor DNA that lacks Flp binding symmetry element b (data not shown and Ref. 35). These results imply that Flp bound to duplex
substrate D1 brings it into a synaptic complex and therefore enhances
the reaction.
A Single-stranded Core Region Also Plays a Crucial Role in
Cleavage-dependent Strand Exchange by Mutant Protein Flp
Ins115--
The experiments described so far have used activated
substrates containing a 3'-phosphotyrosine leaving group. These
substrates do not require the covalent attachment of Flp to the
3'-phosphoryl group to carry out strand exchange and ligation. It was
of interest to know whether single-stranded DNA might also play a role
in a strand exchange reaction where DNA substrates need to be cleaved (cleavage-dependent strand exchange). To answer this
question, we carried out strand exchange assays by Flp Ins115 between
the nicked DNA donor D and a "suicide substrate" (37).
Flp Ins115, like wild-type Flp, is able to cleave and attach covalently
to the linear FRT site but fails to carry out strand exchange between
two linear FRT sites as well as between a linear FRT site and the
nicked DNA donor D shown in Fig. 2
(34).3 We wished to know
whether single-stranded DNA might overcome the defect of this mutant
protein in a cleavage-dependent strand exchange reaction.
As shown in Fig. 8a, suicide
substrates enabled us to couple single-stranded DNA with Flp-mediated
strand cleavage and covalent attachment. Suicide substrates Su-1, Su-2,
and Su-3 contain a nick on the bottom strand of the core region 3, 4, and 5 nucleotides away from the a cleavage site,
respectively. Upon Flp-mediated cleavage and covalent attachment, a
single-stranded region containing 3, 4, and 5 nucleotides was generated
in the core region of Su-1, Su-2, and Su-3, respectively (Fig.
8a). As shown in Fig. 8b, Flp Ins115 was able to
execute strand exchange between suicide substrates and donor D
(lanes 3, 6, and 9). These results
suggest that single-stranded DNA also plays an important role in the
strand exchange reaction where strand cleavage is required. Flp Ins115
was able to use suicide substrates Su-3 and Su-2 for strand exchange
more efficiently than substrate Su-1 (Fig. 8b, lanes
6 and 9 versus lane 3), indicating that the amount of
single-stranded DNA in the core region of the FRT site may also be
crucial for strand exchange. Consistent with the data obtained in Figs.
5 and 6, a 3-nucleotide single-stranded DNA in the core was also able
to suppress the defect of Flp Ins115 in strand exchange where strand
cleavage is required. Wild-type Flp was able to carry out strand
exchange and ligation readily regardless of whether the suicide
substrates contained a nick 3, 4, or 5 nucleotides away from the
a cleavage site (Fig. 8b, lanes 2,
5, and 8). These results are also consistent with the data obtained when 3'-phosphotyrosine containing substrates that
bore 3-, 4-, or 5-nucleotide single-stranded DNA in the core region was
used in strand exchange reactions by wild-type Flp (data not
shown).

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Fig. 8.
Panel a, schematic representation of
strand exchange between the suicide substrates and the nicked full-FRT
DNA donor. Suicide substrates Su-1, Su-2, and Su-3 contained a nick on
the bottom strand 3, 4, and 5 nucleotides away from the a
cleavage site, respectively. After Flp-mediated strand cleavage and
covalent attachment, the short oligonucleotides will diffuse away and
generate various amounts of single-stranded DNA in the core region of
the FRT site. Therefore, the assay specifically measures
cleavage-dependent strand exchange and ligation. The
suicide substrates were 5'-end labeled with 32P on the
bottom strand as indicated by the asterisk. Panel
b, analysis of cleavage-dependent strand exchange and
ligation by Flp proteins. The substrates and protein are indicated
above each lane.
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DISCUSSION |
Although single-stranded DNA is known to play an important role in
homologous recombination, little is known about the role of
single-stranded DNA in site-specific recombination (38, 39). The
studies presented in this paper have addressed the importance of
single-stranded DNA in strand exchange by Flp, a conservative site-specific recombinase. We show that single-stranded DNA in the core
region of the FRT site plays an important role in strand exchange
either dependent on or independent of Flp-mediated strand cleavage and
covalent attachment. Defects in strand exchange exhibited by certain
mutant Flp proteins can be overcome by providing DNA substrates
containing single-stranded core regions.
It has been shown previously that Flp is able to carry out strand
ligation independently of its ability to execute strand cleavage (29).
Using activated FRT substrates bearing a 3'-phosphotyrosine group, we
have developed an assay to monitor specifically the strand exchange
activity of Flp proteins. Whereas wild-type Flp could catalyze strand
exchange, mutant Flp proteins Ins115, Y343F, and Y343S were impaired in
their ability to carry out strand exchange when FRT substrates
contained duplex core sequences (Fig. 2).
Cleavage is required for strand exchange to generate a free 5'-OH end
that can attack a phosphotyrosyl bond in a partner DNA that is cleaved
similarly. We propose that Flp-mediated cleavage and covalent
attachment may induce a conformational change in the protein-DNA
complex which leads to the separation of the strands in the core region
to promote strand exchange. Although Flp Y343F is inactive in strand
cleavage because it lacks the catalytic tyrosine residue, it is able to
carry out strand ligation if both a free 5'-OH end and a 3'-tyrosyl end
are provided (29). However, as shown in this paper Flp Y343F is unable
to carry out efficient strand exchange even if both a free 5'-OH and a
3'-tyrosyl end are provided. It is possible that the inability of this
mutant protein to attach covalently to DNA and to alter the DNA
conformation may impair its ability to carry out strand exchange
(46).
It has been shown that before synapsis, binding of two Flp molecules to
the symmetry elements flanking the core sequence induces a severe bend
(>144°) in the DNA (14, 15). As a result of this DNA bend, it is
possible that the strands of the core region might be separated, and
this might facilitate strand exchange (31). Ins115, which has been
shown to be defective in inducing the type II bend in DNA (34), would
not be able to separate the strands in the core region. We suggest that
the inability to induce a bend may be the reason that Flp Ins115 is
incapable of carrying out strand exchange.
Because Flp Ins115 is able to promote strand cleavage and covalent
attachment but not DNA bending and Flp Y343F is able to promote DNA
bending but not DNA cleavage and covalent attachment, it is likely that
either DNA bending or cleavage and covalent attachment alone may not be
sufficient to separate the strands of the duplex core region in the FRT
site that is required for strand exchange by Flp. For the Y343S
protein, the defects in strand exchange may be a composite of its
inability to induce a bend in the DNA and its inability to cleave and
attach covalently to the DNA.
Using substrates containing a single-stranded core, we first
demonstrated that defects in strand exchange exhibited by mutant Flp
proteins can be partially overcome by the presence of a single-stranded core region of one partner DNA. The presence of single-stranded core
regions in both partner DNAs allowed the mutant Flp proteins to
achieve the levels of strand exchange observed for wild-type Flp. This
suggests that denaturation of the duplex core region might occur during
Flp-mediated recombination. Previous attempts to detect single-stranded
DNA in the core region by chemical probing were unsuccessful, possibly
because of limitations in the techniques used (40). However,
recent studies have revealed that Flp exhibits an intrinsic
single-strand-specific DNA binding
activity.4 Therefore, it is
possible that this specific single-stranded DNA binding activity of Flp
may play a role in facilitating strand exchange.
A 3-nucleotide single-stranded region in the core immediately
adjacent to the cleavage site was sufficient to permit the mutant Flp
proteins to engage in strand exchange. This suggests that the core
region of the FRT site may be only partially separated during strand
exchange. A partially single-stranded core is consistent with the
finding that only a limited amount of branch migration is required for
the resolution of a Holliday junction as well as the 3-nucleotide
swapping model (24, 42-44). Further evidence supporting the idea that
the core region may be partially separated during strand exchange was
presented by Guo et al. (45) who solved the crystal
structure of a synaptic complex of the Cre recombinase covalently
attached to its target DNA sequence. They showed that upon cleavage of
the DNA target by Cre, 3-nucleotide single-stranded segments toward the
core side of the cleavage site were released. Because like Flp, Cre is
also a member of the integrase family of site-specific recombinases, it
is likely that the single-stranded DNA in the core region plays an
important role in strand exchange catalyzed by these enzymes.
We thank Linda Beatty, Rick Collins,
Helena Friesen, Marc Perry, Arkady Shaikh, and John Walker for critical
and insightful comments.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M23380.