From the Division of Molecular Genetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom
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ABSTRACT |
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Xer site-specific recombination functions in the
stable maintenance of circular replicons in Escherichia
coli. Each of two related recombinase proteins, XerC and XerD,
cleaves a specific pair of DNA strands, exchanges them, and rejoins
them to the partner DNA molecule during a complete recombination
reaction. The rejoining activity of recombinase XerC has been analyzed
using isolated covalent XerC-DNA complexes resulting from DNA cleavage
reactions upon Holliday junction substrates. These covalent protein-DNA complexes are competent in the rejoining reaction, demonstrating that
covalently bound XerC can catalyze strand rejoining in the absence of
other proteins. This contrasts with a recombinase-mediated cleavage
reaction, which requires the presence of both recombinases, the
recombinase mediating catalysis at any given time requiring activation
by the partner recombinase. In a recombining nucleoprotein complex,
both cleavage and rejoining can occur prior to dissociation of the complex.
Xer site-specific recombination functions in the stable
segregation of circular replicons in Escherichia coli by
resolving circular multimers that can arise by homologous recombination or rolling circle replication (reviewed in Ref. 1). Recombination occurs at specific target sites and requires the catalytic action of
two recombinase proteins, XerC and XerD (2). Homologues of XerC and
XerD have been identified in many bacteria with circular chromosomes.
The Xer proteins are members of the bacteriophage Integrase family-mediated recombination proceeds via two temporally and
spatially separated pairs of strand exchanges, producing a Holliday
junction as an intermediate (6-11). Each strand exchange involves two
trans-esterification steps; the strand cleavage reaction occurs by a
nucleophilic attack of the active site tyrosine upon the activated DNA
phosphodiester producing a 3'-phosphotyrosyl bond and a 5'-OH. The
second rejoining reaction occurs when nucleophilic attack of a 5'-OH on
the 3'-phosphotyrosyl bond restores the continuity of the DNA strand
and displaces the tyrosine. If the 5'-OH comes from the cleaved DNA
partner, strand exchange has occurred.
Because site-specific recombinases break and rejoin DNA phosphodiester
bonds, these enzymes have evolved mechanisms to ensure that efficient
recombination occurs only on correctly aligned recombination sites that
have been bound by the four recombinase molecules needed to catalyze
the cleavage and rejoining of four DNA phosphodiester bonds. The
structure of Cre bound to Holliday junction DNA (6) provides support
for biochemically based models that suggest DNA conformation, and
protein-protein interactions play a key role in determining which pair
of recombinase molecules within a tetrameric recombination complex are
to be active at any point in time (12-14). The failure of singly bound
XerC or XerD molecules to catalyze efficient DNA cleavage suggests that XerC and XerD each activates catalysis of its partner recombinase when
bound to DNA (12). The inappropriate positioning of the active site
tyrosine for in-line attack on the scissile DNA phosphate in the
crystal structure of a XerD provides a structural basis for both
monomer inactivity and partner recombinase-mediated activation through
correct repositioning of the tyrosine nucleophile (14). Flp recombinase
uses a different strategy to control catalysis; a single active site
contains contributions from two Flp monomers (15).
For two characterized Xer recombination sites, psi from the
plasmid pSC101 (16) and cer from ColE1 (17), recombination occurs with a distinct order of strand exchange, with XerC catalyzing the first pair of exchanges to form the Holliday junction intermediate (18, 19). In psi site recombination, the junction is then resolved to products by XerD-mediated strand exchange (19, 20).
DNA molecules containing Holliday junctions and bearing recombination
core sites have been shown to be substrates for in vitro strand exchange mediated by several different integrase family recombinases (8, 21-25). The use of a Holliday junction-containing substrate allows the analysis of the cleavage, rejoining, and strand
exchange steps, as well as their controlling factors, during a reaction
on the same substrate. Neither XerC nor XerD on its own is active in
DNA cleavage or strand exchange upon Holliday junctions (12, 25, 26),
implying that interaction between the two recombinases is necessary to
produce an active site configuration that is competent for cleavage.
Using either a linear suicide substrate or a supercoiled plasmid
substrate, inefficient cleavage or topoisomerase activity,
respectively, has been observed in the absence of the partner
recombinase (27, 28).
The rejoining reaction is much less well characterized than the
cleavage step in integrase family-mediated recombination. Previous
studies of rejoining have been carried out using the Flp recombinase
with either activated substrates bearing a 3'-phosphotyrosyl bond or a
half-site DNA substrate (29, 30). The homology dependence of rejoining
was also studied using half-sites and full sites with mismatched bases
(31, 32). In this study we have used isolated covalent protein-DNA
complexes resulting from XerC-mediated cleavage of a Holliday junction
to study the rejoining reaction and its requirements for interactions
with partner recombinases. This fully paired linear full site substrate
with a covalently attached wild-type protein should be equivalent to
the true covalent intermediate produced in a wild-type reaction. As
well as providing a wild-type substrate for the study of rejoining, it
also extends the analysis of the rejoining step of recombination to a
second member of the integrase family of recombinases and allows
dissection of the protein interactions necessary for catalysis. A major
conclusion is that a covalently bound XerC molecule can catalyze DNA
rejoining in cis; the presence of XerD does not stimulate
the reaction and therefore is not required to activate XerC-mediated rejoining.
Construction of Labeled Substrates--
Holliday
junction-containing substrates were constructed by annealing four
synthetic oligonucleotides as described elsewhere (25, 26). The
junctions used were HJ11 and
HJ7, based on the cer6 core site (26), and HJ, based on the
dif core (12) or a dif-cer6 hybrid. The
oligonucleotides used for the dif-cer6 hybrid
site are identical to those used for HJ, except for the central region
sequences, and are as follows: strand 1, 5'-GATCCGTGATCACGCTGAAC
GCGTTTTAGC GGTGCGCATA AGGGATGTTATGTTAAATGG CCTAACGCCTAAAGCGGCCGCCTA-3'; strand II, 5'-ATCGTAGGCG
GCCGCTTTAGGCGTTAGGCCATTTAACATAACATCCCTTATGCGCACCCGACCGTTGCCGGATCCG-3'; strand III, 5'-TCGATCGAGA
TCTCAGGATGTCATTTAACATAACATCCCTTATGCGCACCGCTAAAACGCGTTCAGCGTGATCACG-3'; and strand IV,
5'-CGATCGGATCCGGCAACGGTCGGGTGCGCATAAGGGATGTTATGTTAAATGACATCCTGAGATCTCGA. The central region of each sequence is shown in bold.
Before annealing the substrates, one oligonucleotide was radiolabeled
at the 5' end using T4 DNA polynucleotide kinase and [ Purified Proteins--
XerC and XerD proteins were purified as
described elsewhere (12). Construction of maltose-binding protein
fusions was described previously (25), and protein was purified
according to the protocol suggested by New England Biolabs. All
proteins were supplemented with glycerol to 50% and stored at
Holliday Junction Reactions--
Reaction conditions were as
described previously (25). Standard reactions contained ~0.1 pmol of
labeled DNA, 500 ng of poly(dI-dC)·poly(dI-dC), 1 µg of acetylated
bovine serum albumin and ~10 pmol of XerC and ~5 pmol of XerD per
10 µl of reaction. Binding reactions were incubated on ice for 5 min
followed by electrophoresis through 6% polyacrylamide in 1× TBE (89 mM Tris borate, pH 8.3, 2 mM EDTA). Cleavage
reactions were incubated at 37 °C for 30 min and then stopped by the
addition of 2 µl of SDS stop buffer (50 mM Tris-HCl, pH
7.5, 1% SDS). The DNA was then electrophoresed through 4%
polyacrylamide-TBE containing 0.1% SDS at 200 V for 165 min. Gels were
dried and exposed to x-ray film (Genetic Research Instrumentation) or
analyzed quantitatively on a PhosphorImager (Molecular Dynamics).
Where species were to be excised from a gel, ~0.5 pmol of DNA was used.
Isolation of XerC-DNA Covalent Complexes--
Large scale
Holliday junction cleavage reactions were electrophoresed in the
presence of SDS as described above. The wet gel was exposed to x-ray
film, and the bands corresponding to XerC-DNA covalent complexes were
excised. The complexes were electroeluted overnight at 4 °C in 0.1×
TBE and 0.1% SDS at 16 V and were then ethanol-precipitated.
Resuspension was in recombination buffer (25).
Proteinase K Treatment--
A stock solution of proteinase K was
made in aqueous solution to a concentration of 4 mg/ml and stored at
XerC-DNA Covalent Complex Reactions--
Isolated complexes were
supplemented with bovine serum albumin and poly(dI-dC)·poly(dI-dC) to
create identical conditions as in cleavage reactions. An aliquot of the
resuspended covalent complex was transferred to SDS stop buffer before
incubation to prevent any further reaction. Complexes were incubated on
ice for 1 h to aid protein refolding, and then Xer proteins were
added as stated and the reactions incubated for an additional hour at 37 °C. SDS stop buffer was added, and each reaction was divided into
two aliquots; one of these was electrophoresed in the presence of SDS,
and the other was treated with proteinase K, ethanol-precipitated, and
electrophoresed through a 10% polyacrylamide-TBE gel containing 7 M urea. Size markers for denaturing gels were 5'
end-labeled oligonucleotides.
Proteinase K treatment was also carried out after resuspension in
recombination buffer with SDS stop buffer in the experiments described.
The DNA was phenol/chloroform-extracted and ethanol-precipitated. It
was then resuspended in recombination buffer and treated in the same
manner as the above described covalent complex reactions.
XerC Covalently Attached to Linear Duplex DNA Catalyzes DNA
Rejoining in cis--
Reactions of the Xer recombinases with synthetic
Holliday junctions bearing various core recombination sequences produce
both rejoined linear duplex strand exchange products and covalent
recombinase-DNA complexes that have undergone cleavage at the expected
position (12, 25, 26). The levels of unjoined cleavage product observed are higher than those reported for similar reactions mediated by either
Untethered synthetic Holliday junction molecules based on either the
chromosomal dif sequence (33) or the plasmid hybrid cer6 sequence (34) are substrates for XerC catalysis but do not undergo significant XerD catalysis. XerC catalysis is dependent on
the presence of both recombinases (12, 25, 26) and is directed at a
specific phosphodiester on a specific strand (by convention, the
"top" strand (18)). Two different XerC-DNA covalent complexes are
produced upon reaction. One, HJCC, consists of a four-way junction with
XerC covalently joined to one or the other of the two top strands,
whereas the second top strand remains intact. The second, LDCC, is a
covalent complex with linear duplex DNA resulting from Holliday
junction molecules that have undergone XerC-mediated cleavages, without
rejoining, on both top strands. Mixing experiments using a
maltose-binding protein fusion to XerC (MBP-XerC) and the wild-type
protein confirmed that a single molecule of XerC was attached to the
DNA in both types of covalent complex (data not shown). LDCCs are
present in greater quantities than HJCCs (e.g. see Fig.
1A; Refs. 12, 25, and 26).
LDCCs with XerC were isolated in the presence of SDS from reactions
with Holliday junction substrates HJ1 and HJ7, which bear the
cer6 core recombination site. Whereas HJ1 is free to branch migrate through the whole recombination core, HJ7 contains two base
pair changes that constrain the junction branch point to a 6-base pair
region from the right half of the central region into the XerD binding
site (26). HJ7 yields a lower level of rejoined linear duplex product
but a higher level of both covalent complexes when compared with HJ1
(26). The isolated complexes were then assayed for their ability to
catalyze the strand rejoining reaction.
The purified HJ1 LDCCs migrated through polyacrylamide containing SDS
as rather broad bands in which there was some smearing ahead of the
main band, possibly because of some proteolytic degradation during
processing of the sample (Fig. 1A). In the absence of
renaturation, little or no rejoined linear duplex (RLD) was evident
(Fig. 1A, lane SDS; less than 2% of the label was in RLD).
When the covalently attached protein was allowed to renature, RLD was
produced (Fig. 1A, lane "no protein"; 8.5% of the
strand IV label and 6% of the strand I label was in RLD). Analysis of
these reactions on a denaturing gel confirmed that strand rejoining had
occurred and that it occurred with the adjacent 5'-OH within the linear
duplex (Fig. 1B). Note that in the denaturing gel, there is
a significant yet relatively constant amount of 92-nt parental strands.
We believe these are derived by contamination of the LDCCs with
uncharacterized DNA containing full-length parental strands; this may
contain Holliday junction related molecules in which three rather than
four strands have reannealed. When DNA from unreacted Holliday junction
substrate was extracted from a position corresponding to that at which
the proposed contamination runs (i.e. in the LDCC position)
and then incubated with XerC and XerD, no reaction was observed (data
not shown). Therefore, this contamination does not affect our
interpretation of the data (see later). No evidence for rejoining of
the 3'-phosphate to any other 5'-OH was observed. The LDCCs derived
from HJ7 had essentially the same properties as those derived from HJ1
(data not shown).
When isolated covalent complexes were treated with proteinase K, before
incubation in recombination buffer, no increase in the level of
rejoining was seen with or without SDS present (Fig. 1B).
This observation confirms that the strand rejoining reaction is
XerC-catalyzed and occurs slowly, if at all, by spontaneous, noncatalyzed attack of a DNA 5'-OH on an adjacent 3'-phosphotyrosine. We therefore conclude that a XerC molecule covalently bound to its
substrate DNA catalyzes rejoining by the displacement of its own
phosphotyrosyl by the DNA 5'-OH. These results are consistent with the
covalent complexes being trapped reaction intermediates and demonstrate
that cleavage and rejoining can occur in a nonconcerted fashion.
Rejoining Is Stimulated by the Addition of Exogenous XerC, but Not
XerD; This Effect Is Independent of the Active Site Tyrosine on the
Exogenous XerC--
The effect of adding exogenous recombinases to the
covalent protein-DNA complex was also examined (Fig. 1). Addition of
XerD had no significant effect on the amount of rejoined product formed (8% of label in RLD compared with 8.5% RLD when no exogenous protein was added), indicating that interactions between the covalently attached XerC and XerD are not required for the strand rejoining reaction. The addition of XerC led to an increase in the level of
linear duplex observed (16% of strand IV label and 18% of strand I
label as RLD). Addition of a mutant XerC lacking the active site
tyrosine nucleophile, XerCY275F, also led to an increase in linear
duplex production over the reaction with no added protein (15% from
6% of strand I label as RLD).
The addition of XerC and XerD together led to a further increase in the
level of rejoining compared with the addition of XerC alone (Fig.
1B, lane XerC+XerD). This could be a consequence
of XerC+XerD reaction on contaminating Holliday junction-related material and/or a direct stimulation of rejoining on the LDCCs (for
example by stimulating XerC binding to DNA through cooperative XerC-XerD interactions on DNA). Similarly, the increase in 84-nt recombinant product strands in the proteinase K-treated LDCC reaction that was subsequently incubated with with XerC and XerD is likely to
have arisen by stimulation of XerC-mediated rejoining on the proteinase
K-treated LDCC. A similar reaction has been observed with Flp
recombinase (29). The stimulatory effect of exogenous XerC may be the
result of its binding directly to the left half-site and activating the
phosphotyrosyl bond of the covalently attached XerC molecule. This
process would require that the exogenous XerC molecule bind to the left
half-site and correctly position its active site residues involved in
phosphodiester activation, despite the presence of the existing
protein-DNA phosphotyrosyl bond and the rest of the covalently
attached protein. We would expect this to happen only on molecules in
which the covalently bound DNA had failed to renature, because
structural studies of both XerD and Cre suggest that a correctly folded
recombinase molecule covalently bound to DNA is unlikely to allow
access of the active site of a second molecule to the scissile
phosphotyrosyl bond (6, 14, 35). An alternative explanation is that the
action of the exogenous protein is to aid in the refolding of the
covalently attached XerC molecule. This would be an unlikely result
from a non-recombinase co-purified protein, because MBP-XerC, the
maltose-binding protein fusion derivative of XerC, also stimulates
rejoining, and MBP-XerC was purified by a different method than the
wild-type XerC. Furthermore, if the possible refolding were a direct
result of recombinase-recombinase interactions, then XerD would be
expected to exhibit stimulation too.
The addition of exogenous XerC also led to the formation of a species
migrating more slowly than the substrate covalent complex on the
SDS-polyacrylamide gel. This species is probably the result of
disulfide bond formation between the covalently attached XerC and the
exogenous XerC and is removed upon addition of the reducing agent
Denaturing polyacrylamide gels of reactions using bottom strand-labeled
substrates confirmed that the bottom strand remained intact with no
evidence of its cleavage in any of these reactions (data not shown).
Therefore, all of the observed reaction can be attributed to top strand
cleavage and rejoining mediated by XerC.
The failure of exogenous XerD to stimulate rejoining by the covalently
bound XerC contrasts with a normal XerC-mediated DNA cleavage reaction,
which is dependent on activation by XerD. We have proposed that a major
role of the partner in activation is to position the active site
tyrosine correctly for in-line attack on the scissile DNA
phosphodiester (Ref. 14; also see Ref. 6). It is therefore not
surprising that XerC-catalyzed rejoining occurs independently of XerD
because the tyrosine nucleophile is irrelevant to this step. Despite
the fact that in an XerC covalent complex the relevant active site
residues and the DNA 5'-OH must be able to position themselves
correctly for the rejoining reaction, DNA rejoining appears to be
rate-limiting in in vitro reactions on Holliday junction
substrates, with measurable amounts of covalent complex being
detectable at all times within a reaction.
Covalent Complexes Containing MBP-XerC Show the Same Pattern of
Rejoining as Complexes with Wild-type Protein--
Covalent complexes
formed from reactions of MBP-XerC and XerD with HJ1 and HJ7 were also
isolated, purified, and treated in reactions identical to those
described above. The slower electrophoretic mobility of these complexes
means that they should be not contaminated with the same material as
the LDCCs containing normal XerC. The covalently attached MBP-XerC
fusion protein is also proficient in catalyzing the rejoining of the
DNA in cis when refolding is allowed (Fig.
2). As for the wild-type XerC-DNA
complexes, the rejoining of the MBP-XerC-DNA covalent complexes was
stimulated by exogenous XerC (wild-type or MBP-XerC; Fig.
2B).
Some contaminating intact Holliday junction substrate co-purified with
the MBP-XerC-DNA complexes (Fig. 2A; and as parental single
strands in Fig. 2B). We assume that this contamination arose
as a consequence of the smearing of the Holliday junction substrate up
the gel (see the control HJ1 reaction lane in Fig. 1). The
fact that either recombinase on its own does not give any reaction with
a Holliday junction should therefore not confuse the interpretation of
the rejoining reactions of the covalent complexes. To confirm this
hypothesis, unreacted Holliday junction was electrophoresed and gel
chips were excised from positions equivalent to those of the covalent
complexes. DNA purified from a position above that of Holliday junction
was now shown to migrate with the same mobility as the substrate and
showed a reaction only when both XerC and XerD were present (data not shown).
Exogenous XerC and MBP-XerC Do Not Cleave the Existing Protein-DNA
Phosphotyrosyl Bond--
When MBP-XerC was added to an XerC-DNA
complex, no MBP-XerC-DNA covalent complex was produced (Fig.
1A). This result suggests that the exogenous MBP-XerC does
not directly attack the existing phosphotyrosyl bond with its own
catalytic tyrosine but can stimulate the attack of the DNA 5'-OH upon
this bond. Similarly when XerC was added to covalent complexes formed
by MBP-XerC, no covalent complexes containing only wild-type XerC were
produced (Fig. 2A). These observations are consistent with
the idea that cleavage involves attack of the nucleophile in line with
the leaving group: to cleave the protein-DNA phosphotyrosyl, a
nucleophile must approach from the direction opposite the
phosphotyrosyl bond. Because the recombinase proteins make contacts
with the DNA of the central region (2, 36), steric hindrance may render
the phosphotyrosyl bond unable to rotate sufficiently to allow in-line
nucleophilic attack by the tyrosine of the exogenous protein.
Therefore, cleavage of an existing phosphotyrosyl by the tyrosine of an
exogenous protein would not be expected.
DNA Rejoining in Holliday Junction Covalent Complexes Follows the
Same Pattern as for Linear Duplex Covalent Complexes--
Covalent
complexes of XerC and MBP-XerC with HJs were isolated and treated in
reactions similar to those of the linear duplex covalent complexes.
These complexes exist as a mixture of two equivalent forms, with one or
the other top strand having been cleaved. Because the core
recombination site is identical on the two top strands of HJ1, it was
expected that the two forms were equimolar.
Because the HJCC migrates in a polyacrylamide gel at almost the same
position as the linear duplex covalent complex of MBP-XerC, the level
of co-purified contaminating intact junction was similar to that
observed upon extraction of the MBP-XerC LDCCs. Again, only when both
XerC and XerD are present should the co-purified DNA produce any reaction.
Judging by the mobility of the isolated complexes, there does not
appear to have been a great deal of proteolysis or DNA degradation during purification, although they are contaminated with some substrate
Holliday junctions and Holliday junction degradation products (Fig.
3A; the three
"SDS" lanes). Strand rejoining is demonstrated by an increase in the intensity of the Holliday junction band in the non-SDS lanes (for example, with HJ7 the proportion of
total labeled DNA as Holliday junction increases from 15% (+SDS lane) to 34% (no protein lane) and 32% (+XerD
lane).Therefore, the covalently attached XerC is capable of
catalyzing the rejoining reaction in cis, and the addition
of XerD does not stimulate rejoining, paralleling the results with the
linear duplex covalent complexes. Denaturing polyacrylamide gel
analysis of the HJ7 reactions confirmed that rejoining occurred to give
the parental strand size (Fig. 3B).
Holliday Junction Covalent Complexes Are Substrates for XerC
Cleavage Regardless of the Presence of XerD--
Surprisingly, the
incubation of renatured HJCCs in the absence of exogenous recombinase
also produced RLD product (Fig. 3A). The denaturing gel of
these reactions confirmed that these were completed strand exchange
products of the correct size (Fig. 3B). We assume that these
RLDs arise from attack of free XerC (released from HJCCs in rejoining
reactions) on HJCCs; cleavage occurring on the second top strand of
HJCC before rejoining of the initially cleaved strand occurs. Because
XerC-mediated rejoining within a HJCC generates a Holliday junction
that is not a substrate for XerC catalysis in the absence of XerD, the
cleavage and rejoining reactions that generate the RLDs must occur
directly on the HJCCs.
The appearance of RLDs is correlated by an increase in LDCCs, showing
that the rejoining of the two duplexes derived from HJCC cleavage does
not need to be concerted. Furthermore, these results show that XerC can
cleave independently of XerD on a HJCC, whereas no products are seen
when XerC alone is added to intact Holliday junction substrates; this
could be because HJCCs are better cleavage substrates than intact
Holliday junctions or because XerC can cleave in a nonconcerted fashion
at a low frequency on both the intact junction and the covalent
complex. On the HJCC the presence of the existing cleavage on the other
top strand and the lack of protein-protein contacts to keep the
junction together allows the complex to dissociate before rejoining
occurs and yields two linear duplex covalent complexes, one or both of which are then rejoined to product. On the intact junction, two of
these rare cleavage events would be required to occur concurrently for
the junction to dissociate.
Both DNA Cleavage and Rejoining Occur within a Protein-DNA Complex
Derived from a Recombinase-bound Holliday Junction Substrate--
RLD
can be generated only from Holliday junction substrates that have
undergone two recombinase-mediated cleavages. Although it seems likely
that rejoining would occur within this recombinase-DNA complex under
normal conditions, it is possible that the rejoining reaction can occur
after dissociation of these complexes within single LDCCs. These
possibilities are not mutually exclusive, and it is possible that the
observed low level of LDCCs results from rare junction dissociation
events prior to rejoining, whereas rejoined linear duplex strand
exchange products result from cleavage and rejoining reactions that are
completed within a nucleoprotein complex.
To test this hypothesis, it was necessary to ascertain whether LDCCs
are associated with protein-DNA complexes that migrate during
electrophoresis with the mobility of Holliday junction molecules bound
with recombinase or with the mobility of recombinase-bound duplex.
Reactions of three different Holliday junction substrates with XerC and
XerD were electrophoresed on a binding gel. Each reaction yielded two
species, the upper one corresponding to the recombinase-bound junction
DNA and the lower to the bound linear duplex product (data not shown).
DNA from these bands was extracted separately in the presence of SDS
and treated with proteinase K. The DNA was then analyzed on a
denaturing polyacrylamide gel (Fig. 4).
The bound junction complex contains almost all of the detectable
cleaved DNA and a small proportion of the RLD; most of the label is in
the parental size strands as expected. Similar results were obtained
for all three substrates, although the two containing the
cer6 central region gave a higher level of cleaved and
rejoined products. The lower complex is made up almost exclusively of
RLD, consistent with the idea that after rejoining, synaptic complexes
dissociate. Because the majority of cleaved molecules are contained
within LDCCs rather than HJCCs (e.g. see Fig.
1A), we conclude that most of the cleaved DNA is held
together by protein-protein interactions prior to rejoining within the
bound complex. However, we cannot exclude the possibility that SDS
treatment of unreacted recombinase-bound Holliday junctions induces
recombinase-mediated cleavage and some rejoining.
INTRODUCTION
Top
Abstract
Introduction
References
integrase family
of recombinases. Members of this family contain two highly conserved
motifs, containing four invariant residues: a tyrosine, which acts as
the nucleophile during strand cleavage, and two arginines and a
histidine (the RHR triad), which have been implicated in the activation
of the scissile phosphodiester in both the cleavage and rejoining
reactions. The two arginines are thought to stabilize the pentavalent
phosphate transition state, whereas the histidine may act as a general
base catalyst (3-6). Recent structural and biochemical studies have
revealed two further residues that are directly implicated in
catalysis. An invariant lysine is present in a conserved
turn,
lying between motifs I and II, in the recombinases and the structurally
and mechanistically related type 1B topoisomerases. A second histidine in motif II contacts the scissile phosphate and may participate in the
general acid-base catalysis; this histidine, although well conserved,
is not invariant, being a tryptophan in Cre recombinase (5, 6).
EXPERIMENTAL PROCEDURES
-32P]ATP.
70 °C.
20 °C. One µl of this stock was added per 10 µl of reaction.
Proteinase K treatment was carried out at 37 °C for 5 min in the
presence of 0.1% SDS.
RESULTS AND DISCUSSION
integrase or Flp recombinase, indicating that Xer rejoining
reactions are rate-limiting under the assay conditions used. To test
whether the covalent complexes obtained with XerC have the properties
of reaction intermediates and can catalyze strand rejoining, the
covalent complexes were isolated and the requirements for strand
rejoining studied.
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Fig. 1.
Purified HJ1 LDCC is competent for strand
rejoining. A, purified LDCCs were incubated either in
the absence of exogenous protein without renaturation (SDS)
and after renaturation (no protein) or with the indicated
proteins after renaturation. Samples were analyzed by electrophoresis
on a polyacrylamide gel containing SDS. Strand rejoining produces RLD.
Schematics of the structures of the various species are shown alongside
the gel. The bold line between proteins represents a
disulfide bond; note the absence of these bands when
-mercaptoethanol (EtSH) is added. The first
lane in the left-hand panel contains a HJ1 reaction
with XerC and XerD. Substrates were radiolabeled (asterisk)
on strand IV or I. B, purified LDCCs labeled on strand I (92 nt) were analyzed on a denaturing polyacrylamide gel with and without
proteinase K treatment after reaction with no added protein
(lanes SDS (no renaturation) and no protein
(renaturation)) or after the addition of the indicated proteins. The
reactions labeled proteinase K were treated with proteinase
before the addition of the indicated proteins. All reactions were
proteinase K-treated prior to electrophoresis to reveal the cleaved
species.
-mercaptoethanol (Fig. 1A, compare lanes XerC
and XerC+EtSH). The addition of XerD produced no higher
complex, consistent with the lack of cysteine residues in XerD.
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Fig. 2.
Purified MBP-XerC covalent complexes show the
same rejoining characteristics as wild-type XerC complexes.
A, isolated LDCC complexes were reacted as indicated (lane
descriptions as in Fig. 1) and electrophoresed in the presence of SDS.
Note that the contaminating HJ does not decrease in intensity during
reaction, indicating that it is not a substrate for XerC catalysis in
the absence of XerD. B, the same samples electrophoresed on
a denaturing gel.
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Fig. 3.
HJCCs are rejoined to give Holliday junction
molecules and cleaved to yield linear duplex and linear covalent
complexes. A, HJCC reactions were
electrophoresed through polyacrylamide in the presence of SDS. The
lanes are labeled as in Figs. 1 and 2. Rejoining is represented by an
increase in the intensity of the Holliday junction band (HJ;
for example, for the HJ7 substrate, the proportion of total DNA that is
HJ increases from 15% (+SDS) to 34% (no
protein) and 32% (+XerD)). Cleavage produces both LDCC
and RLD in roughly equal proportions. The band marked C is a
contaminant that has arisen by partial denaturation of HJ-HJCC; it is
not a recombination substrate. B, analysis of the HJ7
reactions (A) by denaturing polyacrylamide gel
electophoresis. Strand rejoining is indicated by the decrease in the
abundance of the cleaved strand. The 84-nt strand exchange product is
derived from RLD.
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Fig. 4.
Covalent intermediates are held together by
protein-protein interactions before and following strand exchange.
Reactions of three different Holliday junction substrates with XerC and
XerD were fractionated on a native polyacrylamide binding gel. Each
junction produced two bands, the upper band migrating at the
position of recombinase-bound Holliday junction and the lower
band migrating at the position of recombinase-bound linear duplex.
The protein-DNA complexes were extracted from the binding gel in the
presence of SDS, proteinase-treated, and analyzed by electrophoresis on
the denaturing gel as shown.
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ACKNOWLEDGEMENTS |
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We thank Lidia Arciszewska for providing DNA substrates and advice and Andrew Spiers for supplying purified proteins.
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FOOTNOTES |
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* This work was supported by the Wellcome Trust and the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Microbiology, University of Texas,
Austin, TX 78712.
§ To whom correspondence should be addressed. Tel.: 44-1865-275296; Fax: 44-1865-275297; E-mail: sherratt{at}bioch.ox.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are: HJ, Holliday junction; HJCC, Holliday junction covalent complex; RLD, rejoined linear duplex; LDCC, linear duplex covalent complex; MBP, maltose-binding protein; nt, nucleotide(s).
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REFERENCES |
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