(Received for publication, February 10, 1997)
From the Department of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, United Kingdom
The RusA protein of Escherichia coli
is an endonuclease that resolves Holliday intermediates in
recombination and DNA repair. Analysis of its subunit structure
revealed that the native protein is a dimer. Its resolution activity
was investigated using synthetic X-junctions with homologous cores.
Resolution occurs by dual strand incision predominantly 5 of CC
dinucleotides located symmetrically. A junction lacking homology is not
resolved. The efficiency of resolution is related inversely to the
number of base pairs in the homologous core, which suggests that branch
migration is rate-limiting. Inhibition of resolution at high ratios of
protein to DNA suggests that binding of RusA may immobilize the
junction point at non-cleavable sites. Resolution is stimulated by
alkaline pH and by Mn2+. The protein is unstable in the
absence of substrate DNA and loses ~80% of its activity within 1 min
under standard reaction conditions. DNA binding stabilizes the
activity. Junction resolution is inhibited in the presence of RuvA.
This observation probably explains why RusA is unable to promote
efficient recombination and DNA repair in ruvA+
strains unless it is expressed at a high level.
The RusA protein of Escherichia coli is an endonuclease
that resolves Holliday intermediates made during homologous genetic recombination and DNA repair (1). Proteins of this type were first
described in bacteriophage-infected bacterial cells (2-5), but have
since been found in bacteria (6, 7), yeast (8-10), and mammalian cells
(11, 12). The T4 enzyme, endonuclease VII, cleaves a variety of DNA
secondary structures including Holliday junctions (2), cruciforms (13),
branched structures (14, 15), heteroduplex loops (16), and mismatches
(17). This broad substrate spectrum is consistent with a role both in
recombination and in the removal of branch structures from the phage
DNA prior to packaging (18). RuvC protein of E. coli is
highly selective for Holliday junctions (19-21), which suggests a more
specialized role in recombination. It binds the four-way duplex
structure of a Holliday junction and folds the DNA in an open
configuration that allows symmetrical strands to be located within the
catalytic core of each subunit and cleaved to yield nicked duplex
products (22-24). Strand cleavage has been shown to involve four
acidic residues in the protein (25, 26) and occurs 5 of a T that has
to be located within a certain sequence context for efficient resolution (27, 28). This sequence-dependence reflects both topological factors that dictate folding of the junction (28) and the
need to make specific protein-DNA contacts (29).
RusA was discovered through its ability to promote recombination and DNA repair in strains lacking RuvC. Genetic analysis of this alternative resolvase provided the first indication that RuvC cannot cleave junctions efficiently in vivo without the associated activities of RuvA and RuvB (1, 30). These two proteins assemble at junctions to form a highly specialized RuvAB complex that drives the branch point along the DNA (31-34). They presumably help RuvC to target cleavable sequences by holding the junction in an open configuration (RuvA) and moving it along the DNA (RuvB), possibly through formation of a RuvABC "resolvasome" complex (34, 35). RusA has no requirement for RuvAB. However, efficient recombination and DNA repair does need RecG which like RuvAB drives branch migration of Holliday junctions (30, 36-38). Whether or not this reflects a need to locate junctions at cleavable sequences remains to be determined.
The 14-kDa RusA polypeptide is encoded by a gene (rusA) located within the defective prophage, DLP12, and is probably of bacteriophage origin (36). The rusA gene is normally expressed poorly, if at all, and can be deleted with no apparent effect. However, it can be activated to suppress the ruv mutant phenotype by insertion of either IS2 or IS10 (formerly called rus-1 and rus-2 mutations, respectively) upstream of the gene to promote transcription, or by cloning in a multicopy plasmid (1, 30, 36). Expression from multicopy plasmids is needed to achieve full suppression in the presence of RuvA, which suggests that the binding of Holliday junctions by RuvA can prevent their resolution by RusA (1, 30). In this work, we detail a new purification of RusA and investigate the native form of the protein. We also report its general biochemical properties, its sequence specificity, and the inhibition of its resolution activity by RuvA.
E. coli strain
N3757 (1) is a ruvAC65 eda-51::Tn10
derivative of BL21 (DE3) plysS (39). pAM151 (1) is a
derivative of pT7-7 carrying rusA+ under control
of the phage T7
10 promoter (39).
RuvA (40) and RuvC (41) were purified
as described elsewhere. T4 polynucleotide kinase was from Pharmacia
Biotech Inc., and [-32P]ATP from Amersham. All other
reagents were from Sigma or BDH and were of analytical grade.
Heparin-agarose, Reactive Blue 4-agarose, and double-stranded DNA cellulose were purchased from Sigma. Phosphocellulose P11 was from Whatman, and DEAE Bio-Gel A from Bio-Rad. A pre-packed gel filtration column (Superose 12 HR 10/30) was from Pharmacia. All chromatography was carried out at 4 °C in buffer A (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM DTT,1 10% glycerol). Phenylmethylsufonyl fluoride and KCl or NaCl were added to buffer A as indicated.
Protein Gel ElectrophoresisSDS-PAGE analysis of protein samples was conducted using 15% gels and followed standard procedures (42). Unless stated otherwise, samples were mixed with SDS loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromphenol blue, 20% glycerol) and boiled for 5 min before electrophoresis. Molecular mass markers (Bio-Rad) were rabbit muscle phosphorylase b (97,000), bovine serum albumin (67,000), hen egg white albumin (45,000), bovine carbonic anhydrase (31,000), soybean trypsin inhibitor (21, 500), and hen egg white lysozyme (14,400). Gels were stained with Coomassie Brilliant Blue, or silver-stained as described (43).
Overexpression of RusA500-ml batches of strain N3757
transformed with pAM151 were grown with aeration at 37 °C in
Luria-Bertani broth containing 10 g/liter NaCl, 100 µg/ml ampicillin,
and 20 µg/ml chloramphenicol. At a cell density corresponding to an
A650 of 0.5, RusA was induced by adding
isopropyl-1-thio--D-galactopyranoside to a final
concentration of 2 mM and incubating for a further 3 h. The cells were chilled on ice, harvested by centrifugation,
resuspended in 10 ml of lysis buffer (100 mM Tris-HCl, pH
8.0, 2 mM EDTA, 5% glycerol), and stored at
80 °C
until required. Under these conditions, RusA was induced to 15-30% of
the total cell protein.
Frozen cells (70 ml) from 5 liters of
induced culture were thawed at room temperature and then mixed on ice
with 25 ml of 5 M NaCl, 0.5 ml 200 mM DTT, 1 ml
of 10% (v/v) Triton X-100. Preliminary trials revealed that a high
salt concentration was required during cell lysis to reduce loss of
RusA. Cells were lysed by freezing in liquid nitrogen and rethawing,
and cell debris was removed by centrifugation at 40,000 rpm for 60 min
at 4 °C, using a Kontron TST 41.14 rotor. Despite the presence of
high salt, ~70% of the RusA was removed with the cell debris. All
subsequent steps were carried out at 4 °C. Purification of RusA was
monitored throughout by SDS-PAGE analysis of column fractions and by
assays for junction cleavage activity, using J12 DNA as a substrate.
The cleavage assays also provided a measure of nonspecific nuclease
contamination. The supernatant (85 ml, 978 mg of protein) fraction
containing the soluble RusA was dialyzed overnight against 4 liters of
buffer A containing 1 mM phenylmethylsulfonyl fluoride and
0.5 M KCl, centrifuged at 15,000 rpm for 10 min to remove
traces of insoluble material, and applied to a phosphocellulose column
(2.6 × 8.5 cm, 45-ml bed volume) equilibrated in the same buffer.
The column was washed with 100 ml of the same buffer before eluting
bound proteins with a 450 ml of gradient of 0.5-1.0 M KCl
in buffer A. RusA eluted between 0.7 and 0.9 M KCl. Peak
fractions were pooled and dialyzed against 2 liters of buffer A
containing 0.1 M KCl. SDS-PAGE analysis revealed that RusA
was >90% pure at this stage. Minor protein bands and trace
exonuclease contaminants were removed by fractionation on three further
columns. The dialyzed phosphocellulose pool (144 ml, 47 mg of protein)
was loaded on a DEAE Bio-Gel A column (2.6 × 4.5 cm, 24-ml bed
volume) and washed with 50 ml of the same buffer. Bio-Gel A does not
bind RusA under these conditions, but retains contaminants that do not
fractionate from RusA in later chromatographic steps. The flow-through
(~200 ml, 32 mg of protein) was applied in aliquots of 50 ml to a
double stranded DNA-cellulose column (1.0 × 5.1 cm, 4-ml bed
volume), washed with 8 ml of buffer A containing 0.25 M
KCl, and bound proteins eluted with a 40-ml gradient of 0.25-1.0
M KCl in buffer A. RusA eluted between 0.5 and 0.7 M KCl. Peak fractions from the four aliquots were pooled
(80 ml, 28 mg of protein), dialyzed against 2 liters of buffer A, and
applied to a heparin-agarose column (1.0 × 7.6 cm, 6-ml bed
volume). The column was washed with 12 ml of buffer A containing 0.25 M KCl and bound proteins eluted with a 60-ml gradient of
0.25-1.0 M KCl in buffer A. RusA eluted between 0.5 and
0.7 M KCl. No contaminating bands could be detected in
these fractions with Coomassie Blue or silver-staining of SDS gels. The
peak fractions were pooled and dialyzed into storage buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM DTT, 50% (v/v) glycerol), and stored in aliquots at
80 °C.
All protein concentrations were determined by a modified Bradford method, using a protein assay kit from (Bio-Rad) and bovine serum albumin (Pharmacia) as standard. Amounts of RuvA, RuvC, and RusA are expressed as moles of the monomeric protein.
Glutaraldehyde Cross-linkingA 200-µl sample of RusA (1 mg/ml protein) was dialyzed into TEA buffer (20 mM TEA-HCl,
pH 8.5, 1 mM EDTA, 150 mM NaCl, 0.5 mM DTT, 10% glycerol), mixed with fresh glutaraldehyde
(final concentration 14 mM), and incubated for 1 min at
room temperature before storing at 20 °C.
RusA (500 µg) was dialyzed for 5 h
against buffer A containing KCl at either 0.15 or 1 M and a
300-µl sample applied to a precalibrated gel filtration column.
Molecular mass standards (Bio-Rad) were bovine thyroglobulin (670,000),
bovine -globulin (158,100), chicken ovalbumin (44,000), horse
myoglobin (17,000), and cyanocobalamin (1,350). Protein was detected by
measuring the absorbance at 280 nm.
Oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer using cyanoethyl chemistry. Each oligonucleotide was deprotected, precipitated in ethanol, and purified on a 12% (w/v) polyacrylamide gel containing 7 M urea. The bands containing full-length oligonucleotides were cut out and extracted from the gel by soaking in water overnight.
Construction of Junction DNA SubstratesX-junctions were
made by annealing four partially complementary oligonucleotides, each
of approximately 50 nucleotides in length. The point of strand
crossover is either fixed centrally within the structure (static
junction, J0) or free to branch migrate within a central core of
homology (mobile junctions J2, J3, J4, J11, J12, and J26). The number
after the letter J indicates the size of the homologous core in base
pairs. The sequences of the oligonucleotides used for J0 (35), J2 (26),
J3 (35), J4 (35), J11 (27, 35), J12 (37, 44), and J26 (45) have been
described. J4 was made by annealing oligonucleotides 5, 7, 9, and 10 (35). J0 has been referred to elsewhere as X-static (35) and J11 as
junction A (27). Annealing followed the procedures described (46). One
of the strands was labeled at the 5 end prior to annealing using
[
-32P]ATP and T4 polynucleotide kinase. Junctions were
purified by nondenaturing 10% PAGE and electroelution. Unlabeled
junctions were made as described above, except that oligonucleotides
were annealed in equimolar ratios and the resulting substrates were not
purified by PAGE. Unlabeled linear duplex DNA was made in the same way
using oligonucleotides 1 and 5 described previously (37).
Cleavage of 32P-labeled junction DNA by RusA and RuvC was assayed at 37 °C in buffer CB (25 mM Tris-HCl, pH 8.0, 1 mM DTT, 100 µg/ml bovine serum albumin, 6% (v/v) glycerol) or SCB (25 mM Tris-HCl, pH 8.0, 1 mM DTT, 100 µg/ml bovine serum albumin, 10% (v/v) glycerol), with either MgCl2 or MnCl2 added as indicated (10 mM in standard resolution reactions). Reactions (20 µl final volume) were terminated by adding 5 µl of stop mixture (2.5% SDS, 200 mM EDTA, 10 mg/ml proteinase K) and incubating for a further 10 min at 37 °C to deproteinize the mixture. DNA products were analyzed by native PAGE, using 10% gels in TBE (90 mM Tris borate, pH 8.0, 2 mM EDTA). Gels were dried, and labeled products were detected using a Molecular Dynamics PhosphorImager (Model 425) and by autoradiography. Reactions were quantified using ImageQuant software (Molecular Dynamics) to analyze PhosphorImages. For time courses, 20-µl samples were removed at intervals from bulk reactions and processed as described. To assess activity over a wide range of pH, assays were conducted in a series of different buffers, each covering a narrow range of pH, with a pH overlap between each buffer type.
Mapping Cleavage SitesSeparate preparations of junction
DNA, each 5-32P-labeled in a different strand, were
incubated with RusA in SCB buffer for 10 min at 37 °C. Deproteinized
samples were extracted with phenol, and the DNA was precipitated with
ethanol, resuspended in sequencing gel loading dye (0.3% (w/v)
bromphenol blue, 0.3% (w/v) xylene cyanol, 10 mM EDTA, pH
7.5, 97.5% formamide), and denatured by boiling for 5 min before
analyzing by denaturing PAGE, using 12% gels (42). Sequencing ladders
of each labeled oligonucleotide generated using a Maxam-Gilbert
sequencing kit (Sigma) were loaded on the same gel to provide markers.
Gels were dried, and analyzed by autoradiography and PhosphorImaging.
Cleavage sites were mapped by reference to the sequencing ladder. A
1.5-base allowance was made to compensate for the nucleoside eliminated
in the sequencing reaction.
32P-Labeled junction DNA (0.2-0.6 ng) was mixed with RusA, RuvA, or RuvC, in binding buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1 mM DTT, 100 µg/ml bovine serum albumin, 6% (v/v) glycerol) in a final volume of 20 µl. After 10-15 min on ice, protein-DNA complexes were resolved by nondenaturing PAGE using 4% gels in low ionic strength buffer (6.7 mM Tris-HCl, pH 8.0, 3.3 mM sodium acetate, 2 mM EDTA). Gels were cooled to 4 °C before use, but electrophoresis was at room temperature with continuous buffer recirculation. Gels were dried and analyzed by autoradiography.
In previous work,
we described a recombinant plasmid (pAM151) for the overexpression of
RusA and purified some of the protein from strain N3757 transformed
with this construct (1). To obtain larger quantities of the protein for
physical analyses, we devised a new purification ("Materials and
Methods"). This yielded 24 mg of RusA (in 21 ml) from 5 liters of
induced cells. The recovery was much lower than expected given that
RusA accounted for 15-20% of the total cell protein. A large fraction
of the induced protein was in an insoluble form that was removed with
the cell debris during centrifugation of the lysed cells. Subsequent
studies revealed that solubility can be improved by growing cells and
inducing RusA at 25 °C (data not shown), allowing much better
recovery of the induced protein by the same method. The protein
purified was free of any visible contaminants as judged by Coomassie
Blue and silver staining of SDS gels (Fig. 1A,
lane c, and data not shown).
To investigate the native form of RusA, 11 µg of the protein was analyzed by SDS-PAGE with and without prior boiling in SDS sample loading buffer. The boiled sample showed a single band of RusA (Fig. 1A, lane c). It migrates as a 15-kDa species, which is close to its predicted molecular mass of 13.8 kDa (1). Without boiling, a second band is clearly visible. Its migration suggests a molecular mass of about 30 kDa, which is consistent with a dimer of RusA. A further sample of the protein was treated with glutaraldehyde before boiling in sample loading buffer containing SDS. Fig. 1B shows a 30-kDa protein species in the treated sample (lane b). The only other band visible corresponds to the monomer species seen in the untreated control (lane c). The absence of any other band suggests that glutaraldehyde specifically cross-links a dimer of RusA.
The ability to detect a dimer on denaturing SDS gels without prior cross-linking (Fig. 1A, lane b) suggests that a substantial fraction of the purified protein is in this form. To determine the molecular mass, a sample of the native protein was dialyzed against buffer A containing 1 M KCl and applied to a Superose 12 HR column. The elution profile (Fig. 1C) showed a single sharp peak. SDS-PAGE analysis of the fractions confirmed that this peak coincided with the peak of RusA (data not shown). By comparison with protein standards analyzed using the same buffer, the molecular mass of the protein at the peak was determined to be 24 kDa. This is nearly twice the value of 13.8 kDa predicted from the amino acid sequence of the protein (36). Given the high salt conditions, this result strongly supports the idea that RusA is a dimer in solution. We also analyzed RusA in the presence of 0.15 M KCl. A substantial fraction of the protein eluted faster than the 670-kDa marker, suggesting a tendency to form aggregates.
Autoinhibition of Junction CleavagePrevious studies (1)
showed that RusA resolves X-junctions to nicked duplex products.
Resolution can be monitored by electrophoresis of the reaction products
on a nondenaturing polyacrylamide gel (Fig.
2A). Using this simple assay, we investigated
the resolution activity of RusA over a range of protein concentrations
in reaction buffer containing 10 mM MgCl2 or
MnCl2. Fig. 2B shows the result of a typical
experiment. Resolution is clearly sensitive to the concentration of
RusA, peaking sharply at 50 nM in the Mn2+
reactions and at 200 nM in the Mg2+ reactions.
Above these levels, the activity of RusA is very clearly inhibited. At
subinhibitory concentrations, RusA is substantially more active in
Mn2+ than it is in Mg2+ (Fig. 2B,
inset). This difference disappears as RusA becomes inhibitory.
Furthermore, even with 5 µM RusA in the reaction, resolution is not totally abolished. Similar results were obtained using junction J3 (data not shown), although the fraction of junction resolved was generally higher (see below). Autoinhibition in this case
reached a lower limit of ~30% junction resolution (data not shown).
Buffer Requirements and Time Course of Resolution
To
determine the optimal conditions for resolution by RusA, we
investigated the activity under a variety of reaction conditions, using
J12 with RusA at 100 nM. RusA showed a requirement for
divalent metal ions with a broad optimum of 10-30 mM for
Mg2+ (Fig. 3A). Replacing
Mg2+ with Mn2+ stimulated resolution in the
2-10 mM range, but at 20 mM and above
Mn2+ was substantially less effective than Mg2+
(data not shown). No activity was detected with Zn2+,
Ca2+, or Cu2+. RusA was insensitive to NaCl up
to 200 mM, but was inhibited at higher concentrations and
inactive at 500 mM (Fig. 3B). Activity in
Mg2+ buffer was stimulated at alkaline pH, and increased
with increasing pH throughout the range tested (Fig.
4A). In contrast, the activity in
Mn2+ buffer was optimal at pH 7.5. Up to pH 8.5, it was
also significantly higher than in Mg2+ buffer. Fig.
4B shows the time course of the reaction in the presence of
10 mM Mg2+ under two different buffer
conditions, one at pH 7.5 in Tris buffer, and one at pH 9.5 in glycine
buffer. As expected, significantly more of the junction was resolved at
the higher pH. In both cases the reaction was over within the first
10-15 min even though substantial fractions of the substrate remained
unresolved. The failure to cleave all the junction molecules is
surprising given that the protein (in terms of dimers) was present in
at least a 50-fold molar excess. It means either RusA is inactivated
rapidly during the reaction or that a fraction of the junction DNA,
determined by the pH, is resistant to cleavage. It could also reflect a
combination of these factors.
Sequence Specificity of Junction Cleavage
RusA resolves J12
by cleaving strands 1 and 3 (Fig. 5) 5 of the CC
dinucleotide located symmetrically within the homologous core (1). The
same site is targeted by RuvC (47). This coincidence could reflect some
special feature of J12 because subsequent studies revealed that RuvC
has a preference for cleaving strands 3
of a T within the context
(A/T)TT
(G/C), where
identifies the cleavage point (27). To see
if resolution by RusA is sequence-specific, we therefore investigated
its ability to resolve junction J11. This junction (Fig. 5) is resolved
very efficiently by RuvC, with >90% of strand cleavage at ATT
G,
which matches the consensus target for RuvC (27). Fig. 6
shows that J11 is resolved by RusA to the same extent as J12. In
contrast, RuvC cleaves J11 much more efficiently than J12.
The inability of RusA to respond to the RuvC target sequence in J11
suggested that it had a different requirement for efficient junction
cleavage. We decided therefore to map the strand cleavages in J11. Four
preparations of J11, each 32P-labeled in a different
strand, were incubated with RusA under standard cleavage conditions in
the presence of either Mg2+ or Mn2+. The
products were resolved by electrophoresis on denaturing polyacrylamide
gels and the cleavage sites in each strand mapped as described under
"Materials and Methods." Fig. 5 summarizes the results of the
mapping, while the amount of cleavage detected at each site is
presented in Table I. The data revealed that RusA
resolves J11 by symmetrical strand cleavage within the homologous core.
In Mg2+, >90% of the cleavage was 5 of the CC in strands
2 and 4 (site 1). Very minor cleavage was also detected at a second
site in strands 1 and 3 (site 2). Reactions done in Mn2+
showed slight stimulation of cleavage at site 2, but site 1 was still
strongly preferred. No cleavage was detected at the RuvC target site in
strands 1 and 3.
|
The base 5 of the major RusA cleavage site differs between J11 (G) and
J12 (T). Both junctions are resolved equally by this protein,
suggesting the difference is of no consequence. However, the homologous
cores of J11 and J12 differ substantially in sequence (Fig. 5), which
could mask a base-specific effect at this position. We therefore
analyzed cleavage of J26. The central region of this junction is
identical in sequence to J12, but the flanking regions are different
and extend the homologous core by 7 bp on either side (Fig. 5).
Fortuitously, each of these regions contains a CC dinucleotide, one
with a 5
T and the other with a 5
G. RusA resolves J26 with about the
same efficiency as J12 (Fig. 4, Table I, and data not shown). In
Mg2+ buffer, substantial cleavage was detected at two sites
(Fig. 5 and Table I, sites 1 and 2). Both are 5
of a CC dinucleotide. Site 1, which accounts for about two-thirds of the resolution activity,
has a 5
T, and is identical to the major cleavage site in J12. Site 2, which accounts for most of the remainder, has a 5
G. Minor cleavages
occurred at the remaining CC dinucleotide (site 3) and 5
of a C at the
right end of the homologous core as drawn (site 4). The low cleavage at
site 3 is surprising given that it has a T 5
of the CC and, like site
1, is located in strands 1 and 3. A similar observation has been seen
with RuvC, which cleaves the ATTG target in strands 2 and 4 with high
efficiency, but hardly touches the same sequence in strands 1 and 3 or
the TTTG in strands 2 and 4, which also matches the consensus target sequence (48). The failure to cleave at these sites indicates that the
general sequence context is also important for resolution. RusA
activity was stimulated by Mn2+, with increased strand
cleavage at sites 1, 3, and 4 at the expense of site 2. Additional
cleavage was also seen at a new position (site 5, Fig. 5 and Table
I).
The failure of RusA to target site 3 efficiently raises the possibility
that resolution is not sequence-specific in terms of protein-DNA
interactions, but is targeted to particular locations where the point
of strand crossover might accumulate due to the local sequence context.
In theory, the junction in the substrates used is free to branch
migrate within the confines of the homologous core. However, previous
studies with J26 and other similar junctions revealed that a major
fraction of the molecules have the junction located at rather few
positions, sometimes only one (24, 47, 48). This non-random
distribution might have a significant bearing on the apparent
sequence-specificity of resolution. We therefore tested RusA on J2,
which has the junction constrained to a homologous core of two A:T base
pairs, and also on J0, a static junction with G:C base pairs at or near
the crossover point (Fig. 5). No nicked duplex products were detected
(data not shown), which means that RusA cannot resolve either
structure. However, a low level of cleavage was detected 5 of the CC
dinucleotide in strand 3 of J0 (Table I). These data support the
conclusion that junction resolution by RusA involves sequence-specific
interactions between the protein and the DNA and that it occurs by a
dual incision mechanism targeted predominantly 5
of CC
dinucleotides.
The results described in the
previous sections revealed that factors other than the presence of a
target sequence can have an overriding effect on the efficiency of
resolution by RusA. The CC dinucleotide target identified using J11,
J12, and J26 is located within an homologous core ranging from 11 to 26 bp (Fig. 5). To see if branch migration could be a limiting factor, we
constructed two new junctions with homologous cores of either 4 or 3 bp, each containing the sequence TCC as a target for strand cleavage
(Fig. 5, J4 and J3). Both are resolved very efficiently, with ~68%
of J4 and ~73% of J3 converted to nicked duplex products within 10 min in standard Mg2+ buffer at pH 7.5 (Fig.
7A, lanes b, c, e, and f, and data
not shown). As expected, cleavage occurs specifically 5 of the CC dinucleotide, although there may be some minor cleavage between the two
cytosines (Fig. 7B, lanes c, d, k, and l; Table
I). The levels of resolution seen with these two junctions are
substantially higher than with J11, J12, and J26, which reached a
maximum of 40-50% (Fig. 4B, Table I, and data not shown).
Resolution of J3 and J4 could be stimulated by Mn2+ (Table
I), but the effects were modest compared with those seen with J12 or
J26 (Fig. 4A, Table I). These data suggests that the length
of the homologous core has a substantial effect on the fraction of
substrate molecules that can be cleaved by RusA.
Stability of RusA
To gain further insight into the factors
governing RusA activity, we monitored the time course of resolution of
J3 at three different concentrations of protein, and compared the
results with the time courses for resolution of J2 by RuvC under
similar conditions. At 1 nM, RusA resolved ~4% of the J3
DNA within 2 min of starting the reaction (Fig.
8A). No further activity was detected over
the remaining period of incubation. Increasing RusA 10-fold resulted in
resolution of about 54% of the DNA, an increase of ~13-fold. Again,
most of the resolution occurred within the first 2 min, but significant
activity was detected for up to 5 min. At 100 nM RusA, 76%
of the junction was resolved, and activity was detected over an even
longer period (up to 15 min). However, the initial rate was slow
compared with that seen with 10 nM RusA. We assume this
reduced rate of cleavage is a further reflection of the autoinhibition
seen with RusA at high concentrations (Fig. 2). The fact that
resolution reached a clear plateau in all three cases, and that the
activity was detected over a longer time scale the higher the initial
concentration of protein, suggested that RusA is rapidly inactivated
during the incubation. The results contrast sharply with those observed
with RuvC. First, the initial rate of cleavage was roughly proportional
to the concentration of protein over the 10-100 nM range
tested. Second, junction resolution continued over the 30-min period of
incubation. Indeed, the time courses at 10 and 20 nM
protein suggest that RuvC loses very little of its activity during this
period.
To investigate the stability of RusA more directly, the protein (at 10 nM) was incubated in reaction buffer lacking either DNA or
Mg2+, or both DNA and Mg2+, before adding the
missing ingredients to assay for resolution activity. No loss of
activity was detected over 60 min at 0 °C under any of the
conditions tested (Fig. 9, and data not shown). In the
absence of DNA, ~80% of the resolution activity was lost within 1 min at 37 °C (Fig. 9). The presence of Mg2+ made little
difference (data not shown). However, when RusA was incubated for 30 min at 37 °C in the presence of junction DNA (without
Mg2+), it retained >90% of its resolution activity. This
protection by substrate DNA was seen even when the concentration of
RusA was increased to 100 nM, without increasing the amount
of DNA. 91.8 ± 3.7% of the initial resolution activity was
present after 30 min preincubation. At 1 nM protein, 100%
of the initial activity was retained over this time scale. Junction
molecules were present at a concentration of only 1 nM in
these studies. It is surprising that this amount could stabilize a
50-fold molar excess of RusA dimers. Although RusA binds preferentially
to junctions, it has quite a high affinity for linear duplex DNA, which
enables it to bind the arms of the junction to give a series of
protein-DNA complexes that appear as a ladder of retarded species in
bandshift assays (1) (see Fig. 11B). To see if this could
help to stabilize RusA, we incubated the protein at 37 °C with
unlabeled linear duplex DNA before adding labeled J3 and
Mg2+ to assay resolution activity. Resolution activity
decayed rapidly under these conditions, but less so than in the absence
of DNA (Fig. 9), which confirms that binding to linear duplex DNA
affords some protection.
The instability of RusA activity means that inactive protein accumulates rapidly during the course of a resolution reaction. This inactive protein could interfere with further activity. To investigate this possibility, a sample of RusA was inactivated by incubation for 10 min at 37 °C in the absence of DNA. Its inactivation was confirmed by assaying resolution activity. A sample of the inactive protein was analyzed by SDS-PAGE. Only one band of protein was detectable on the gels, even with silver staining (data not shown). This band had the same mobility as the native species, which suggests that inactivation is not caused by proteolysis. A further sample was used to test for the ability to bind 32P-labeled J12 DNA in a standard bandshift assay (see "Materials and Methods"). No bandshifts were detected with 12-100 nM protein in the reaction (data not shown), which established that the inactive protein can no longer bind DNA. Over the range of protein tested, native RusA forms a series of well defined protein-DNA complexes (1) (see Fig. 11B). The idea that inactive RusA binds unproductively to junction DNA can therefore be eliminated. However, we have not eliminated the possibility that inactive protein interferes with resolution by interacting directly with the native protein.
Junction Turnover by RusAThe rapid inactivation of RusA when
free of DNA would be expected to limit its ability to cycle from one
junction to another during the course of a reaction. To gain some
indication of junction turnover, resolution of 32P-labeled
J3 DNA was assayed in reactions containing a 40-fold excess of
unlabeled J12 DNA. RusA was used at protein to junction (total) ratios
in the range of 0.025-0.5. Fig. 10 shows that the amount of J3 resolved is reduced compared with control reactions done
in the absence of unlabeled J12 DNA, when the protein to junction
ratios ranged from 1 to 20. If RusA functions as a dimer, a single
turnover would be expected to resolve from 1.25 to 25% of the J3 DNA,
which is very close to the range observed (1.6-35%, Fig. 10).
However, the expected values are based on the assumption that all
molecules of J3 are equally cleavable and that the preparation of RusA
used is 100% active. Both of these assumptions are likely to be
incorrect given the sequence specificity of strand cleavage and the
instability of the protein. If the four possible locations for the
strand crossover in J3 (Fig. 5) are equally represented, and only
junctions at one of these positions can be cleaved, RusA would have to
cycle through 4 molecules on average before finding one it could
cleave. We conclude therefore that RusA probably acts catalytically.
However, the number of junctions resolved on average by each enzyme
molecule is likely to be rather low (6 from the data in Fig. 10)
unless of course the RusA preparation has a substantial fraction of
inactive protein and/or the distribution of the strand crossover point
is skewed unfavorably.
Inhibition of RusA Resolution Activity by RuvA
Previous studies revealed that the ability of RusA to fully suppress the recombination, and DNA repair-deficient phenotypes of ruvB and ruvC mutants requires the absence of RuvA protein (30). One plausible explanation for this effect is that RuvA sequesters Holliday intermediates and blocks their resolution. To investigate whether RuvA can block RusA directly, we monitored resolution activity in the presence of increasing amounts of RuvA. We used junction J11 for this purpose so that RuvC could be used as a control. Both proteins were used at a final concentration of 100 nM. Fig. 11A shows the results of a typical experiment of this type. In the absence of RuvA, 70% of the junction was resolved by RuvC (lane b) and 40% by RusA (lane j). Titrating RuvA into the reaction had a substantial inhibitory effect on both RuvC (lanes c-i) and RusA (lanes k-q), with >90% inhibition achieved at 100 nM and 400 nM RuvA, respectively (lanes e and o). A bandshift assay showed that RusA and RuvC bind J11 with approximately equal affinities. RusA (Fig. 11B, lanes c-g) gives a ladder of retarded complexes, which is quite characteristic of the protein (1), whereas RuvC (Fig. 11B, lanes j-n) gives a single well defined complex (19). These complexes were not detected in the presence of inhibitory concentrations of RuvA (data not shown). They were replaced by a well defined RuvA-junction complex (complex II) that sandwiches the junction point between two tetramers of RuvA (35, 49). We assume this complex prevents RusA or RuvC from gaining access to the junction. From the results shown in Fig. 11A, its formation in vivo would certainly explain the inhibition of RusA's ability to function during recombination and DNA repair (30).
In this paper we have described an improved scheme for the purification of RusA and investigated the subunit structure of the protein by gel filtration, glutaraldehyde cross-linking, and SDS-PAGE. These studies revealed that the native protein is a dimer. It is stable in 1 M NaCl but is dissociated to monomers by boiling. However, without boiling, a substantial fraction of the dimers remain intact during SDS-PAGE, which suggests that the constituent monomers are tightly bound. A dimeric structure for RusA is consistent with its ability to resolve Holliday intermediates, an activity that requires dual strand incision at the junction point (24, 29). Several of the Holliday junction resolvases studied to date have been shown to bind junctions as dimers (29, 50, 51). In the case of RuvC, the target strands are folded into the catalytic core of each subunit and cleaved in a coordinated and sequence-dependent manner (22, 23, 24, 27).
Using small synthetic X-junctions, we found that RusA also cleaves
junctions at specific sequences. Our previous studies with J12 had
revealed that RusA introduced symmetrically-placed nicks in strands of
the same polarity (1). Remarkably, the vast majority of the incisions
were at the same site cleaved by RuvC (GTCC). However, this proved
to be a coincidence reflecting the fact that RuvC cleaves 3
of a T and
RusA 5
of a CC dinucleotide. When we analyzed resolution of junctions
(J11 and J26) containing the RuvC target sequence, ATT
G, we found
that RusA did not cleave this site at all. The junctions were resolved
efficiently, but in both cases >90% of the activity was targeted 5
of a CC dinucleotide located symmetrically within the homologous core,
either at G
CC (J11) or both G
CC and T
CC (J26). There was very
little evidence of cleavage at non-symmetrical sites, which supports
the notion that RusA acts as a dimer.
From the junctions analyzed, a CC dinucleotide is essential for
efficient resolution by RusA; a single C is not enough. It may be
significant therefore that RuvC prefers to cleave 3 to a TT. Previous
studies of T4 endonuclease VII and of T7 endonuclease I revealed that
each of these enzymes resolves J26 predominantly by symmetrical strand
cleavage at one of three sites within the homologous core (45). None of
these sites is common to both enzymes. Cleavage at all six sites occurs
next to a pyrimidine, and in five out of the six occurs between two
pyrimidines. The CCE1 resolvase of Saccharomyces
cerevisiae also targets cleavage between two pyrimidines (51).
This preference for cleaving the DNA between or next to a pyrimidine
dinucleotide by five different resolvases suggests that some general
feature of these dinucleotides, or their purine complements, affects
folding of the junction and its interaction with the protein. RuvC has
been shown to bind junctions in an open configuration and even then
will resolve the structure only when the target sequences are in the
non-crossover strands (22, 23). The fact that four of the enzymes
tested on J26 target different pyrimidine dinucleotide sites indicates that each enzyme also has to make specific contacts with bases flanking
the cleavage site. It reinforces the fact that both topological factors
and sequence-dependent effects determine the efficiency of
resolution (28, 29).
By constraining a TCC target to homologous cores of 4 and 3 base pairs, we found that the efficiency of junction resolution by RusA depends not only on DNA sequence but also on the number of base pairs in the homologous core. For instance, RusA resolves J11 and J12 with approximately equal efficiency, whereas under the same reaction conditions it resolves nearly twice as much of J3. Furthermore, J26 is cleaved as efficiently as J11 or J12 despite having more than twice the length of homologous core. However, it also has two sequences that are targeted efficiently by RusA, as opposed to one in both J11 and J12. These observations raise the possibility that branch migration may be a limiting factor for RusA, although we cannot rule out other junction-specific effects.
Under the conditions used, RusA rarely resolved more than 85% of the substrate DNA even when the junction was constrained. This limitation can be attributed in part to the instability of the protein. Most (~80%) of its resolution activity is lost within 1 min at 37 °C when incubated in the absence of junction DNA. This means the protein is likely to have difficulty cycling from one molecule to another without losing activity. The X-junctions used in this work that are cleaved by RusA have homologous cores of 3-26 base pairs within which the junction itself can branch migrate. If the junction point is distributed and RusA, like RuvC (24), resolves molecules only when the crossover is at a target sequence, then branch migration would be required to achieve 100% resolution. Previous studies have shown that junctions fold into an antiparallel stacked-X in the presence of divalent metal ions such as Mg2+, and have to unstack before branch migration can occur (15, 52-55). RuvA protein, which acts with RuvB to catalyze branch migration, solves this problem in vivo by holding the DNA in an open configuration that enables branch migration to be driven over many base pairs in a single step (31, 34). With naked junctions, the stacking and unstacking of the DNA between each base pair of branch migration is rate-limiting (56).
Branch migration of free DNA is unlikely to be a significant factor in determining the efficiency of resolution by RusA over the time scale of our experiments. The spontaneous rate of junction branch migration in Mg2+ (56) is considerably faster than the rate of decay of RusA. Therefore, even if each RusA dimer was limited to cleaving one junction, it should be possible to resolve all the junction molecules by having a large excess of protein over DNA. What we actually found was that beyond a certain level, increasing the protein to DNA ratio reduced resolution activity, which is contrary to expectation. In the two cases examined, there was a lower limit to the amount of junction resolved at high protein concentrations. The limit was 10-15% for J12 and 30% for J3, which suggests that it is related to the length of the homologous core. We suspect therefore that the autoinhibition observed is due to a mass effect of protein binding, which would limit the junction folding and unfolding needed to allow branch migration. This "freezing" of the junction would limit resolution to those molecules located at a cleavable sequence. Given the instability of the free protein, binding of at least some of the junctions at non-cleavable positions readily explains the ceiling on junction resolution at more modest ratios of protein to DNA.
We found that resolution could be increased substantially by replacing Mg2+ in the buffer used with Mn2+, or by increasing the pH. This could be due to some specific effect on the enzyme. However, very similar findings were reported previously for RuvC (41, 48), raising the possibility that it might be related to a more general effect on protein-DNA interaction. Furthermore, the stimulation of RusA by Mn2+ was much greater with J12 than with J4 or J3, again suggesting some possible relationship with branch migration. Mn2+ and alkaline pH may destabilize the interaction of RusA with junction DNA, thereby allowing more freedom for branch migration before the protein decays. The fact that very little of the additional resolution in Mn2+ involved cleavage at new sites argues against any general reduction in cleavage specificity.
The similarity between RusA and RuvC revealed by these studies is striking. However, there are significant differences, apart from the sequence specificity. RusA seems much more active in resolution than RuvC. This was particularly noticeable when RusA was used at a concentration that did not cause autoinhibition (Fig. 8). It is difficult to quantify because the sequence-specificity requires the use of different junctions for the two proteins. However, it is likely to be substantial given that RusA is also prone to decay, which limits its turnover of junctions. RuvC appears to be particularly stable under standard reaction conditions and can withstand prolonged incubation at room temperature in the absence of substrate DNA (41). It is clear from genetic studies that RuvC cannot function efficiently in vivo without RuvAB (1, 30, 36). It most probably functions as part of a RuvABC resolvasome complex assembled on junction DNA (35). In vitro resolution reactions containing RuvC alone may therefore not reflect its true activity. RusA on the other hand appears to function alone, although its ability to promote recombination and DNA repair does require the presence of RecG (30). It can certainly do without RuvAB; that is how it was discovered (30). Indeed, its activity is inhibited by RuvA both in vivo (30) and in vitro (this work). The reason why RecG activity is needed is less clear. There is no evidence for a direct interaction between the two proteins. One possibility is that RecG helps RusA by moving junctions located at non-cleavable sites.
The ability of RusA to act alone is consistent with its bacteriophage origin (36). This may also explain both its high activity and instability. Presumably, a short burst of potent resolvase activity would be needed late in the infection cycle to resolve junctions prior to packaging DNA. There would be no need to evolve a protein that retained its activity over a long time scale. In this respect, RusA may resemble other bacteriophage enzymes such as bacteriophage T4 endonuclease VII. However, its sequence specificity and failure to resolve a static junction is more characteristic of a specialized Holliday junction resolvase than of an enzyme required to remove branch structures from DNA.
We thank Gary Sharples and Peter McGlynn for their advice, Akeel Mahdi for pAM151, and Carol Buckman for excellent technical support.