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J. Biol. Chem., Vol. 275, Issue 33, 25540-25546, August 18, 2000
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From the Department of Biochemistry, University of Dundee,
Dundee DD1 5EH
Received for publication, April 21, 2000, and in revised form, May 31, 2000
Holliday junction resolving enzymes are
ubiquitous proteins that function in the pathway of homologous
recombination, catalyzing the rearrangement and repair of DNA. They are
metal ion-dependent endonucleases with strong structural
specificity for branched DNA species. Whereas the eukaryotic nuclear
enzyme remains unknown, an archaeal Holliday junction resolving enzyme,
Hjc, has recently been identified. We demonstrate that Hjc manipulates
the global structure of the Holliday junction into a 2-fold symmetric X
shape, with local disruption of base pairing around the point of
cleavage that occurs in a region of duplex DNA 3' to the point of
strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic metal ion binding domain in Hjc that
has been identified previously in several restriction enzymes. The
roles of catalytic residues conserved within this domain have been
confirmed by site-directed mutagenesis. This is the first
example of this domain in an archaeal enzyme of known function
as well as the first in a Holliday junction resolving enzyme.
Holliday junction resolving enzymes play a role in the pathway of
homologous recombination, recognizing and cleaving the four-way DNA
junctions that arise from strand exchange between homologous duplex DNA
species. Junction resolving enzymes are ubiquitous in nature. These
proteins have been identified in Eubacteria (RuvC (1, 2) and RusA (3)),
bacteriophage (T4 endonuclease VII (4) and T7 endonuclease I (5, 6)),
fungal mitochondria (Cce1 (7)), and most recently Archaea (Hjc and Hje
(8, 9)). Whereas activities have been detected in nuclear extracts from yeast (10) and mammalian cells (11, 12), the relevant genes have yet to
be identified. Resolving enzymes function as dimers, resolving the
four-way DNA junction by the introduction of paired nicks in opposing
strands with a magnesium-dependent endonuclease activity.
Despite these functional similarities, the junction resolving enzymes
are structurally diverse with no detectable sequence similarity among
any of the known examples. Structural studies have highlighted this
diversity because the crystal structures of RuvC (13), T4 endonuclease
VII (14), and T7 endonuclease I1 have radically different
folds. These observations have led to the suggestion that resolving
enzymes have arisen several times during the course of evolution,
perhaps by recruitment of nucleases with other cellular roles. This is
almost certainly the case for the eubacterial enzyme RuvC, which shares
a fold and metal binding site with members of the RNase HI superfamily
(13, 15).
The Archaea constitute a third domain of life that is distinct from
both the Eubacteria and the Eucarya. Whereas the Archaea resemble their
fellow prokaryotes in most respects, they share many similarities with
the Eucarya in the information processing pathways including DNA
replication, transcription, and translation (reviewed in Ref. 16), and
the archaeal processes constitute a useful model system for the much
more complex eucaryal equivalents. We are investigating the pathway of
homologous recombination in the Archaea and have detected two Holliday
junction resolving enzymes, Hje and Hjc, in the Crenarchaeote
Sulfolobus solfataricus (8, 17). The gene for Hjc has been
identified and is conserved in all Archaea for which extensive genome
sequence is available (8, 9). We have cloned S. solfataricus
Hjc and overexpressed the recombinant enzyme in Escherichia
coli (8). Here we demonstrate manipulation of the global structure
of the four-way junction substrate by Hjc and disruption of base
stacking near the cleavage site. We report the identification of a
structural motif in Hjc that is shared by a diverse group of nucleases
and constitutes a binding site for the catalytic metal ions. Parallels
between Holliday junction resolving enzymes and restriction enzymes
that were first pointed out at a biochemical level for the yeast
mitochondrial enzyme Cce1 (18) now achieve a firm structural basis in
the archaeal Hjc enzyme.
Oligonucleotide Synthesis and Assembly of DNA Junctions
Oligonucleotides were synthesized and four-way DNA junctions
assembled as described previously (18) using the sequences described in
the following paragraphs.
Junction 3--
This fixed four-way junction was prepared with
arms of 15 or 25 bp2 as
described previously (45). Comparative gel electrophoresis experiments utilized a version of junction 3 with four arms of 60 bp in length. Six forms of junction 3 with two long and two short arms were derived from this junction as described previously (46).
Junction 1--
This is a fixed junction with 20 bp in each arm
assembled from four oligonucleotides, each of 40 nucleotides in
length as described previously (47).
Junction Z28--
This junction is utilized for
permanganate-probing experiments. The junction has arms of 15 bp
including eight thymine residues centered around the point of strand
exchange on the r strand: b, 5'-TCCGTCCTAGCAAGGAGTCTGCTACCGGAA;
h, 5'-TTCCGGTAGCAGACTAAAAGGTGGTTGAAT; r,
5'-ATTCAACCACCTTTTTTTTAACTGCAGCAG; x,
5'-CTGCTGCAGTTAAAACCTTGCTAGGACGGA.
Expression and Purification of Recombinant Hjc Protein
Recombinant Hjc protein was expressed in E. coli
strain BL21 (DE3) CodonPlus RIL (Stratagene), and the protein was
purified as described previously (8). In brief, Hjc was purified by chromatography on an SP-Sepharose high performance 26/10 column (Hi-Load, Amersham Pharmacia Biotech) equilibrated with Buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol). A 500-ml linear gradient of 0-1000
mM NaCl was used to elute cationic proteins. Fractions
corresponding to a distinct absorbance peak were analyzed by
SDS-polyacrylamide gel electrophoresis, pooled, concentrated, and
loaded onto a 26/70 gel filtration column (Superdex 200 Hi-Load,
Amersham Pharmacia Biotech) and developed with Buffer A containing 300 mM NaCl. Active fractions were pooled and shown to contain
essentially homogeneous Hjc protein. This protein was used for all
subsequent analyses.
Site-directed Mutagenesis
Site-directed mutagenesis of Hjc was carried out in the plasmid
pUC119 using the QuikChange method (Stratagene). After mutagenesis, DNA
sequencing was used to confirm that no spurious mutations had been
introduced. The Hjc mutants were subcloned into pET19b and were
expressed and purified similar to the wild-type enzyme.
The oligonucleotides used to introduce the mutations are listed
as follows: E12Qfor, 5'-GGAAAGGTTCCGCAGTACAACGAAATATTGTGAG; E12Qrev,
5'-CTCACAATATTTCGTTGTACTGCGGAACCTTTCC; D42Nfor,
5'-GACCCTATACCGAATATTATCGCT; D42Nrev, 5'-AGCGATAATATTCGGTATAGGGTC;
E55Qfor, 5'-CGTTATTATTTTAATTCAGATGAAGAGTAG; E55Qrev,
5'-CTACTCTTCATCTGAATTAAAATAATAACG; K57Afor,
5'-TTTTAATTGAGATGGCGAGTAGAAAGG; K57Arev,
5'-CCTTTCTACTCGCCATCTCAATTAAAA.
Comparative Gel Electrophoretic Retardation Analysis of Hjc-DNA
Junction Interactions
Samples of purified Hjc protein (1 µM) were
incubated with radioactive 5'-32P-labeled four-way DNA
junction 3 in binding buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1 mg/ml bovine serum albumin, and 0.2 mg/ml calf
thymus duplex competitor DNA) including either 1 mM EDTA or
0.1 mM MgCl2 in a 10-µl total volume for 5 min at 20 °C, prior to addition of loading buffer (0.25% bromphenol
blue, 0.25% xylene cyanol FF, 15% Ficoll type 400) at a dilution of
1:6 (v/v). Samples were loaded onto 5% polyacrylamide gels and
electrophoresed in Tris-borate-EDTA buffer or Tris-borate and
0.1 mM MgCl2 with buffer recirculation. After
electrophoresis, gels were dried on Whatman 3MM paper and exposed
to x-ray film for documentation.
Equilibrium Binding of Wild-type and Mutant Hjc Proteins
Binding affinity was measured by gel electrophoretic retardation
analysis using radioactive 5'-32P-labeled junction 1 in
EDTA binding buffer as described previously (8).
Assay of Hjc Activity
Assays were carried out using 1 µM purified
recombinant Hjc protein in reaction buffer (20 mM Tris-HCl,
pH 7.5, 50 mM NaCl, 15 mM MgCl2)
using 80 nM 5'-32P-labeled junction as a
substrate. Calf thymus DNA (0.2 mg/ml) was added as a competitor to
minimize nonspecific endonuclease activity and DNA-binding proteins.
Reactions were initiated by the addition of magnesium to the assay mix
in a 5-µl total volume and incubated at 60 °C. At set time points
aliquots were removed, and the reactions were stopped by the addition
of 4 µl of formamide/EDTA loading mix with heating to 95 °C.
Products were analyzed by denaturing gel electrophoresis and phosphor
imaging as described previously (48).
Modification of DNA by Potassium Permanganate
Radioactive 5'-32P-labeled four-way DNA junction Z28
was incubated at room temperature for 5 min in the presence or absence of 1 µM Hjc protein in binding buffer. The total reaction
volume was 20 µl. Reactions were initiated by the addition of 2 µl
of freshly dissolved 25 mM KMnO4 and were
stopped after 1 min with the addition of 1.5 µl of
Manipulation of the Global Structure of the Four-way DNA Junction
by Hjc--
The Holliday junction assumes a conformation known as the
"stacked X structure" in the presence of divalent metal ions,
folding by coaxial stacking of pairs of helices in an antiparallel
2-fold symmetric cross (reviewed in Ref. 19). This structure has been studied by a wide variety of techniques and has recently been confirmed
by x-ray crystallography (20, 21). Comparative gel electrophoresis has
proven a powerful technique for the analysis of the global
configuration of the four-way junction, both in solution and complexed
with proteins. Using this technique, all Holliday junction binding
proteins studied to date have been shown to alter the conformation of
the Holliday junction on binding (reviewed in Ref. 22).
We examined the effect of Hjc binding on the global conformation of the
four-way junction J3 using comparative gel electrophoresis. In EDTA,
the free junction species adopted the four-slow/two-fast pattern
characteristic of the 4-fold symmetric junction with arms related by
90°. In the protein-DNA complex, this pattern was altered significantly with bound species adopting a distinctive "butterfly" (slow-fast-intermediate-intermediate-fast-slow) pattern (Fig. 1). When the experiment was repeated in
the presence of 0.1 mM Mg2+, the free junction
adopted a slow-medium-fast-fast-medium-slow "smile" pattern, as
observed previously for J3, which folds with B upon X and H upon R
stacking (23). Again, the Hjc·DNA complexes adopted the
butterfly pattern. These observations suggest that the four-way
DNA junction is bound by Hjc in a 2-fold symmetric conformation with
the B/X arms and H/R arms related by acute angles and the B/H and R/X
pairs related by obtuse angles (Fig. 1). This X shape is reminiscent of
that induced on J3 by the resolving enzyme RuvC, although in that case
the structure deviated only slightly from 4-fold symmetry and tended
toward acute angles between the B/H and R/X pairs (24). Whereas we
cannot quantify the angles between the pairs of arms, the large
differences in mobility of the 6 S species suggest that the angles
deviate significantly from the 90° observed for Cce1 and RuvA and may
approach or surpass a 120°/60° relationship for the obtuse and
acute angles.
Using a single turnover kinetic assay, we measured the rates of
cleavage of each of the four strands of junction J3 individually. Cleavage was found to be biased, with a 5-fold faster rate observed for
the b and r strands compared with the h and x strands (Fig. 2). Relating this to the global structure
of J3 in complex with Hjc, the more strongly cleaved strands are
situated in the strands exchanging with acute angles, 3 nucleotides 3'
of the branch point (Figs. 1 and 2). Cleavages in the other two strands
probably reflect a minor population of the junction that has been bound
by Hjc in the other possible conformation with the B/X and H/R pairs of
arms related by acute angles.
Permanganate Probing Suggests Disruption of Base Pairing at the
Cleavage Site--
Thymine residues in DNA are susceptible to
modification by potassium permanganate but are protected from
modification in the context of fully stacked duplex DNA. This
modification renders the phosphodiester backbone susceptible to
cleavage by piperidine. Modification by potassium permanganate is
therefore a sensitive probe for base stacking in duplex DNA. Holliday
junction binding proteins such as Cce1 and RuvA increase the
sensitivity of thymines at the branch point to permanganate
modification in the presence of magnesium ions, and this has been
interpreted as evidence that the proteins unstack the DNA junction and
possibly disrupt base pairing at the point of strand exchange (24,
25).
Permanganate probing was used to analyze base stacking in the context
of the Hjc-junction complex with junction Z28, which has eight thymines
flanking the point of strand exchange on the r strand. In the absence
of Hjc protein, all eight thymines were subject to modification by
potassium permanganate, with the strongest reactivity at the 2 nucleotides flanking the point of strand exchange and symmetrical
distribution with respect to the junction center (Fig.
3A, lane 3). The presence of
magnesium in the reaction reduced the sensitivity of the flanking
thymines to permanganate, consistent with junction stacking under these
conditions (Fig. 3A, lane 6). In the presence of saturating
concentrations of the Hjc protein, we observed a marked increase in
reactivity with potassium permanganate in both EDTA and magnesium (Fig.
3A, lanes 4 and 7).
Quantification of the increase in reactivity for each of the thymine
residues (from positions A Conserved Motif in Hjc Resembles the Active Site of Type II
Restriction Enzymes--
Analysis of the residues conserved in a
multiple alignment of all known Hjc sequences revealed a motif
containing three acidic residues: Glu-12, Asp-42, and Glu-55 followed
by lysine (Lys-57), similar to a conserved motif present in type II
restriction enzymes (28) (Fig. 4). This
motif constitutes part of the active site of the restriction enzymes,
with the three acidic residues forming the metal binding pocket for the
two catalytic magnesium ions (29) and the conserved lysine predicted to
play a role in stabilizing the transition state during catalysis
(26).
To investigate the predicted secondary structure of Hjc in this region,
we submitted a multiple alignment of seven Hjc proteins to the
PredictProtein structure prediction program (30). The results
are outlined in Fig. 4 with the conserved domain of Hjc aligned with
the corresponding domains of EcoRV, PvuII, and
BglI. The predicted secondary structure for Hjc consists of
an Roles of Conserved Residues Probed by Site-directed
Mutagenesis--
To test our model for the metal binding site of the
Hjc protein, we created site-directed mutants of the three conserved
acidic residues (E12Q, D42N, E55Q) and the conserved lysine (K57A). The mutant proteins were expressed in E. coli and purified as
for the wild-type enzyme. The D42N mutant was poorly expressed in E. coli, and only small amounts of protein could be
purified. The other three mutants were expressed at similar levels to
the wild-type protein and retained the ability to bind specifically to
the four-way junction J1 with an affinity comparable with the wild-type
Hjc protein (Fig. 5A). To
compare the catalytic activities of the wild-type and mutant enzymes,
we incubated the proteins with the four-way junction substrate Z28 in
cleavage buffer at 60 °C and separated cleavage products from uncut
substrate by denaturing gel electrophoresis (Fig. 5B). A
weak residual activity in the mutant K57A represented a 1000-fold
decrease in activity compared with the wild-type enzyme. Mutants E12Q,
D42N, and E55Q displayed no detectable catalytic activity. All four of
these mutations thus appeared to have had a specific effect on
catalysis rather than on substrate binding, consistent with the
functional assignment made by analogy to the equivalent residues in
EcoRV and other nucleases.
Hjc contains a motif first identified as constituting part of the
active site of the type II restriction enzyme EcoRV.
Subsequent crystal structures of the restriction enzymes
PvuII (32) and BglI (33) demonstrated that the
metal binding pocket is structurally conserved in an We have shown by site-directed mutagenesis that mutations in residues
Glu-12, Asp-42, Glu-55, and Lys-57 of Hjc all result in at least a
1000-fold decrease in catalytic activity, though the mutants retain the
ability to bind the four-way junction. By comparison, mutagenesis of
the equivalent residues in EcoRV results in similar large
decreases in catalytic activity without significantly affecting
substrate binding (40-44). This is the first example of this
domain in a Holliday junction resolving enzyme and the first in an
archaeal protein of known function.
Using the sequence alignments shown in Fig. 4, we used the crystal
structure of the metal binding domain of EcoRV to generate a
model of the catalytic domain of Hjc (Fig.
6A). The model encompasses the
N-terminal half of the Hjc protein consisting of an In conclusion, we have demonstrated that the archaeal Holliday junction
resolving enzyme Hjc manipulates the global structure of the four-way
DNA junction into a 2-fold symmetric X conformation on binding. Paired
nicks in the phosphodiester backbone are introduced on the minor groove
face in a region of duplex DNA 3 bp from the point of strand exchange,
accompanied by local distortion of base stacking in the duplex. Hjc
utilizes an We thank Steve Halford for pointing out the
sequence similarity between Hjc and the catalytic domain of the
restriction enzymes.
*
This work was funded by the Biotechnology and
Biological Sciences 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.
Published, JBC Papers in Press, June 6, 2000, DOI 10.1074/jbc.M003420200
1
S. E. V. Phillips and D. M. J. Lilley,
personal communication.
The abbreviation used is:
bp, base pairs.
A Conserved Nuclease Domain in the Archaeal Holliday Junction
Resolving Enzyme Hjc*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol. After ethanol precipitation, DNA samples were
reacted with 100 µl of 1 M piperidine for 30 min at
95 °C. Piperidine was removed by vacuum desiccation, and the pellets
were washed three times with 30 µl of water and were vacuum
desiccated after each water addition. The dried samples were
resuspended in formamide loading mix and analyzed on 15% denaturing
polyacrylamide gels.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Comparative gel electrophoretic analysis of
the global structure of the four-way junction upon binding of Hjc.
Six species of radioactive 5'-32P-labeled junction 3 were
assembled from 16 oligonucleotides such that each species had two long
arms of 60 bp and two short arms of 15 bp. A,
analysis in the absence of magnesium ions. The six junction species
were incubated with Hjc in the presence of 1 mM EDTA, and
the products were analyzed by electrophoresis on a 6% polyacrylamide
gel containing 1 mM EDTA. The free junction exhibits the
slow-fast-slow-slow-fast-slow pattern characteristic of the extended
square configuration of the four-way junction in the absence of metal
ions (top left). The complexed species exhibit a
slow-fast-intermediate-intermediate-fast-slow butterfly pattern,
indicating an alteration in the global junction conformation by Hjc.
This pattern can be interpreted as a 2-fold symmetric conformation,
with the BH and RX pairs of arms related by an acute angle and the BX
and HR pairs related by an obtuse angle. B, analysis in the
presence of 100 µM magnesium ions. The junction species
were incubated with Hjc in the presence of 100 µM
magnesium ions, followed by gel electrophoresis in the presence of 100 µM magnesium ions. The free species migrate in the
slow-intermediate-fast-fast-intermediate-slow pattern indicative of the
stacked X structure with B upon X coaxial stacking (bottom
left). The bound species exhibit the
slow-fast-intermediate-intermediate-fast-slow pattern, as seen in the
presence of EDTA, indicating that the bound junction conformation is
independent of the presence or absence of magnesium ions. The following
junction species were electrophoresed in both A and
B. Lane 1, species with long B and H arms;
lane 2, species with long B and R arms; lane 3,
species with long B and X arms; lane 4, species with long H
and R arms; lane 5, species with long H and X arms;
lane 6, species with long R and X arms; lanes
7-12, same as lanes 1-6 with the addition of Hjc
protein.
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Fig. 2.
Comparison of cleavage rates of the four
strands of J3. Single turnover kinetic analysis of the rates of
cleavage of the four strands of junction J3 by Hjc. Paired nicks are
introduced by Hjc 3 nucleotides 3' of the junction center. Cleavage of
the b and r strands is 4-5-fold faster than for the h and x strands.
The natural logarithm of the ratio (total junction/uncleaved junction)
is plotted against time of cleavage. Data points are the
means of triplicate measurements with error bars
showing S.E. The first order rate constant (kc) was
calculated from the gradient of a line fitted to the data by linear
regression through the origin. The inset shows J3 in the X
conformation suggested by comparative gel electrophoresis with
arrows indicating the positions of strong cleavage in the b
and r strands 3 nucleotides 3' of the junction center. b strand
cleavage ( 
) rate is 19 ± 0.3 × 10
5
s1. r strand cleavage (
) rate is 15 ± 0.5 × 105 s1. h strand cleavage (
) rate is
3.4 ± 0.2 × 105 s1. x strand
cleavage (
) rate is 4.1 ± 0.1 × 105
s1.
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Fig. 3.
Base unstacking probed by potassium
permanganate. Hjc binding to four-way junction Z28 induces
distortion of base stacking that is asymmetrical with respect to the
point of strand exchange and correlates with the position of strand
cleavage. A, junction Z28, containing four thymine residues
on either side of the branch point of strand r, was
5'-32P-labeled on the r strand. The junction was incubated
in the presence of 1 mM EDTA or 0.1 mM
MgCl2 with or without 1 µM Hjc protein in
binding buffer and was reacted with potassium permanganate as described
under "Materials and Methods." Reactivity to permanganate was
observed at all eight thymine positions, and this was enhanced
by the addition of Hjc protein. The position of the point of strand
exchange is indicated by the white triangles. Lane
1, junction Z28 strand r A+G markers; lane 2, junction
Z28 C+T markers; lanes 3 and 4, junction in EDTA
reacted with permanganate in the absence and presence of Hjc,
respectively; lane 5, control showing junction cleavage
background without permanganate modification; lanes 6 and
7, junction in magnesium reacted with permanganate in the
absence and presence of Hjc, respectively. B, denaturing gel
electrophoretic analysis of the cleavage of each of the four strands of
junction Z28 by Hjc. Strong cleavage was observed in the h and x
strands, 3 nucleotides 3' of the point of strand exchange (indicated by
arrows in the schematic representation of the junction),
with little or no cleavage observed in the b or r strands. The point of
cleavage in the h strand opposes the area of increased reactivity to
permanganate observed in the r strand. C, the enhancement in
reactivity to permanganate was calculated for each of the 8 nucleotide
positions in the r strand of junction Z28 by phosphor imaging, and the
data from the mean of duplicate experiments were plotted as a
histogram. Black bars represent reactivity in EDTA and
gray bars reactivity in the presence of magnesium. A bias in
the reactivity toward the thymines 5' of the point of strand exchange
is apparent.
4 to +4) revealed a pronounced bias in the
reactivity toward the 5' side of the junction center (Fig.
3B), suggesting the protein unstacks the duplex DNA in this region. Analysis of the cleavage of junction Z28 by Hjc reveals that,
similar to junction J3, cleavage is biased, occurring predominantly in
the h and x strands 3 nucleotides 3' of the branch point (Fig. 3C). The position of cleavage of the h strand coincides with
the area of enhanced reactivity toward permanganate detected on the r
strand, suggesting that Hjc may introduce structural distortion in the
DNA helix near the cleavage site. The base pairing in this region of
the DNA duplex may be weakened or disrupted by Hjc on binding. Such
distortions are common features of DNA recognition by restriction
enzymes such as EcoRV (26) and EcoRI (27).
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Fig. 4.
A conserved metal binding domain in Hjc.
A, Sulfolobus Hjc sequence from residues 1-60 is
shown aligned with that of three type II restriction endonucleases,
EcoRV, BglI, and PvuII. Four residues
absolutely conserved in all Hjc sequences (Glu-12, Asp-42, Glu-55, and
Lys-57) are highlighted in black. These align with residues
in all three restriction endonucleases that are known to constitute the
binding pocket for the catalytic metal ions at the active site. The
secondary structural elements of the three restriction enzymes taken
from the crystal structures and the predicted secondary structure of
Hjc generated by the program PredictProtein (30) using a
multiple alignment of all the Hjc proteins are shown below the
respective sequences. H, 
-helix (light gray);
E, -sheet (dark gray). There is a good match
between the known domain structures of the three restriction enzymes
and the predicted structure of the first 60 residues of the Hjc
protein. B, cartoon showing common structural elements of
EcoRV and Hjc. The positions of the catalytic residues in
EcoRV are indicated, with the corresponding numbering for
Hjc residues in parentheses.
-helix containing the first acidic residue followed by three
-sheets, in remarkable agreement with the known secondary structures
of the three restriction enzymes (31).
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Fig. 5.
Characterization of site-directed mutants of
Hjc. Mutant forms of the Hjc protein containing the mutations
E12Q, D42N, E55Q, and K57A were constructed and purified and tested for
their ability to bind to and cleave the four-way DNA junction.
A, electrophoretic analysis of DNA junction binding
affinity. Radioactive 5'-32P-labeled junction 1 (1 nM) was incubated with increasing concentrations of
wild-type or mutant Hjc at room temperature in binding buffer in the
presence of 1 mM EDTA. Free junction and DNA-junction
complexes were separated by electrophoresis in 5% acrylamide gels. The
fraction of DNA-junction bound to protein was calculated for each
concentration of Hjc by phosphor imaging and plotted against the
logarithm of the protein molarity (with Hjc assumed to be dimeric). The
data were fitted to a model for the binding process (see "Materials
and Methods"), from which the binding affinities were calculated. All
measured equilibrium dissociation constants agreed to within a factor
of 3. , wild-type Hjc; , mutant E12Q; , mutant E55Q; ,
mutant K57A. B, activity of wild-type and mutant Hjc enzymes
is shown. Mutants E12Q, D42N, and E55Q gave no detectable cleavage
products after incubation with junction Z28 labeled on the h strand for
30 min at 60 °C (lanes 1-3). Very weak activity was
detected for mutant K57A under the same conditions (lane 4)
whereas the wild-type (w-t) enzyme displayed strong cleavage
activity after a 20-s incubation (lane 5). Lanes
1-4 show junction Z28H that was incubated for 30 min at
60 °C with mutants E12Q, D42N, E55Q, and K57A, respectively.
Lanes 5 and 6 show junction Z28H that was
incubated at 60 °C with wild-type Hjc for 20 s and 30 min,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-barrel
catalytic domain in the three enzymes, even though the overall folds
are very different. The type II restriction enzymes EcoRI,
BamHI, and Cfr101 also possess this -barrel
domain (reviewed in Refs. 34 and 35), and more recently it has been
identified in MutH (35), 
exonuclease (36), and type I and III
endonucleases (37). All of these enzymes have a catalytic (usually
N-terminal) domain in the form of an -barrel with different
domains that mediate subunit interactions and DNA recognition (35, 38).
The quaternary structures and functions of these proteins are
remarkably divergent. Type II restriction enzymes are dimeric and
recognize and cleave specific nucleotide sequences. MutH is monomeric,
and its activity is regulated by the MutL and MutS proteins following
mismatch DNA recognition (35). However, -exonuclease, a processive
5'
3' exonuclease that degrades one strand of a DNA duplex after the
formation of a double strand break, has a toroidal homotrimeric
structure (36). Perhaps the most striking example of the separation of
recognition and catalysis is seen in the restriction enzyme
FokI, which has the catalytic -barrel domain separated
from the DNA recognition domain by a linker (39). Nature has thus
repeatedly utilized a catalytic metal ion binding domain efficient in
phosphodiester bond hydrolysis coupled with an array of specific DNA
and protein recognition elements to solve many diverse problems in
nucleic acid metabolism.
-helix and a
three-stranded antiparallel -sheet. The three acidic residues group
in reasonable positions to act as ligands for two magnesium ions
(magenta spheres in the model), and the orientation of the conserved lysine is consistent with a role in catalysis. By analogy with the other nucleases containing the metal ion binding domain, the
C-terminal half of the protein, which is less conserved in Hjc proteins
from different archaeal species, may play a role in dimerization
and/or DNA recognition. Fig. 6B shows a model for the
complex of the catalytic domain of Hjc, positioned on the minor groove
side of a four-way DNA junction, which is represented in the X
conformation suggested by comparative gel electrophoresis. The presumed
catalytic magnesium ion has been positioned within 5 Å of the known
site of cleavage on the phosphodiester backbone. Using this constraint,
the model suggests a possible mode of binding of the Hjc protein with
the four-way DNA junction substrate. Basic residues in Hjc are in
reasonable proximity to the negatively charged phosphodiester backbone,
and in particular, the model suggests possible roles for two arginines,
Arg-13 and Arg-28. These are the only basic residues other than the
catalytic Lys-57 that are absolutely conserved in all Hjc sequences,
and in the model of the complex they exist in suitable positions to
form salt bridges with phosphates on an adjacent arm of the junction. On the basis of the model and the conservation of these residues we
predict that they are important for DNA binding and possibly for
recognition of the branched structure of the four-way DNA junction.
View larger version (42K):
[in a new window]
Fig. 6.
Model of the catalytic domain of Hjc and its
interaction with the Holliday junction. Catalytic domain models
were generated using the program MODELLER (49) with the structure
generated based on homology to the requisite domain of EcoRV
(1B94). Twenty model structures were generated and averaged using the
program X-PLOR (50). The junction was generated within INSIGHT II
(Molecular Simulations, Inc.). The complete structure was
assembled with the rigid body minimization using distance restraints
between the catalytic metal ions, the conserved arginine residues, and
the DNA backbone. A, model of the catalytic domain of Hjc.
The structure is represented by a cartoon of the predicted secondary
structure. Proposed catalytic residues and metal ions are shown as
stick and space filling representations,
respectively. B, model of the Hjc-junction complex. Model
catalytic domains are represented by ribbons with the
positions of the magnesium ions and the conserved residues Arg-13 and
Arg-28 indicated as stick representations. The model DNA
junction structure is shown in gray with a backbone
ribbon highlighting the position of junction cleavage in
red.
-barrel domain for the chemical step in the
resolution of four-way DNA junctions, with four conserved catalytic
residues found in many other nucleases of diverse function. The
C-terminal half of the Hjc protein may complete the -barrel
domain and provide an interface for dimerization. Thus the Hjc dimer
may consist of two nuclease domains capable of introducing nicks in
duplex DNA on either side of the branch point of a four-way junction,
with other structural elements providing structural specificity and
forming the dimer interface.
ACKNOWLEDGEMENT
FOOTNOTES
A Royal Society University Research Fellow. To whom correspondence
should be addressed: Center for Biomolecular Sciences, St. Andrews
University, North Haugh, St. Andrews KY 16 9ST, United Kingdom. Tel.:
01334-463432; Fax: 01334-462595; E-mail: mfw2@st- andrews.ac.uk.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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