Biochemical Characterization of a Structure-Specific
Resolving Enzyme from Sulfolobus islandicus
Rod-Shaped Virus 2
Andrew F. Gardner*, Chudi Guan, William E. Jack
New England Biolabs, Inc., Ipswich, Massachusetts, United States of America
Sulfolobus islandicus rod shaped virus 2 (SIRV2) infects the archaeon Sulfolobus islandicus at extreme temperature (70uC–
80uC) and acidity (pH 3). SIRV2 encodes a Holliday junction resolving enzyme (SIRV2 Hjr) that has been proposed as a key
enzyme in SIRV2 genome replication. The molecular mechanism for SIRV2 Hjr four-way junction cleavage bias, minimal
requirements for four-way junction cleavage, and substrate specificity were determined. SIRV2 Hjr cleaves four-way DNA
junctions with a preference for cleavage of exchange strand pairs, in contrast to host-derived resolving enzymes, suggesting
fundamental differences in substrate recognition and cleavage among closely related Sulfolobus resolving enzymes. Unlike
other viral resolving enzymes, such as T4 endonuclease VII or T7 endonuclease I, that cleave branched DNA replication
intermediates, SIRV2 Hjr cleavage is specific to four-way DNA junctions and inactive on other branched DNA molecules. In
addition, a specific interaction was detected between SIRV2 Hjr and the SIRV2 virion body coat protein (SIRV2gp26). Based
on this observation, a model is proposed linking SIRV2 Hjr genome resolution to viral particle assembly.
Citation: Gardner AF, Guan C, Jack WE (2011) Biochemical Characterization of a Structure-Specific Resolving Enzyme from Sulfolobus islandicus Rod-Shaped Virus
2. PLoS ONE 6(8): e23668. doi:10.1371/journal.pone.0023668
Editor: Jean-Pierre Vartanian, Institut Pasteur, France
Received June 2, 2011; Accepted July 22, 2011; Published August 17, 2011
Copyright: ? 2011 Gardner et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by New England Biolabs, Inc (www.neb.com). The funders had no role in study design, data collection and analysis, decision
to publish, or preparation of the manuscript.
Competing Interests: The authors are employed by New England Biolabs, Inc. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing
data and materials. This study was conducted in the Research Department and is not currently intended for product development.
* E-mail: email@example.com
Holliday junction resolving enzymes are structure-specific
endonucleases that catalyze key steps during DNA homologous
recombination and replication . Resolving enzymes have been
identified in all domains of life including bacteria (RuvC), archaea
(Hjc), and eukarya (Human GEN1) . Resolving enzymes have
attracted intense interest as a model to understand the molecular
basis for substrate recognition and cleavage of four-way junctions
. Variations in resolving enzyme sequence bias, cleavage
pattern, and substrate specificity suggest that a variety of
[3,4,5,6,7,8,9,10,11]. Sulfolobus islandicus rod shaped virus 2
(SIRV2) infects the archaeon Sulfolobus islandicus at extreme
temperature (70uC–80uC) and acidity (pH 3) and encodes a
14 kD Holliday junction resolving enzyme (SIRV2 Hjr). Hjr
protein sequences are conserved among rudiviruses including
Acidianus Rod-Shaped virus 1 (ARV1), Stygioglobus rod-shaped virus
(SRV), and Sulfolobus islandicus rod-shaped viruses 1 (SIRV1) and 2
(SIRV2), and have been proposed as key enzymes in rudivirus
genome replication . Specifically, during the last stage of
SIRV2 replication, multiple double-stranded SIRV2 genomes are
catenated. At the junctions between genome monomers, opposing
inverted terminal repeats can be extruded to form hairpin four-
way junctions. SIRV2 Hjr is proposed to introduce symmetrical
nicks across this junction to resolve the concatamers, producing
monomer copies with linear hairpin ends . Consistent with its
cleave four-way junctions
proposed biological role, SIRV2 Hjr was previously shown to
cleave four-way junctions in vitro [13,14]. SIRV2 Hjr is related to
the well-studied resolving enzymes from Sulfolobus, Hje and Hjc
[4,9,15,16,17,18,19]. Sulfolobus Hje and Hjc are homodimeric
enzymes that recognize and cleave four-way junctions by paired
nicks on four-way junction arms [4,18]. Even though a significant
amount of data is available on Sulfolobus resolving enzymes, much
less is known about the molecular mechanism of SIRV2 Hjr
specificity and cleavage. Therefore, this study demonstrates a
unique SIRV2 Hjr four-way junction cleavage pattern, the
minimal requirements for four-way junction cleavage, and
substrate specificity. Based on the biochemical analysis of SIRV2
Hjr, this study also addresses characteristics that support a role for
SIRV2 Hjr in resolution following genome replication.
Materials and Methods
‘‘Hjr’’ will be used throughout to refer to the product of the
allele from S. islandicus SIRV2 Hjr (or the codon-optimized allele
described herein), and ‘‘MBP-Hjr’’ will refer to its fusion to
Maltose Binding Protein below. Where necessary for clarity, the
attribution will be expanded.
All restriction endonucleases, modifying enzymes, DNA poly-
merases, nucleotides, DNA ladders, and expression vectors were
PLoS ONE | www.plosone.org1 August 2011 | Volume 6 | Issue 8 | e23668
from New England Biolabs. Purified Sulfolobus solfataricus Holliday
junction endonuclease (Sso Hje) was kindly provided by Dr.
Malcolm White, University of St. Andrew’s, UK.
E. coli strains for cloning (NEB 5 alpha) and expression (NEB
Turbo and NEB T7 Express) were from New England Biolabs.
MBP-SIRV2 Hjr gene synthesis, cloning and purification
To improve protein expression, a synthetic Hjr gene was codon
optimized to reflect the codon usage of E. coli rather than the
native S. islandicus. Hjr gene was synthesized by PCR amplification
of overlapping oligonucleotides .
To assemble a template for Hjr gene synthesis, an equimolar
amount (1 mM) of each overlapping oligonucleotide (Table S1) was
combined in 16Standard Taq Buffer (10 mM TrisHCl, pH 8.3,
50 mM KCl, 1.5 mM MgCl2) and then serially diluted by two-
fold. PCR reactions (50 mL) were assembled as follows: 16
Phusion Master Mix (containing dNTPs, HF reaction buffer, and
Phusion DNA polymerase), 0.5 mM Forward Primer (Table S1,
primer 1), 0.5 mM Reverse Primer (Table S1, primer 10), and Hjr
gene synthesis oligonucleotide template mixtures. Reactions were
cycled in a PCR instrument (98uC 2 minutes followed by 25 cycles
of 98uC 10 seconds, 65uC 15 seconds, 72uC for 30 seconds,
followed by a final extension step at 72uC for 30 seconds). A band
corresponding to the Hjr gene (405 bp) was gel purified. The Hjr
codon optimized PCR product was cloned into expression vector
pMAL-c4X (New England Biolabs) digested with XmnI to create a
construct (pEPD) encoding an N-terminal Maltose Binding
Protein (MBP) – SIRV2 Hjr fusion protein. The sequence of
plasmid pEPD was verified by DNA sequencing.
For MBP-Hjr expression and purification, NEB Turbo E. coli
was transformed with plasmid pEPD. A 1 liter NEB Turbo E. coli/
pEPD culture was grown at 37uC to mid-log phase (OD600=0.5),
whereupon protein expression was induced by addition of 0.4 mM
IPTG. Cells were then incubated at 37uC for five hours, and were
collected by centrifugation. The cell pellet was suspended in 0.2 L
Buffer A (20 mM TrisHCl, pH 7.5, 0.2 M NaCl, 1 mM EDTA)
and lysed by sonication. Cell debris was removed by centrifugation
and the supernatant applied to a 15 mL amylose column. The
column was washed with 0.15 L Buffer A. MBP-SIRV2 Hjr was
eluted with 30 mL Buffer A containing 10 mM maltose. MBP-Hjr
purification was monitored by 4–20% SDS-PAGE analysis.
Fractions containing MBP-Hjr were pooled, dialysed against
storage buffer (0.1 M KCl, 10 mM Tris-HCl, pH 7.4 @ 25uC,
1 mM dithiothreitol, 0.1 mM EDTA, 50% Glycerol) and stored at
220uC. A portion of MBP-Hjr was proteolysed by Factor Xa
protein to separate the MBP binding domain from SIRV2 Hjr and
not further purified. Activity at 55uC was comparable between the
intact and proteolyzed proteins, so the MBP-Hjr fusion was used
in most experiments. However, it should be noted that even
though no apparent differences in overall activity were observed
using the MBP-Hjr fusion, steps in the reaction pathway may be
influenced by the presence of the MBP-fusion.
MBP-Sulfolobus islandicus Holliday junction endonuclease
(Sis Hje) gene synthesis, cloning and purification
The gene encoding a Holliday junction endonuclease from
Sulfolobus islandicus strain Y.N.15.51 (Sis Hje)  was synthesized
using the methods described above using the overlapping
oligonucleotides presented in Table S1. Sis Hje E. coli codon
optimized PCR product was cloned into expression vector pMAL-
c4X digested with XmnI to create a construct encoding an N-
terminal Maltose Binding Protein (MBP) – Sis Hje fusion protein.
SIRV2gp26 gene synthesis, cloning and purification
A gene encoding the SIRV2gp26 coat protein was synthesized
using the methods described above, with the overlapping
oligonucleotides listed in Table S1. The SIRV2gp26 E. coli codon
optimized PCR product was cloned into expression vector pMAL-
c4X digested with XmnI to create a construct encoding an N-
terminal Maltose Binding Protein (MBP) – SIRV2gp26 fusion
protein. A portion of the MBP-SIRV2gp26 was treated with
Factor Xa protease to separate MBP and SIRV2gp26 and heated
to 65uC for 20 minutes to inactive the protease.
Two plasmids containing hairpin four-way junctions were
constructed to assay resolving enzyme activity. pUC(AT) is a
derivative of pUC19 containing an inverted repeat of twenty A
and T dinucleotides ((AT)20) between the EcoRI and PstI sites that
forms a hairpin four-way junction upon supercoiling [22,23]
(Figure S1A). Plasmid pEMM2 is derived from pNEB206A (NEB,
Ipswich, MA) and contains an insert corresponding to the
expected four-way junction formed between SIRV2 genome
dimers during genome replication (Figure S1B). pEMM2 was
constructed by annealing overlapping SIRV2 four-way junction
oligonucleotides in 16 Standard Taq Buffer (10 mM TrisHCl,
pH 8.3, 50 mM KCl, 1.5 mM MgCl2). This oligonucleotide
cassette with 39 overhangs was ligated to complementary ends
on pNEB206A vector linearized by XbaI and Nt. BbvCI (New
England Biolabs) to create pEMM2. The correct pEMM2
sequence was confirmed by DNA sequencing.
Synthetic oligonucleotide substrates were also used to charac-
terize resolving enzyme specificity and activity. Four-way Junction
3 (J3) was constructed by annealing strands (25 mM each) b, h, r,
and x in 16 Standard Taq Buffer. In addition, fluorescently
labeled J3 four-way junctions were prepared by annealing one 6-
carboxyfluorescein (FAM)-labeled strand and three unlabeled
strands. A SIRV2 four-way junction was constructed by annealing
strands (25 mM each) 1, 2, 3, and 4.
In addition to four-way junction DNA, Hjr activity was assayed
on alternate DNA structures, including single- and double-
stranded DNA, bulged DNA, hairpin, and three-strand Holli-
day-like junctions. Schematics representing the DNA structures
used in these experiments are depicted in Figure 1. Oligonucle-
otides for DNA substrates are listed in Table S2. Double-stranded
DNA was formed by annealing FAM-labeled top strand to an
unlabeled complement in 16 Standard Taq Buffer. A FAM-
labeled hairpin DNA mimicking the SIRV2 genome end was
formed by self-annealing. Heteroduplex bulged DNA (25 mM
stock) was constructed by annealing two oligonucleotides in 16
Standard Taq Buffer to create an unpaired central region flanked
by complementary base pairing. A three-strand Holliday-like
junction was prepared by annealing four-way junction J3 strands
(FAM)-b, h, r.
Assays for resolving enzyme activity
Four-wayjunctionresolutionwasmonitored bycleavage ofeither
four-way junction containing plasmids (pUC(AT) or pEMM2) or
fluorescently labeled synthetic four-way junctions. Typically in a
50 mL reaction, plasmids (11 nM) were incubated with 20 nM
resolving enzyme in 16 ThermoPol Buffer (20 mM Tris-HCl,
10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton X-
100, pH 8.8@ 25uC) at 55uC for one hour. Reaction products were
separated by agarose gel electrophoresis. Synthetic four-way
junctions were constructed as described above and one strand was
fluorescently labeled on its 59 end. In a 20 mL reaction, a synthetic
four-way junction (J3 or SIRV2 at 100 nM) was incubated with
Characterization of SIRV2 Hjr
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20 nM resolving enzyme in 16ThermoPol Buffer (unless otherwise
noted) at 55uC for one hour. FAM-labeled 16-mer and 18-mer were
size standards during electrophoresis (Table S2). Reaction products
were separated by 20% denaturing PAGE and quantified using the
fluorescence detected by a GE Typhoon scanner.
Requirements for SIRV2 Hjr cleavage
Hjr cleavage activity was assayed with synthetic DNA four-way
junction J3 under a variety of reaction conditions to determine
requirements for cleavage. Four-way junction J3 (FAM-labeled on
strand b) (100 nM) and MBP-Hjr (20 nM) were incubated in
reaction buffer at 55uC for 30 minutes. Reaction buffers varied in
pH (4–10), divalent cation (MgCl2, MnCl2, ZnSO4, CaCl2,
CoCl2), or concentrations of NaCl (0–500 mM), NH4SO4 (0–
200 mM), or MgCl2(0–100 mM). Sodium acetate (10 mM) was
used in the pH range of 4.0–6.0 and TrisHCl (10 mM) was used in
the pH range of 7.0–10. Reactions were halted by addition of 50%
formamide and 5 mM EDTA. Reaction products were separated
by 20% denaturing PAGE and fluorescence detected on a GE
Hjr substrate specificity
To determine Hjr substrate specificity, a panel of DNA
substrates including single- and double-stranded DNA, bulged
DNA, hairpin, and three-strand Holliday-like junctions were
prepared as described above. MBP-Hjr (20 nM) or T7 endonu-
clease I (7.5 nM) (New England Biolabs) was incubated with
0.10 mM DNA in 16ThermoPol Buffer at 55uC for 30 minutes.
Reaction products were separated by 20% denaturing PAGE and
quantified using the fluorescence detected by a GE Typhoon
Characterization of Hjr four-way junction cleavage
In a 0.10 mL reaction, pUC(AT) (25 nM) was incubated with
20 nM MBP-Hjr in 16 ThermoPol Buffer at 55uC. Linearized
products were gel purified by QiaPrep PCR purification kit
(Qiagen) and eluted in 0.10 mL of EB buffer. Linearized products
(50 mL) were treated with 400 U T4 DNA ligase in 16T4 DNA
ligase buffer for one hour at room temperature to seal hairpin
nicks. Lambda exonuclease treatment was carried out in a 30 mL
reaction by mixing 3 mL 106 lambda exonuclease buffer, 5 U
lambda exonuclease and 26 mL of linearized products, and
incubated at 37uC for one hour to degrade DNA with free 59
termini. Reaction products were separated by agarose electropho-
Protein extracts of SIRV2-infected S. islandicus were prepared
from 0.25–0.5 L cultures after concentration of the cells by
centrifugation, suspension of the cells in 25 mL of 150 mM NaCl,
20 mM TrisHCl, pH 7.5, and 1 mM EDTA and lysis by
sonication. Cell debris was removed by centrifugation and the
supernatant centrifuged a second time to further remove cell
debris. The resulting clarified cell-free lysate was used for further
studies. In a 0.5 mL reaction, 10 mg MBP-Hjr and incubated with
,1 mg SIRV2-infected S. islandicus extract at 4uC for 16 hours
with shaking in NEBuffer 3 (100 mM NaCl, 50 mM Tris-HCl,
10 mM MgCl2, 1 mM dithiothreitol, pH 7.9 @ 25uC). Anti-MBP
magnetic beads were added and affinity complexes were
magnetically separated and washed five times with 1.0 mL of
NEBuffer 3 to elute non-specifically bound protein. The remaining
specific protein complexes were eluted by boiling in 16 SDS-
PAGE loading buffer for 5 minutes and analyzed by SDS-PAGE.
Identification of interacting proteins by Mass
Proteins eluted from MBP-Hjr capture experiments were
digested into peptides with trypsin and run on an LC/MS-MS
for peptide analysis at the New England Biolabs Proteomic
Facility. Peptide masses were compared to a database of S.
islandicus strain Y.N.15.51 (accession: NC_012623 ) and
Figure 1. SIRV2 Hjr cleavage is specific to four-way junction DNA. Substrates (A: single-stranded DNA, B: double-stranded DNA, C:
heteroduplex double-stranded DNA, D: hairpin DNA, E: four-way junction J3, F: three-way Holliday-like junction) were constructed as described in
Materials & Methods and 59 labeled with fluorescence for detection (as indicated by a black circle). Distilled water (-), Hjr (S) (20 nM) or T7
endonuclease I (T7) (7.5 nM) was incubated with 100 nM substrate in 16ThermoPol Buffer at 55uC for 30 minutes. Reaction products were separated
by 20% denaturing PAGE and fluorescence detected by a GE Typhoon scanner.
Characterization of SIRV2 Hjr
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SIRV2 protein sequences using Spectrum Mill software (Agilent
Technologies). Peptides that did not exactly match the amino acid
sequences in the database, had alternatively charged states, or
were modified by phosphorylation or glycosylation were not
scored as positives. The sequence of host S. islandicus LAL 14/1 is
not publicly available, forcing the use of data from closely related
strains, so the list of identified proteins is expected to be an
underestimate of true positive interactors in the actual extract.
Data was filtered for quality with an MS/MS Score cut off of 15.
Proteins from S. islandicus or SIRV2 were identified by sequence
comparison using a BLAST  similarity score cut off of E,0.1.
Interacting proteins identified by mass spectrometry were
tabulated and grouped according to their presumed functional
Interaction between Hjr and SIRV2gp26 coat protein
To directly test the interaction between Hjr and SIRV2gp26
coat protein in vitro, anti-MBP::MBP-Hjr magnetic beads (hereaf-
ter called MBP-Hjr affinity beads) were prepared by mixing 10 mg
MBP-Hjr with 0.1 mg anti-MBP magnetic beads pre-equilibrated
in NEBuffer 3 (New England Biolabs). Bead complexes were
mixed thoroughly and incubated at 4uC with shaking for 1 hour.
A magnet was applied and supernatant was decanted and bead
complexes were washed 5 times with 16NEBuffer 3. These MBP-
Hjr affinity beads (10 mg) were incubated with approximately
10 mg SIRV2gp26 prepared as described earlier at 25uC for
2 hours with shaking in 0.5 mL 16 NEBuffer 3. Affinity
complexes were separated by a magnet and washed five times
with 1.0 mL of NEBuffer 3 to remove any non-specifically bound
protein. Protein complexes were eluted by boiling in 16 SDS-
PAGE loading buffer for 5 minutes and analyzed by SDS-PAGE.
Background binding was assessed using MBP affinity beads
prepared with unfused MBP (anti-MBP::MBP5).
SIRV2 Hjr cleaves four-way junction but not branched
Previously described viral resolving enzymes cleave a variety of
branched DNA structures formed during replication [11,25,26].
To test if Hjr substrate requirements parallel those of other viral
resolving enzymes, Hjr and bacteriophage T7 endonuclease I
activities on the panel of DNA substrates depicted in Figure 1 were
compared. In contrast to T7 endonuclease I, Hjr only cleaves four-
way junction DNA and is inactive on single- or double-stranded
DNA, hairpin DNA, heteroduplex loops (bulges), and three-way
Holliday-like junctions (Figure 1). S. solfataricus Hje shows a
similarly narrow substrate range and only cleaves four-way
junction DNA structures .
Hjr activity on four-way junction DNA
To identify DNA structural elements and sequences required for
Hjr cleavage, activity on a variety of four-way junction DNAs was
examined. First, Hjr was tested on plasmid substrates containing
junctions that resemble hairpin four-way junction substrates
formed during SIRV2 replication in vivo. Plasmid pUC(AT)
contains an AT-rich hairpin four-way junction and has been used
as a substrate to study T7 endonuclease I and fowlpox resolving
enzyme (Figure S1A) [23,27]. Plasmid pEMM2 includes a hairpin
four-way junction that mimics the sequence formed at the SIRV2
concatamer junctions during replication (Figure S1B). Hjr cleaves
the pUC(AT) four-way junction to convert closed circular
pUC(AT) to a linear form (Figure 2A). Hjr also cleaves the four-
way junction structure in plasmid pEMM2 to a linear form (data
not shown). The Hjr-cleavage site was mapped by restriction
fragment length analysis using XmnI and HindIII and shown to be
specific for the DNA four-way junction (Figure S2). Relaxed
nicked and linear pUC(AT) and pEMM2 are not substrates,
presumably because the four-way junctions do not form in the
absence of supercoiling (data not shown). In addition, plasmids
lacking four-way junctions are also not substrates (data not shown).
Hjr cleavage was then tested on a synthetic four-way junction
designed to mimic possible structures formed during SIRV2
replication. The SIRV2 cruciform sequence allows four-way
junction motion along the duplex and as the four-way junction
migrates along the substrate, the crossover points may vary. The
Hjr dimer recognizes four-way junction structures and makes a
nick on pairs of four-way junction strands . Hjr nicks strands 1
and 3 (Lanes 1, 3) at three paired sites and makes two paired nicks
on the strands 2 and 4 of SIRV2 four-way junction DNA (Lanes 2,
4) (Figure 2B, C). The observed multiple cleavage sites could
reflect different four-way junction configurations due to migration,
each cleaved at a fixed distance from a crossover point but at
different positions relative to the end or alternatively multiple
cleavage sites at a single crossover point.
After initial characterization of Hjr activity with the mobile
SIRV2 four-way junction, an alternate well-characterized fixed
four-way junction DNA substrate (Junction 3 (J3)) was used for
further detailed characterization. By utilizing the well-defined
junction J3 as a substrate for Hjr cleavage, comparisons can be
made to other resolving enzyme cleavage patterns described in the
literature that use the same J3 substrate [9,28,29]. J3 is composed
of four hybridized DNA strands: two DNA strands are
continuously stacked and the other two strands base pair with
one continuous strand then switch to base pair with the other
continuous strand on the adjacent helix (Figure 3A) . The pair
of DNA strands that maintain base stacking through the junction
are referred to as the continuous strands (strands h and x) while
strands that exchange between helices are the exchange strands
(strands b and r) (Figure 3A), and bases at the junction are stacked
with different strands on the two faces . Local sequence
properties change the probability of adopting the stacking switch.
The four-way junction J3 favors an isoform conformation with
strands h and x as continuous strands and strands b and r as
exchange strands .
Four-way junction J3 was fluorescently labeled on either the b,
h, r, or x strand. Both Sso and Sis Hje cleave preferentially on the
continuous strands (h, x) of the J3 four-way junction (Figure 3B). In
contrast, Hjr preferentially cleaves the exchange strands (b, r) of
this same four-way junction (Figure 3B).
Requirements for SIRV2 Hjr four-way junction cleavage
The reaction conditions for Hjr cleavage were investigated in
detail using the synthetic J3 four-way junction. Despite the acidic
growth environment, the internal pH of Sulfolobus islandicus is
neutral. The pH optimum for Hjr activity reflects its native
cytosolic environment with optimal activity between pH 7 to 9,
partial activity at pH 6 and 10 (80% or 70% activity, respectively)
and no activity below pH 5 (Figure S3A). Consistent with previous
studies, a divalent cation (MgCl2 or MnCl2) is required for
resolving enzyme activity (Figure S3C) . Hjr activity is optimal
between 0.5 and 20 mM MgCl2(Figure S3B). Hjr is minimally
active with CaCl2and not active with cofactors ZnSO4or CoCl2
(Figure S3C). NaCl or (NH4)2SO4are not required for Hjr activity
and do not stimulate cleavage (Figure S3D, E). However, Hjr
activity is inhibited by higher concentrations of NaCl (.250 mM)
and (NH4)2SO4(.10 mM) (Figure S3D, E).
Characterization of SIRV2 Hjr
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SIRV2 Hjr four-way hairpin junction cleavage results in
linear DNA with hairpin termini
The last stage of SIRV2 replication is envisioned as a
concatamer joined by a hairpin four-way junction. Hjr is proposed
to introduce symmetrical nicks across this junction to resolve the
concatamers, producing single molecules with linear hairpin ends
. To test this model, a plasmid-encoded hairpin four-way
junction was cut with SIRV2 and the nature of the ends was
determined as schematically depicted in Figure 4A. First, the four-
way junction containing plasmid, pUC(AT), was cleaved with Hjr
to generate a linear fragment and then incubated with buffer alone
or with T4 DNA ligase (Figure 4A). Lambda exonuclease was
added to digest DNA with free 59 ends. As predicted, linear
fragments are sensitive to lambda exonuclease digestion in the
absence of ligation due to free 59 ends (Figure 4B). However, linear
fragments treated with T4 DNA ligase are resistant to lambda
exonuclease cleavage suggesting that ends are nicked hairpins that
can be ligated to form contiguous covalently closed linear DNA.
Therefore, these data support a model in which Hjr cleaves
genome concatamers at hairpin four-way junctions to produce
single genome copies having nicked hairpin ends that can then be
sealed by DNA ligase to form covalently closed molecules.
SIRV2 Hjr interacts with host DNA binding proteins and
SIRV2 coat protein (gp26)
To investigate what protein partners might function with Hjr,
immunoprecipitation was used. MBP-Hjr was incubated with a
cell-free extract of SIRV2-infected S. islandicus, containing protein,
lipid, and nucleic acids. Components bound to the resolving
enzyme were captured by magnetic anti-MBP beads and washed
to reduce non-specific binding. Treatment of the washed beads
with Factor X protease released Hjr and bound proteins. Several
proteins in the 10–20 kD range and the 40–50 kD range were
observed by SDS-PAGE (data not shown).
Proteins captured by MBP-Hjr immunoprecipitation were
identified by mass spectrometry and are listed according to their
presumed functional role (Table S3). MBP-Hjr forms complexes
with proteins including the DNA binding proteins, Sso10b, Sso7d,
Cren7 and Alba (Table S3). Interactions between MBP-Hjr and
these DNA binding proteins could be a result of direct
protein:protein interactions or may result from binding between
MBP-Hjr and a nucleic acid that is in turn bound by a DNA
binding protein. Most strikingly, an interaction between Hjr and
the coat protein, SIRV2gp26 was identified with a high confidence
value (Table 1). Further studies using purified proteins were
implemented to verify this interaction with the SIRV2 coat
SIRV2 Hjr and SIRV2 coat protein (gp26) interact in vitro
The putative direct interaction between purified MBP-Hjr and
SIRV2gp26 was tested in vitro. As detected by SDS-PAGE,
magnetic anti-MBP::MBP-Hjr beads pulled down SIRV2gp26
(Figure 5). Control reactions with MBP5 alone did not pull down
SIRV2gp26 suggesting that the interaction is specific for Hjr
rather than for the MBP domain of the fusion protein.
To address the molecular basis for substrate recognition and
cleavage, we characterized the resolving enzyme from the
Figure 2. SIRV2 Hjr cleaves four-way DNA junctions. (A) Cleavage of plasmid pUC(AT) by Hjr was monitored over time by agarose gel
electrophoresis. The mobility of nicked pUC(AT) was established by treating the plasmid with the nicking enzyme Nt. BstNBI (Lane N), and that of the
linear form by digestion with HindIII (Lane L). The NEB 1 kb DNA ladder (M) was used as a reference. (B) A four-way junction sequence and structure is
shown with uppercase nucleotides correspond to native SIRV2 sequence. Strands are designated 1–4. SIRV2 Hjr cleavage sites are noted by triangles.
(C) A four-way junction DNA substrate corresponding to the SIRV2 concatamer junction sequence (shown in B) was constructed by annealing four
oligonucleotides, three unlabeled and one FAM-labeled; differently-labeled substrates are designated 1, 2, 3, or 4. MBP-Hjr was incubated with these
(Lanes 1–4 respectively) at 55uC for 1 hour in 16ThermoPol Buffer. Reaction products were separated by denaturing 20% PAGE and quantified using
a phosphoimager. Fragment sizes are indicated.
Characterization of SIRV2 Hjr
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Figure 3. S. solfataricus and S. islandicus Hje and SIRV2 Hjr cleaves of four-way junction J3 with opposite polarity. (A) Resolving enzyme
cleavage sites are shown on a schematic representation of four-way junction J3 with continuous strands colored blue (x) and black (h) and exchange
strands colored red (b) and green (r). Sso Hje and Sis Hje cleave four-way junction J3 junction two base pairs 39 from the junction in a symmetrical
fashion on the h and x continuous strands while Hjr cleaves two base pairs 39 from the junction in a symmetrical fashion on the b and r exchange
strands. (B) Four-way junction J3 DNA (100 nM) 59 FAM-labeled either on the h, b, r, x strand was incubated with 20 nM S. solfataricus (Sso Hje), S.
islandicus Hje (Sis Hje), or Hjr in 16ThermoPol Buffer at 55uC for 60 minutes. Reaction products were separated by denaturing 20% PAGE. S and P
represent substrate (34-mer) and product (18-mer) bands, respectively.
Figure 4. SIRV2 Hjr cleaves hairpin four-way junctions to produce linear fragments with nicked hairpin ends. (A) Hjr was used to cleave
the hairpin four-way junction containing plasmid, pUC(AT), to generate linear fragments with nicked hairpin ends. The linear fragments were then
incubated with either buffer alone or T4 DNA ligase to seal nicks. Lambda exonuclease (gray shape) was then added to degrade DNA having free 59
ends (dotted lines). (B) Hjr cleaved pUC(AT) products were incubated with buffer alone (-) (Lane 1), with lambda exonuclease (Lane 2), with T4 DNA
ligase (Lane 3) or with T4 DNA ligase then lambda exonuclease (Lane 4) and separated by agarose gel electrophoresis.
Characterization of SIRV2 Hjr
PLoS ONE | www.plosone.org6 August 2011 | Volume 6 | Issue 8 | e23668
hyperthermophilic virus, SIRV2. Hjr cleaves four-way junctions
without cleaving other related DNA structures and contrasts with
the bacteriophage resolving enzymes T4 endonuclease VII and T7
endonuclease I that recognize a wide range of DNA structures
including four-way junctions, Y-structures, heteroduplex loops,
single-strand overhangs, nicks, gaps, apyrimidinic sites, and base
mismatches [32,33,34]. T4 endonuclease VII and T7 endonucle-
ase I have large electropositive areas on the protein surface that
bind to the four-way junction DNA backbone over a large area
and are flexible and broad enough to allow binding and cleavage a
variety of branched DNA substrates [35,36]. Unlike bacteriophage
resolving enzymes, Sso Hje, Sso Hjc, and SIRV2 Hjr substrate
specificity is narrow and limited to X-shaped four-way junctions.
The Sso Hje three-dimensional structure provides a model to
explore elements that determine the narrow substrate range of Sso
Hje and SIRV2 Hjr. Electropositive patches are arranged on the
Sso Hje and Sso Hjc DNA binding surface in an X-shaped pattern
[4,18]. Presumably, this pattern of electropositive residues favors
binding of X-stacked four-way junctions by Sso Hje and Hjc (and
by extension, SIRV2 Hjr). Further structural studies of SIRV2 Hjr
in complex with DNA will further reveal if positively charged
surface amino acids are arranged in a pattern to bind X-stacked
four-way junction substrates to confer substrate specificity.
SIRV2 Hjr has a unique strand cleavage preference that may
reflect fundamental differences in Holliday junction recognition
and cleavage. This preference is observed even while sharing
sequence similarity with Sso Hje and Sso Hjc. Sso Hje is specific
for cleavage of continuous strands in four-way junction J3 while
Sso Hjc cleaves both continuous and exchange strands. SIRV2
Hjr presents a third cleavage pattern by nicking exchange strand
pairs. Previous studies of Sso Hje and Hjc have suggested a
structural basis to account for differences in resolving enzyme
specificity and cleavage patterns (1). Sso Hje and Sso Hjc are both
homodimers stabilized along the dimer interface by interactions
between monomer amino acids (Figure 6A). Even thought the
dimer interface is distal to the DNA binding region, Middleton
and coworkers have argued that positioning of the dimers
influences positioning of adjacent catalytic residues on the DNA.
When Sso Hje and Sso Hjc three-dimensional structures are
superimposed, the main chain Ca positioning is structurally well
conserved with the largest differences observed at the dimer
interface (Figure 6A). Specifically, an insertion of three large
hydrophobic residues (M77, F78, M80) in a loop between helix a2
Table 1. MS/MS data for SIRV2gp26 coat protein captured by SIRV2 Hjr immunoprecipitation.
% AA coveraged
SIRV2gp26 coat protein
aA single MS spectral scan of a defined mass range may contain many distinct peaks, each with a characteristic mass/charge ratio – m/z. MS/MS spectra are the total number of peptide spectra generated by MS/MS from a single
distinct peak observed in a MS spectrum which match the predicted in silico fragments from a translated database of the SIRV2 genome (accession number: NC_004086 ).
bTotal number of unique spectra detected and assigned to one peptide.
cSpectra were scored by Spectrum Mill and assigned a quality score based on peak match and quality. Matching spectra were associated with a peptide sequence. A total MS/MS score was generated by summing only the highest
scoring MS/MS spectrum for each peptide.
dPercent of identified peptide amino acids matching total protein amino acids sequence.
eProtein accession number from translated SIRV2 genome; accession number: NC_004086 ).
fProteins were annotated by BLAST similarity (e,0.1) .
gSIRV2gp26 peptide identified by mass spectrometry.
hSIRV2gp26 amino acid sequence highlighting putative trypsin digestion sites (underlined) and peptide identified by mass spectrometry (bold).
Figure 5. SIRV2 Hjr interacts with SIRV2gp26 coat protein in
vitro. The interaction of MBP-Hjr and SIRV2gp26 coat protein was
confirmed by immunoprecipitation. Protein complexes were eluted and
analyzed by SDS-PAGE stained with Coomasie Blue dye: Lane 1:
SIRV2gp26. Lane 2: MBP-Hjr. Lane 3: anti-MBP beads:MBP-Hjr. Lane 4:
anti-MBP beads. Lane 5: anti-MBP beads:MBP5+SIRV2gp26. Lane 6: anti-
Characterization of SIRV2 Hjr
PLoS ONE | www.plosone.org7August 2011 | Volume 6 | Issue 8 | e23668
and beta strand bE of each Sso Hje monomer shifts the dimer
conformation and may position catalytic residues for cleavage of
continuous strands of a four-way junction. In contrast to Sso Hje, a
relatively short Sso Hjc loop connecting helix a2 and beta strand
bE may confer additional flexibility to allow cleavage of both
exchange and continuous strands in a four-way junction. SIRV2
Hjr, like Sso Hje, also contains an insertion in the equivalent loop
although the sequences diverge. Most notably, SIRV2 Hjr
contains three cysteines (C77, C82, C84) in the insertion loop.
These cysteines may form intra- or inter-monomer disulfide bonds
to alter resolving enzyme conformation and thereby position
adjacent catalytic residues to favor cleavage of exchange rather
than continuous strands (Figure 6B). Therefore, variations in
resolving enzyme dimer interfaces may uniquely position catalytic
residues for pairwise nicking on either the continuous (Sso Hje),
exchange (SIRV2 Hjr), or both (Sso Hjc).
The SIRV2 Hjr substrate cleavage specificity has the charac-
teristics to function as a replication resolving enzyme in a poxvirus-
like mode of viral DNA replication. In such a mechanism, linked
genome concatamers are resolved via cleavage during replication
. SIRV2 Hjr action on such hairpin four-way junction
concatamer DNA would produce linear DNA products with
nicked hairpin termini, which in turn could serve as nicked
templates for further replication. Therefore, the SIRV2 resolving
enzyme likely plays a central role in the final stages of SIRV2
replication and functions in the general mechanism for separating
concatamers during replication of linear viral genomes.
Finally, we have shown that in addition to cleaving four-way
junctions, Hjr may have a role in viral assembly. It is tempting to
signal for assembly of the coat protein into a superfilament that
surrounds the resolved SIRV2 genome, thereby forming the virion
body. Known signals for viral packaging include DNA sequences,
proteins to initiate assembly [37,38,39,40,41,42,43,44,45,46,47,48,49].
For instance, vaccinia virus nucleates virion assembly via a transient
interaction between a hairpin terminus-binding protein, I6, and the
vaccinia virus coat protein [48,49]. Therefore, like vaccinia virus I6
resolved, single genome copies rather than concatamers are packaged
into virus particles. In addition, because both Hjr and coat protein
sequences are highly conserved among rudiviruses, this nucleation
model could also serve as a general strategy for virion packaging in
related rudiviruses, Acidianus Rod-Shaped virus 1 (ARV1), Stygioglobus
rod-shaped virus (SRV), and Sulfolobus islandicus rod-shaped virus 1
(SIRV1). Given the similarity between archaea and eukarya, it is also
conceivable that similar encapsidation mechanisms may also exist in
quences. (A) A poly(AT)20cassette in pUC(AT) can form a four-
way junction structure. (B) pEMM2 contains a four-way junction
sequence at the junction of SIRV2 concatamers. These figures
represents one of many conformations of possible mobile four-way
junction structures. The four-way junction center may shift from
what is represented. Gray regions are vector sequence.
Four-way junction DNA structures and se-
pEMM2. Plasmid pEMM2 contains a four-way junction
sequence mimicking the four-way junction formed at SIRV2
concatamer junctions. pEMM2 (100 nM) was incubated with T7
endonuclease I (7.5 nM) (Lane 2), MBP-Hjr (20 nM) (Lane 3), or
MBP-Hjr (2 nM) (Lane 4) at 37uC (T7 endonuclease I) or 55uC
(MBP-Hjr) for 30 minutes. Then reaction products were then
digested with 10 Units of XmnI at 37uC for one hour and
separated by agarose gel electrophoresis. As a control (Lane 1),
1 mg pEMM2 was digested with 10 Units of XmnI and HindIII
(proximal to the four-way junction) to generate a 921 bp and
1851 bp fragment. pEMM2 was digested with XmnI alone (Lane
5), XmnI and HindIII (proximal to the four-way junction).
Plasmid not cleaved by T7 endonuclease I or SIRV2 Hjr and then
cleaved with XmnI will generate a linear 2772 bp band. As
expected, a double stranded break generated from T7 endonu-
clease I and MBPNSIRV2 Hjr cleavage mapped to the cruciform
region of pEMM2 to generate a ,875 bp and 1897 bp fragment
(Lanes 2, 3, 4). (B) A plasmid map of pEMM2 illustrating HindIII,
XmnI and cruciform cleavage site.
Mapping resolvase cleavage site on plasmid
tion cleavage. Hjr cleavage activity was assayed with DNA four-
way junction J3 under a variety of reaction conditions to
determine requirements for cleavage. Four-way junction J3
(FAM-labeled on strand b) (100 nM) and Hjr (20 nM) were
incubated in reaction buffer at 55uC for 30 minutes. Reaction
buffers varied pH (4–10), divalent cation (MgCl2, MnCl2, ZnSO4,
CaCl2, CoCl2), concentration of NaCl (0–500 mM), NH4SO4(0–
200 mM), or MgCl2(0–100 mM). Reactions were quenched by
addition of 50% formamide and 5 mM EDTA. Reaction products
were separated by 20% denaturing PAGE and fluorescence
quantified using a GE Typhoon scanner. S and P represent
substrate (34-mer) and product (18-mer) bands, respectively.
Requirements for SIRV2 Hjr four-way junc-
Figure 6. Structural variation at the Sso Hje and Hjc dimer
interface modulates cleavage specificity. (A) Sso Hje (accession:
1OB8) and Sso Hjc (accession: 1HH1) three-dimensional structures were
aligned using MacPymol. Sso Hje monomers (red and blue) are
visualized by a surface view to highlight the dimer arrangement. A
domain formed by helix alpha-2 and beta strand beta-E form part of the
dimer interface (arrow). In insertion in the loop between helix alpha-2
and beta strand beta-E (arrow) (Sso Hje: red and blue; Sso Hjc: green)
shifts dimer conformation and may confer strand cleavage specificity.
(B). The amino acid sequence of the Sso Hje, Sso Hjc, and SIRV2 Hjr
dimerization interface helix alpha-2 and beta strand beta-E were aligned
by Clustal W and the insertion loop is underlined. Amino acids
conserved between Sso Hje, Sso Hjc, and SIRV2 Hjr are shaded in black
and those conserved between Sso Hje and SIRV2 Hjrare shaded in gray.
Characterization of SIRV2 Hjr
PLoS ONE | www.plosone.org8 August 2011 | Volume 6 | Issue 8 | e23668
islandicus Hje gene synthesis.
Oligonucleotides for SIRV2gp26, SIRV2 Hjr, and S.
Oligonucleotides for DNA substrates.
Proteins that interact with MBP-SIRV2 Hjr.
We are grateful to Jack Benner and Casey Madinger at the New England
Biolabs for providing mass spectrometry analysis. We also thank Malcolm
White at University of St. Andrews for providing Sulfolobus islandicus Hje
and for helpful discussions; David Prangishvili for helpful discussions; Tom
Evans, Fran Perler, and Lise Raleigh for critical review of this manuscript.
We are also indebted to Don Comb for fostering a supportive research
environment at New England Biolabs.
Conceived and designed the experiments: AFG CG WEJ. Performed the
experiments: AFG. Analyzed the data: AFG CG WEJ. Contributed
reagents/materials/analysis tools: AFG CG. Wrote the paper: AFG CG
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