Nucleic Acids Research, 2007, Vol. 35, No. 18Published online 13 September 2007
Catalytic domain of restriction endonuclease BmrI as
a cleavage module for engineering endonucleases
with novel substrate specificities
Siu-hong Chan, Yongming Bao, Ewa Ciszak, Sophie Laget and Shuang-yong Xu*
New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA
Received December 14, 2006; Revised and Accepted August 12, 2007
Creating endonucleases with novel sequence spec-
ificities provides more possibilities to manipulate
DNA. We have created a chimeric endonuclease
(CH-endonuclease) consisting of the DNA cleavage
domainof BmrI restriction
C.BclI, a controller protein of the BclI restriction-
modification system. The purified chimeric endonu-
clease, BmrI198-C.BclI, cleaves DNA at specific
sites in the vicinity of the recognition sequence of
C.BclI. Double-strand (ds) breaks were observed at
two sites: 8bp upstream and 18bp within the C-box
sequence. Using DNA substrates with deletions of
C-box sequence, we show that the chimeric endo-
nuclease requires the 50half of the C box only for
specific cleavage. A schematic model is proposed
for the mode of protein–DNA binding and DNA
cleavage. The present study demonstrates that
the BmrI cleavage domain can be used to create
combinatorial endonucleases that cleave DNA at
specific sequences dictated by the DNA-binding
partner. The resulting endonucleases will be useful
in vitro and in vivo to create ds breaks at specific
sites and generate deletions.
Restriction endonucleases (REases), particularly of Type
IIP that recognize palindrome sequences and cleave within
them, are indispensable tools for DNA manipulation
because of their high sequence and cleavage specificity.
Substrate specificity, which is always coupled to the
catalytic core for Type IIP REases, is known to rely on
intricate interactions between the amino acid residues
of the REase and the bases and the backbone phosphates
of the substrate DNA. However, similar DNA-binding
specificity is rarely reflected in amino acid sequence
homology among REases. BamHI, for example, recog-
nizes G#GATCC and cuts between the first two Gs,
whereas KpnI, a REase isolated from an evolutionary
unrelated bacterium that shares very low sequence
similarity with BamHI, recognizes GGTAC#C and cuts
between the last two Cs. This leaves protein engineers no
obvious means to identify patterns or recognition modules
within the amino acid sequences of Type IIP REases that
recognize similar DNA sequences. Engineering Type IIP
REases has to resort to genetic screening systems
specifically designed for each of the specificities of interest
or sophisticated computational design based on the
atomic structure of the enzymes concerned. Variants of
EcoRV that prefer cleavage sites flanked by AT or GC
have been identified by random mutagenesis within
specific regions of the EcoRV REase (1). Partial successes
have been reported for engineering Type IIP REases that
recognizes degenerate sequences. BstYI (R#GATCY) has
been engineered to cleave AGATCT (2) and BsoBI
(C#YCGRG) to cleave CCCGGG preferentially (3).
Recently, alternative specificity (GC#TGCCGC) has
been introduced to NotI (GC#GGCCGC) through
Computational redesign of homing endonucleases based
on their crystal structures had resulted in a variant of
I-MsoI with altered sequence specificity (5) and a fusion
protein created by swapping domains of I-DmoI and
I-CreI and optimization of domain interface (6). Tailored-
made specificities had been achieved through genetic
screening of I-CreI at specific amino acid residues
that make direct or indirect contact with substrate
Certain types of REases, such as Types I, IIS, IIG and
III endonucleases, cleave substrate DNA outside their
recognition sequences. Biochemical and structural studies
had shown that these endonucleases consist of separate
Yongming Bao, Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, P. R. China.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
*To whom correspondence should be addressed. Tel: þ1 978 380 7287; Fax: þ1 978 921 1350; Email: email@example.com
? 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
DNA recognition and cleavage domains. It has been
suggested that these endonucleases evolved through
recombination of their cleavage domains with DNA-
binding domains of different sequence specificity (14,15).
This uncoupling of substrate specificity and cleavage
activity opens the door to creating combinatorial endo-
nuclease of novel substrate specificity through fusing
DNA cleavage domains to DNA recognition proteins or
FokI is a Type IIS REase that cuts 9 and 13bp
downstream of the recognition sequence of GGATC on
the top and bottom strand, respectively (GGATC
N9/N13). Combinatorial endonucleases have been created
by fusing the DNA cleavage domain of FokI to a few
DNA-binding domains. Fusing the FokI cleavage domain
to the Drosophila Ultrabithorax (Ubx) homeodomain
resulted in a chimeric endonuclease that binds and cuts
outside the Ubx-binding site (17). Chimeric endonucleases
were also created by fusing the FokI cleavage domain to
the Z-conformation-specific Za domain of human RNA
adenosine deaminase (18), Gal4 (19) and natural zinc-
finger motifs (20–25), generating novel substrate specifi-
cities in vitro. Directed-evolution of sequence specificity
of Zn fingers has brought us closer toward engineering
substrate specificity of endonucleases (26,27). Zn finger/
FokI chimeric endonucleases (Zinc finger nucleases,
ZFNs) have been shown to generate deletions in target
sequences in the germline of Drosophila in vivo (28–30).
ZFNs injected into nuclei of Xenopus oocytes induced
efficient insertion of an extra-chromosomal DNA through
homologous recombination (28–31).
With increasing number of sequenced and characterized
Type IIS REases, we set out to find other cleavage
domains that can be used in this combinatorial approach
to creating endonucleases with novel substrate specificity.
BmrI is a Type IIS REase that recognizes the asymmetric
6-bp sequence ACTGGG and cleaves 5 and 4bp down-
stream on the top strand and bottom strand, respectively
(ACTGGG N5/N4). It is highly homologous to its
isoschizomer BfiI in amino acid sequence. BmrI and BfiI
are unconventional REases in that they do not require
divalent metal ions for DNA cleavage (32). They consist
of a DNA-binding domain and a non-specific cleavage
domain consisting of a HKD catalytic motif of the
phospholipase D family (33). The two domains are
joined by a relatively flexible linker sequence (15,34),
which is believed to allow domain movements that are
needed in the transition from recognition to DNA
cleavage activity (35,36). Structural and biochemical
studies suggest that BfiI recognizes its target sequence
with a single DNA recognition domain and makes ds
breaks sequentially (37). The modular structure and
sequential cleavage of ds DNA suggest that the DNA
cleavage domains of BfiI and BmrI can be connected to
other DNA-binding domains to generate novel substrate
C.BclI is the controller protein of the BclI R-M system.
C.BclI binds to a 12-bp inverted repeats upstream of its
own open reading frame (ORF) with a dissociation
constant in the nanomolar range in vitro (38). It represses
the expression of the MTases of the BclI R-M system
in vivo (38). C.BclI contains a helix–turn–helix (HTH)
domain for binding to its target DNA sequence as
predicted by homology modeling of the crystal structure
of C.BclI to that of Cro repressor of phage 434 (38).
The 12-bp inverted repeats in the C-box suggests that
C.BclI binds to its target sequence as homodimer or
Here we make use of the modular property of BmrI
endonuclease and the DNA-binding specificity of C.BclI
to generate a combinatorial endonuclease of novel
sequence specificity. A chimeric endonuclease was con-
structed by linking the cleavage domain of BmrI to C.BclI
through a 14-amino acid linker. The chimeric endonu-
clease requires the 50half of the C box only for making
specific double-strand (ds) breaks 13–16bp downstream of
the binding site.
MATERIALS AND METHODS
The cloning and expression of BmrI R-M system are
described in Higgins et al. (manuscript submitted). The
DNA fragments that encode residues 1–198, 1–204, 1–209
of BmrI were amplified from the cloned BmrI system by
PCR and ligated to pET21a (Novagen). The gene of the
controller protein of the BclI R-M system (bclIC) has been
cloned, and the C.BclI protein has been expressed and
purified previously (38). The chimeric endonucleases of
BmrI-C.BclI were constructed such that two modules were
connected by a linker of 14 amino acid residues. Codons
of a 6? His tag were added to the 30end of the ORF of the
fusion proteins to facilitate purification. The coding
sequence of the chimeric endonuclease was ligated to
pET21a under the control of T7 promoter. All constructs
were sequenced to confirm the absence of mutations.
For small-scale expression, Escherichia coli strain T7
Express (NEB) was transformed separately by each of the
constructs of pET21a carrying fusions between C.BclI and
each of the BmrI truncation variants. The transformed
cells were cultured in 100ml of LB with 0.1mg/ml of
ampicillin at 378C at 200r.p.m. until OD600reached 0.9.
Fusion protein production was induced by adding IPTG
to 0.5mM final concentration and cultured at 16–208C for
3h. Ten milliliters of the culture were harvested and cells
were lyzed by sonication. Five microliters of the soluble
fraction of the lysate were used in cleavage activity assay
described below. For large-scale expression and purifica-
tion, E. coli strain T7 Express was transformed by the
The transformed cells were cultured in 1l of LB with
0.1mg/ml of ampicillin at 378C at 200 r.p.m. until OD600
reached 0.9. Expression was induced by adding IPTG to
0.5mM and cultured for 3h under the same condition.
The culture was harvested and stored at ?208C until lysed
for protein purification.
Purification and refolding
The cell pellet derived from 1l of IPTG-induced culture
(?6g) was re-suspended in 60ml of lysis buffer (50mM
NaH2PO4, 300mM NaCl, pH 8.0, 1mg/ml lysozyme).
Nucleic Acids Research, 2007, Vol. 35,No. 18 6239
The lysate was kept on ice for 30min and then centrifuged
at 15000g for 20min at 48C. The fusion protein was
mainly found in the insoluble fraction (inclusion bodies).
The inclusion bodies were resuspended in 30ml of washing
buffer (2M urea, 100mM NaH2PO4, 10mM Tris–HCl,
pH 8.0) and kept on ice for 30min After centrifugation
at 15000g for 20min at 48C, the inclusion bodies were
re-suspended in 30ml of denaturing buffer (8M urea,
100mM NaH2PO4, 10mM Tris–HCl, pH 8.0) and kept on
ice for 30min. Thedenatured
was centrifuged at 15000g for 20min at 48C. The
supernatant was loaded onto four 1ml Ni-NTA columns
(Qiagen). After washing with wash buffer (8M urea,
100mM NaH2PO4, 10mM Tris–HCl, pH 6.3), the bound
protein was eluted using elution buffer (8M urea, 100mM
NaH2PO4, 10mM Tris–HCl, pH 4.5). A successful
Protein Refolding Kit (US Biological). The eluted protein
(?12ml) was then dialyzed in 500ml of refolding
buffer (50mM Tris–HCl, pH 8.5, 10mM NaCl, 0.4mM
KCl, 2mM MgCl2, 2mM CaCl2, 0.4M sucrose, 0.5%
Triton X-100, 0.05% PEG 3350, 1mM GSH, 0.1mM
GSSH) at 48C overnight. The refolded protein was
dialyzed against a storage buffer (10mM Tris–HCl,
250mM NaCl, 1mM DTT, 0.1mM EDTA, 0.5mg/ml
BSA, pH 7.4) and stored at 48C.
DNA cleavage reactions
Litmus28-bclIC (38) that contains the wild-type C box and
C.BclI ORF sequences was used as the substrate in DNA
cleavage assays. Litmus28 (NEB), from which Litmus28-
bclIC is derived, was used as a negative control for specific
cleavage. 0.4mg (12 pmol) of purified BmrI198-C.BclI
fusion protein was generally used on 125mg (67 fmol) of
substrate DNA in designated buffers and temperatures in
20ml reaction mixtures. Substrate DNA was either pre-
linearized with DraIII or DraIII was added to the cleavage
reactions along with the BmrI198-C.BclI fusion protein as
indicated. Cleavage products were analyzed by electro-
phoresis through 1% agarose gels in 1? TBE buffer.
The intensities of the DNA bands in ethidium bromide-
containing agarose gels were quantified by QuantityOne
software (BioRad). Reaction buffers used included: Buffer
1 (10mM Bis Tris Propane–HCl, 10mM MgCl2, 1mM
DTT, pH 7.0), Buffer 2 (10mM Tris–HCl, 50mM NaCl,
10mM MgCl2, 1mM DTT, pH 7.9), Buffer 3 (50mM
Tris–HCl, 100mM NaCl, 10mM MgCl2, 1mM DTT, pH
7.9), Buffer 4 (20mM Tris–acetate, 10mM magnesium
acetate, 50mM potassium acetate, 1mM DTT, pH 7.9),
EDTA buffer (50mM Tris–HCl, 100mM NaCl, 10mM
EDTA, 1mM DTT, pH 7.9), high salt buffer (10mM
Tris–HCl, 150mM NaCl, 10mM MgCl2, 1mM DTT, pH
7.9), EcoRI buffer (100mM Tris–HCl, 50mM NaCl,
10mM MgCl2, 0.025% Triton X-100, pH 7.5), Mgþþ
buffer (50mM Tris–HCl, 100mM NaCl, 10mM MgCl2,
1mM DTT, pH 7.9), Caþþbuffer (50mM Tris–HCl,
100mM NaCl, 10mM CaCl2, 1mM DTT, pH 7.9) and
Znþþbuffer (50mM Tris–HCl, 100mM NaCl, 2mM
ZnSO4, 1mM DTT, pH 7.9).
To map the cleavage sites, Litmus28-bclIC was digested
with the indicated REases and 125mg of the linear DNA
was then incubated with 0.4mg of purified BmrI198-C.BclI
fusion protein in Buffer 3at 378C for 1h in 20ml reaction
Cleavage sites of BmrI198-C.BclI were determined by
incubating Litmus28-bclIC with DraIII and BmrI198-
C.BclI in Buffer 3at 378C for 1h. The cleaved fragments
(?2.1 and ?1.2kb) were gel-purified and sequenced.
The presence of an extra A and a reduction of peak
intensity of the proceeding peaks in the electropherograms
are indicative of cleavage sites.
The thermal stability of cleavage activity was tested
by incubating BmrI198-C.BclI in Buffer3at the designated
temperatures for 20min in the absence of substrate DNA.
After returning to room temperature, substrate DNA was
added and the reaction mixtures were incubated at 378C
for 1h. To determine the optimal reaction temperature,
BmrI198-C.BclI was pre-heated to the designated tempera-
ture for 2min in Buffer 3 before adding substrate DNA.
The reaction mixtures were then incubated at 378C for 1h.
To determine the mode of binding to the C box,
the EcoRI/AccI fragment (50half of C box plus 32bp
upstream sequence) and the AccI/EcoNI fragment (30half
of C box plus 262bp downstream sequence) were deleted
from Litmus28-bclIC. The truncation plasmid variants
Litmus28-bclIC?50and Litmus28-bclIC?30were used
as substrate for cleavage reaction and run-off sequencing
as described previously.
Construction and expression of BmrI198-C.BclI
CH-endonucleases consisting of N-terminal nuclease
domain of BmrI and C.BclI were constructed and
sequenced. The CH-endonucleases consist of amino acid
residues 1–198, 1–204 and 1–209 of BmrI followed by
a 14-amino acid linker and C.BclI sequence (Figure 1A).
These fusion proteins contain the HKD motif of BmrI and
the natural linker sequence (aa 170–198, colored in yellow;
(Figure 1) between the cleavage domain and the DNA
recognition domain. Small-scale expression experiments
were done and SDS-PAGE analysis showed that all three
fusion proteins expressed mostly as inclusion bodies in
E. coli. DNA cleavage assay showed that the lysate
supernatant derived from the clone that expressed the
fusion protein consists of amino acid residues 1–198 of
BmrI has specific cleavage activity without affecting the
growth of the host cells (data not shown). This fusion
protein was named BmrI198-C.BclI and was used in the
following studies (Figure 1B).
Although lowinduction temperature
increased the yield of soluble protein, the yield of soluble
BmrI198-C.BclI was too low to generate enough protein
for further study (data not shown). Therefore, the
inclusion bodies were unfolded in a buffer containing
8M urea, purified through a nickel-charged metal chela-
tion column and then refolded. The best refolding
Nucleic Acids Research, 2007, Vol. 35, No. 18
condition was found by screening 15 refolding buffers. The
purified and refolded chimeric endonuclease BmrI198-
C.BclI is shown in Figure 1C. The yield of soluble
BmrI198-C.BclI was 0.2mg/g wet cells after refolding.
Plasmid Litmus28-bclIC contains the ORF of C.BclI and
the C-box sequence (38). BmrI198-C.BclI cleaves DraIII-
linearized Litmus28-bclIC but not Litmus28 into 2
fragments of ?2.1 and ?1.2kb (Figure 2A). The sizes of
the fragments are consistent with the position of the
C box on DraIII-linearized Litmus28-bclIC. Restriction
mapping using HpaI, ScaI, BsaI and AlwNI further
confirmed that the cleavage site is in the vicinity of the
C box (Figure 2B). Control experiments using supercoiled
DNA as substrates showed that the CH-endonuclease
had similar level of nicking activity on specific (Litmus28-
bclIC) and non-specific (Litmus28) DNA. The CH-
endonuclease displayed significantly higher ds cleavage
activity on specific DNA than non-specific DNA (data not
The cleavage sites of BmrI198-C.BclI on Litmus28-
bclIC were determined by sequencing the cleavage
products directly. The 2.1 and 1.1kb cleavage products
were gel-purified and subjected to DNA sequencing using
primers running from either upstream or downstream of
the C box. During sequencing reactions, when the DNA
polymerase reaches the end of the template DNA, it ‘runs
off’ from the template. Thus, a sharp decrease in peak
intensity is observed in the electropherograms. In addi-
tion, an extra A is added to the end of the sequence due to
the template-independent terminal transferase activity of
the DNA polymerase. Therefore, the presence of an
aberrant A accompanied by a sharp drop of peak intensity
of proceeding peaks in the electropherograms is inter-
preted as the end of the template and hence the cleavage
site. The height of the aberrant A peak is also suggestive of
the population of the template terminated at that site.
DNA sequencing of the cleaved DNA fragments
indicated that there is a mixture of molecules cut at two
major sites: ?8#?9 and þ19#þ20 of C-box sequence
(# indicating the cleavage site, Figure 2C). On the upper
strand, BmrI198-C.BclI makes two major and one minor
cuts at ?8 to ?11 outside the C box and one major
cut at þ19#þ20 within the C box. On the bottom strand,
it makes one major cut at ?11#?12 outside the
C box and two major cuts at þ17#þ18 and þ18#þ19.
At the ?8 site, blunt ends and 1–3bp 30overhangs are
generated whereas at the þ20 site, 1–2bp 30overhangs
are generated. It is likely that some of the substrate
molecules are cleaved at both sites [similar to the BcgI-
type REases (39,40)] while some are cleaved at either site.
Interestingly, the positions of the two cleavage sites do not
align with the symmetry of the C box. The ?8#?9 site lies
upstream of the C box whereas the þ19#þ20 site lies
within the C box.
Substratesequence requirement forspecific cleavage
The asymmetry of the cleavage site relative to the
C box suggests that the BmrI198-C.BclI fusion protein
Figure 1. (A) Amino acid sequence alignment of BmrI and BfiI
endonucleases. Identical residues are shown in the consensus sequence.
Conserved residues are colored in gray. The N-terminal cleavage
domain is boxed in red, C-terminal DNA-binding domain in blue.
The inter-domain intrinsic linker sequence is colored in yellow. Putative
catalytic residues of the HKD motif (His105, Lys107, Asn125) are
colored in green. Residues 198, 204 and 209 are numbered and marked
with triangles. Domain assignment was made according to the crystal
structure of BfiI (34). (B) Schematic diagram of the CH-endonuclease
BmrI198-C.BclI. Peptide sequence containing residues 1–198 of BmrI
was linked to C.BclI full-length sequence through a 14-amino acid
residues linker sequence that contains Gly, Ala and Ser. The cleavage
domain of BmrI is colored in red, the natural inter-domain linker
sequence of BmrI in yellow and the HKD catalytic motif in light blue.
C.BclI is colored in gray with the helix–turn–helix (HTH) motif
highlighted in green. (C) Purification of BmrI198-C.BclI. Inclusion
bodies isolated from the induced E. coli culture was unfolded in
a buffer containing 8M urea (lane 1). The unfolded BmrI198-C.BclI
was purified by a Ni-charged metal chelate column (Ni-NTA) (lane 2)
and refolded (lane 3).
Nucleic Acids Research, 2007, Vol. 35,No. 186241
does not occupy the whole C box. We deleted the 50
half (Litmus28-bclIC?50) or the 30
bclIC?30) of BclI C box respectively from Litmus28-
bclIC and verified the effect of these binding blocks on
the cleavage activity of BmrI198-C.BclI. Using the
same amount of enzyme, the chimeric endonuclease
as the wild-type substrate Litmus28-bclIC, whereas only
minor cleavage was observed with the substrate Litmus28-
bclIC?50(Figure 3A). This indicates that the nucleotides
deleted in Litmus28-bclIC?30are not involved in the
specific binding and cleavage for the chimeric endonu-
clease, and that the nucleotides deleted in Litmus28-
bclIC?50are necessary for specific binding.
Figure 3B shows the run-off sequencing of the cleavage
productsfrom Litmus28-bclIC?30. Itshows that
Figure 2. Specific cleavage activity of BmrI198-C.BclI. (A) Litmus28-bclIC contains the C box and ORF of C.BclI. When linearized, the C box is
located 1221 and 2042bp away from the DraIII site (upper panel). Litmus28-bclIC and Litmus28 (67fmol) were linearized by DraIII and cleaved by
12, 24 or 48pmol of BmrI198-C.BclI (lanes 2 and 6, 3 and 7, 4 and 8, respectively). Specific cleavage products (2.1 and 1.2kb) were observed in
Litmus28-bclIC (lower panel). (B) Restriction mapping of BmrI198-C.BclI cleavage site. Litmus28-bclIC was pre-cut by the designated REases and
then digested by BmrI198-C.BclI (upper panel). The position of the C box with respect to the restriction sites on Litmus28-bclIC are shown in the
lower panel. In lane 6, the plasmid was incubated with DraIII in the absence of BmrI198-C.BclI (linearized). (C) Run-off sequencing of cleavage
products. After cleavage by DraIII and BmrI198-C.BclI, the cleavage products were subjected to DNA sequencing from both directions.
Electropherograms of sequencing reactions for the top and bottom strands were shown. C-box sequences are boxed in gray. DNA sequences are
shown on top of the electropherograms. The nucleotides are numbered relative to the start (þ1) of the C box sequence. Down arrows indicate the
cleavage sites marked by a drop of the peak intensity and an aberrant A, which are boxed in red line and numbered. Large and small down arrows
indicate major and minor cleavage, respectively. The lower panel shows the ds DNA sequence in the vicinity of the C box. The arrows and the
red boxes correspond to those in the electropherograms. Nucleotides 30to the cleavage sites are numbered.
Nucleic Acids Research, 2007, Vol. 35, No. 18
BmrI198-C.BclI makes major cuts (large down arrows) on
at the same sites (?8#?9 and
þ19#þ20 sites) as it cleaves the wild-type C box on
Litmus28-bclIC (Figure 2C). However, variations of
minor cuts (small down arrows) were observed. On the
top strand, the minor cut between ?10 and ?11
(TAT#TAT) in wild-type substrate was not observed in
duplicated sequencing reactions of the variant. The
variant has two minor cuts (þ20#þ21; þ22#þ23) at the
top strand of the þ20 site, which were not found in wild-
type substrate. The fact that BmrI198-C.BclI makes the
same major cuts on the wild-type Litmus28-bclIC and
the deletion variant of Litmus28-bclIC?30, and that the
chimeric endonuclease does not cut the C box when the
first 15bp of the C box is deleted demonstrate that the first
15bp of the C box is sufficient for specific cleavage by the
Kinetics ofDNA cleavage
The sequence specificity of BmrI198-C.BclI most likely
derives from the DNA-binding HTH motif of C.BclI.
The CH-endonuclease appears to possess a low turnover
rate on interaction with target site. In fact, titration of the
CH-endonuclease against DNA substrate shows that
a ?300-fold molar excess of the fusion protein over
DNA substrate is required to make specific cleavage
(Figure 3A). Therefore, the kinetics of the DNA cleavage
reaction was studied in single-turnover condition. A time
course for the cleavage reaction is shown in Figure 4.
The intensities of the substrate (3.3kb) and cleavage
products (2.1 and 1.2kb) were quantified after agarose
gel electrophoresis and their quantities were estimated
through correlation to that of the input substrate (DraIII
only; Figure 4). The amount of the substrate and the sum
of that of the cleavage products were plotted against time.
In 120min, ?80% of the substrate was cleaved and
the production of the product leveled off. The increase in
the amount of the cleavage products correlated with the
decrease in that of the substrate. The sum of the quantity
of substrate and products added up to the quantity of
input substrate at each time point (data not shown),
suggesting that most of the substrate cleaved was
transformed into the specific products and that non-
specific cleavage activity is not significant within this time
frame. Complete cleavage of the substrate was achieved
with extended incubation times but at the expense of
increasing non-specific cleavage (data not shown).
Ionic strength and magnesiumion requirement
Cleavage activity of the fusion enzyme was tested in
buffers containing different concentrations of NaCl
Figure 3. Cleavage of the C-box deletion variants. (A) DraIII-linearized WT (lanes 1–4), ?30(lanes 5–8) and ?50(lanes 9–12) variants of Litmus28-
bclIC were cleaved with increasing amount of BmrI198-C.BclI under the same condition. The lower panel shows the sequences of the C box and
flankingsequencesfor the substrates.(B) Run-offsequencingof
Annotation scheme is identical to that of Figure 2C.
Nucleic Acids Research, 2007, Vol. 35,No. 186243
and Tris–HCl (Figure 5). Smears of DNA with ?200bp
and smaller resulting from non-specific cleavage were
found in Buffer 1, 2 and 4. Specific cleavage was
only observed in Buffer 3, high salt buffer and EcoRI
buffer. Buffer 1, 2 and 4 contain 50mM or less NaCl,
whereas Buffer 3, high salt buffer and EcoRI buffer
contain 100mM or higher concentration of chloride ion
(NaCl or Tris–HCl). High ionic strength may provide an
electrostatic screen for non-cognate hydrogen bonds and/
or electrostatic interactions between the HTH DNA-
binding motif of C.BclI and the substrate DNA.
This is consistent with the buffer condition (10mM Tris–
HCl, 100mM NaCl, 4mM CaCl2, 5% glycerol, pH 7.5)
with which specific binding of C.BclI to C box was
demonstrated (38). The presence of 0.025% Triton X-100
in EcoRI buffer did not increase non-specific activity,
the primary interactions required for cleavage specificity.
Although BmrI and the truncation mutant BmrI198
do not require magnesium ions for cleavage (data not
shown), the removal of magnesium ions from the cleavage
reaction promotes non-specific cleavage of BmrI198-
C.BclI. Quenching Buffer 3, high salt buffer and EcoRI
buffer with 10mM EDTA (Figure 5, lanes 8–10)
or replacing 10mM MgCl2with 10mM EDTA in Buffer
3 (EDTA buffer; Figure 5, lane 1) resulted in high non-
specific cleavage activity. It is possible that magnesium
ions decrease the DNA cleavage activity of the BmrI
cleavage domain, or they are required for the interactions
between the HTH DNA-binding motif of C.BclI and
the target DNA. The absence of divalent ions in the
? repressor-target DNA structure (PDB entry 1LMB)
and the decrease in ‘star’ activity of wild-type BmrI in
the presence of magnesium ions (unpublished data)
support the notion that magnesium ions increase specific
cleavage activity of the fusion protein by inhibiting DNA
cleavage activity of the BmrI DNA cleavage domain.
Thermostability andreaction temperature
BmrI is isolated from a mesophilic bacterium (Bacillus
megaterium). Its cleavage activity decreased ?60% after
heating at 558C for 20min and was completely destroyed
at 658C (data not shown). Surprisingly, BmrI198-C.BclI
exhibited higher thermal stability. Specific cleavage
was impaired after pre-incubation at 648C for 20min
(data not shown). The increased thermostability of the
CH-endonuclease may be contributed by the removal of
the C-terminal DNA recognition
is probably more susceptible to irreversible thermal
denaturation or due to the addition of a thermostable
binding partner C.BclI. The BclI producing strain Bacillus
caldolyticus is a moderately thermophilic strain with
growth temperature up to 708C (REBASE).
The optimal reaction temperature
endonuclease was determined to be 37–408C. Non-specific
cleavage activity increased above 408C. Non-specific
Figure 4. Time course of BmrI198-C.BclI cleavage. Litmus28-bclIC was
linearized by DraIII and gel-purified. One hundred ninety nanograms
(87fmol) of the DNA were incubated with 24pmol of BmrI198-C.BclI
for 120min at 378C. The intensity of the 3.3kb DraIII-linearized
substrate and the 2.1 and 1.2kb cleavage products were quantified and
correlated to the quantity of input DNA (DraIII only). The quantity of
the substrate (filled square) and the sum of those of the cleavage
products (filled triangle) were plotted against time. The reactions were
carried out under single-turnover condition.
Figure 5. Effect of NaCl and EDTA on specific cleavage activity.
Cleavage reactions were carried out using DraIII-linearized Litmus28-
bclIC in different buffers (lanes 1–7) or in buffer 3, high salt (HS)
buffer and EcoRI buffer quenched with 10mM EDTA (lanes 8–10).
B1, Buffer 1; B2, Buffer 2; B3, Buffer3; B4, Buffer 4.
Nucleic Acids Research, 2007, Vol. 35, No. 18
BmrI and BfiI closely resemble each other in amino acid
sequence. They have 358 amino acid residues and share
79.6% sequence identity (Figure 1A). They belong to the
phospholipase D family that is characterized by the HKD
catalytic motif. Two copies of the HKD motif fold to form
a single catalytic site where one of the histidine residues
forms the phosphohistidine intermediate and the lysine
residues are required for the positioning of the phosphate
group being attacked (41). Because one of the histidine
residues also acts as a nucleophile, the HKD catalytic
motif does not require divalent metal ions for hydrolysis.
Unlike most of the phospholipase D family members that
contains two copies of the HKD catalytic motif, BfiI and
BmrI have only one copy. Presumably, dimerization
is required for BfiI and BmrI to form a functional
catalytic site at the dimerization interface. Structural and
biochemical data suggest that homodimers of BfiI cut the
bottom strand first and then undergo conformational
rearrangement with respect to the nicked DNA inter-
mediate to cleave the top strand (34,37). Top-strand
cleavage activity of BfiI was inhibited at pH 6.5, thereby
converting BfiI into a bottom-strand nicking endonuclease
(NEase) (37). Experimental evidence suggests that low pH
protonates the 50phosphate at the new nick in the bottom
strand and inhibits the rearrangement and/or chemistry
that is required for the sequential top-strand cleavage.
Conformational rearrangement and sequential cleavage of
the two strands of DNA may be utilized by other Type IIS
REases. This makes Type IIS REases an attractive source
of modular DNA cleavage domains for creating combi-
Here we have successfully created a sequence-specific
endonuclease with novel substrate specificity by fusing
a DNA recognition domain to the cleavage domain of
BmrI. The primary hurdle to cross when creating
a combinatorial endonuclease is to attain high specific
cleavage and low non-specific cleavage. Factors that can
contribute to non-specific cleavage include: (i) the affinity
of DNA-binding domain toward target DNA sequence
versus non-specific cleavage activity of the cleavage
domain and (ii) the position of the cleavage domain
relative to the DNA-binding domain. The DNA cleavage
domain and the DNA-binding domain can be envisioned
as counteracting in terms of specific cleavage: the DNA
cleavage domain tends to capture random DNA sequences
and make breaks, whereas the DNA-binding domain
samples the whole DNA molecule for the target sequence.
To make specific cuts, the DNA-binding domain has to
find its target sequence before the cleavage domain cuts
of magnesium ions appears to attenuate non-specific
cleavage activity of the fusion protein by decreasing the
DNA cleavage activity of the BmrI DNA cleavage domain.
In order to make cuts at specific sites, the cleavage
domain of the chimeric endonuclease has to be in contact
domain binds to the target sequence. In the current study,
this was achieved by connecting the two domains with
a flexible linker sequence consisting of Gly, Ala and Ser
residues (GSGGGGSAAGASAS). The linker is expected
to allow the BmrI cleavage domain to adapt a range of
orientations such that it can cut the substrate after the
C.BclI domain binds to the target sequence.
C.AhdI (PDB entry 1Y7Y), the controller protein of the
AhdI R-M system, and C.BclI (PDB entry 2B5A)
are highly homologous in 3D structure and their target
C-box sequences are also similar. Although with lower
sequence homology, C.BclI is also structurally similar
to proteins involved in genetic switches, namely, repressor
proteins from phage ? (42) and phage 434 (43), and BldD-
N, the N-terminal DNA-binding domain of BldD from
Streptomyces coelicolor repressor (44).
The C box of AhdI R-M system has been proposed
to consist of four 5-bp binding blocks: two each in the 50
half and 30half. A homodimer of C.AhdI binds to two
binding blocks of either 50or 30half where each monomer
interacts with one of the 5-bp blocks (Figure 6). It has
been shown that C.AhdI binds the 50half with higher
affinity than the 30half, and that binding of C.AhdI on the
50half induces cooperative binding at the 30half by
another dimer of C.AhdI (45). Our results show that
BclI198-C.BclI cleaves the wild-type C box and the
30deletion variant at the same sites, showing that
the chimeric endonuclease does not bind to the 30half of
the C box. This is consistent with the higher binding
affinity of C.AhdI toward the 50half of its C box.
The BmrI198-C.BclI homodimer or tetramer bound to the
50half of the C box may exclude another homodimer or
tetramer binding to the 30half of the C box. This may
explain the cleavage sites are predominantly located
within the right half of the C box (þ19#þ20) instead of
being located outside of þ24 position.
Based on the published results of C.AhdI and our
BclI C-box deletion results, we propose a scheme of
DNA binding and cleavage by BmrI198-C.BclI. The
BclI C box consists of four binding blocks of AGACTT
and its variant sequences TCACTT, AGGCTA (Figure 6,
sequences indicated in orange, BLK1 to 4). A 6-bp
binding block is considered in contrast to the 5-bp version
of C.AhdI. Because BfiI forms stable homodimers in vitro
(15) at their DNA cleavage domain (34), it is likely that
the CH-endonuclease dimerizes before binding to the
DNA substrate. Therefore, two homodimers of BmrI198-
C.BclI molecules (pairs of homodimer in green or orange,
(Figure 6) would bind to the binding block 1 and 2 of
substrate DNA (boxed in green or orange lines, respec-
tively) and form a tetramer at the dimerization interface of
C.BclI (37,46). Each of the two DNA-bound BmrI198-
C.BclI molecules would interact with one of the binding
blocks via its HTH motif—the BmrI198-C.BclI subunit
(color in green) that binds to BLK1 (in green box) would
extend its DNA cleavage domain 13–15bp downstream to
the þ20 cleavage site (indicated by green lines) whereas the
other subunit (colored in orange) would bind to BLK2
(boxed in orange lines) with its DNA cleavage domain
reaching 13–16bp upstream onto the ?8 site (indicated
by orange lines) (Figure 6). Thus, a nick is made at the ?8
BmrI cleavage domain at each site. At this point, similar
to the mechanism proposed for BfiI, the nicked DNA
site, respectively,throughthe dimeric
Nucleic Acids Research, 2007, Vol. 35,No. 186245
substrate (37), which is likely to have adopted a different
conformation, would induce a conformational change of
the DNA-bound chimeric endonuclease, possibly affected
by the intrinsic flexible linker within BmrI198 plus the
14-amino acid engineered linker (thin lines connecting
the BmrI cleavage domain and C.BclI). This would allow
the cleavage domain of the dimeric DNA-bound chimeric
endonuclease to make contacts with the opposite strand
and repeat the cleavage there. Because the DNA cleavage
domain would have to traverse a longer distance around
the DNA helix to reach the opposite strand, the cuts at the
opposite strand are closer to the binding site. The flexible
spanning across the ds DNA such that alternative
cleavage sites 1–3bp apart are generated. The enzyme
cleaves 1bp further from the binding site at the ?8 site
(13–16bp) than the þ20 site (13–15bp). This is probably
caused by bending of the DNA substrate upon binding of
the fusion endonuclease,
in protein–DNA interactions, particularly with those
involved in gene regulations (47–49).
Our work demonstrates that the BmrI cleavage domain
is useful in the combinatorial approach to creating novel
endonucleases that cleave specifically in the vicinity
of DNA regulatory region. A fusion of C.BclI to the
DNA cleavage domain of BmrI introduces specific
ds breaks at the regulatory sequence of the BclI R–M
system (C box). Potentially, the BmrI DNA cleavage
domain can be fused to other DNA-binding proteins such
as transcription factors to cut specifically at the regulatory
elements of gene expression. Such cleavage has been
demonstrated to induce DNA repair resulting in deletion
of genes in vivo (28–31). We have also fused the BmrI
nuclease domain to a cleavage-deficient variant of NotI
such that the fusion protein binds and cleaves outside
of NotI site, thus creating a NotI neoschizomer in a
related study (Zhang et al., in press, PEDS).
Type IIS REases are useful to create combinatorial
endonucleases because they consist of a DNA-binding
domain that contributes to their substrate specificity and
a separate DNA cleavage domain, which cuts within
or outside the recognition sequences. A well-known
example making use of this modular property is FokI.
The DNA cleavage domain of FokI (GGATC N9/N13)
has been linked to several DNA-binding proteins (18,19)
and zinc-finger motifs (24–27) to create endonucleases of
novel substrate specificities. In particular, a few ZFNs
have been created that possess specific cleavage activity
in vitro (20–23) and in vivo (28–31,50,51). The nuclease
domain of BmrI itself can also be used to generate small
fragments of DNA (data not shown).
The current version of CH-endonuclease remains to
be optimized. The BmrI cleavage domain itself does not
fold properly when expressed in E. coli and requires
refolding. In addition, the presence of two dimerization
interfaces, one on the DNA-binding domain and the other
on the cleavage domain, may lead to oligomerization that
sequesters the catalytic site and attenuates specific activity
of the chimeric endonuclease. This may account for the
high molar ratio of BmrI198-C.BclI over substrate DNA
required for specific cleavage (340:1 for the time course
in Figure 4, with 67% of the substrate being cleaved
in 1h). Although complete cleavage of substrate DNA can
be achieved by higher concentrations of BmrI198-C.BclI
or longer incubation time, these reaction conditions also
a single-chain endonuclease by fusing two identical
DNA cleavage domains in tandem can avoid the problems
arise from the presence of two different dimerization
interfaces. A single-chain variant of PvuII has been
created by linking two monomers of the wild-type PvuII
through a 4-amino acid linker sequence such that a single
molecule of this single-chain PvuII can generate ds breaks
may adopt different conformationswhen
whichis often observed
Figure 6. Proposed scheme of DNA binding and cleavage by BmrI198-
C.BclI. Upper panel: the C box of AhdI system consists of four 5-bp-
binding blocks. Dimers of C.AhdI interact with either BLK1 and
BLK2, or BLK3 and BLK4. C.AhdI has a higher affinity towards
BLK1 and BLK2, and binding of C.AhdI dimers to BLK1 and BLK2
induces synergetic binding of C.AhdI dimers to BLK3 and BLK4.
Middle panel: in C.BclI, the C box can be subdivided into four 6-bp
blocks by the conserved and symmetric sequences 50WGARTW 30
(W¼A or T; R¼A or G; colored in red). Cleavage sites (?8 and þ20
sites) depicted in Figure 2C are shown in terms of distance (bp) from
BLK 1 and BLK 2. We propose that one of the BmrI198–C.BclI
homodimers binds to BLK1 (green box) and cuts 13–15bp downstream
(distance indicated by green lines) and the other homodimer binds to
BLK2 (orange box) and cuts 13–16bp downstream (indicated by
orange lines). The C-box sequence is shown in gray box. Lower panel: a
cartoon of the proposed scheme of binding. The homodimer of
BmrI198–C.BclI that binds BLK1 and cuts at the þ20 site is colored in
green; the homodimer that binds BLK2 and cuts at the ?8 site is
colored in orange. Molecules in dark green or orange are in front side
of the DNA; those in light green or orange are behind the DNA. The
BmrI intrinsic linker plus the 14-amino acid linker connecting the BmrI
cleavage domains to C.BclI-binding domains are shown as solid lines.
DNA sequences of BLK1 and BLK2 are colored in red.
Nucleic Acids Research, 2007, Vol. 35, No. 18
without resorting to dimerization (52). The length and
flexibility of the linker sequence connecting the BmrI
nuclease domain and the DNA-binding protein may be
further optimized for efficient and precise cleavage.
The strategy to couple a DNA-binding protein to a
nuclease domain can also be applied to the construction of
site-specific nicking endonucleases, for example, by fusing
a DNA-nicking domain from I-HmuI to sequence-specific
We thank I. Murray and J. Samuelson for critical reading
of the manuscript and G.K. Balendiran for discussion.
This work was partly supported by an SBIR grant to S.Y -
X (1 R43 GM073345-01, NIH). We also thank Z. Zhu for
providing the plasmid Litmus28-bclIC and Don Comb for
support. Funding to pay the Open Access publication
charges for this article was provided by New England
Conflict of interest statement. None declared.
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