Site- and strand-specific nicking of DNA by
fusion proteins derived from MutH and I-SceI
or TALE repeats
Lilia Gabsalilow, Benno Schierling, Peter Friedhoff, Alfred Pingoud and
Institute for Biochemistry, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany
Received October 10, 2012; Revised January 14, 2013; Accepted January 22, 2013
Targeted genome engineering requires nucleases
that introduce a highly specific
break in the genome that is either processed by
homology-directed repair in the presence of a hom-
ologous repair template or by non-homologous
end-joining (NHEJ) that usually results in insertions
or deletions. The error-prone NHEJ can be effi-
ciently suppressed by ‘nickases’ that produce a
single-strand break rather than a double-strand
produced by engineering of homing endonucleases
and more recently by modifying zinc finger nucle-
ases (ZFNs) composed of a zinc finger array and
the catalytic domain of the restriction endonuclease
FokI. These ZF-nickases work as heterodimers in
which one subunit has a catalytically inactive FokI
domain. We present two different approaches to
engineer highly specific nickases; both rely on the
mismatch repair endonuclease MutH which we
fused to a DNA-binding module, either a catalytically
inactive variant of the homing endonuclease I-SceI
or the DNA-binding domain of the TALE protein
AvrBs4. The fusion proteins nick strand specifically
a bipartite recognition sequence consisting of the
MutH and the I-SceI or TALE recognition sequences,
respectively, with a more than 1000-fold preference
over a stand-alone MutH site. TALE–MutH is a pro-
activityof the DNA
Precise gene targeting requires custom-designed highly
specific nucleases. Two basically different approaches are
being pursued for this purpose, (i) protein engineering of
homing endonucleases, which results in ‘meganucleases’
of predefined specificity (1,2) and (ii) the fusion of a pro-
grammable DNA-binding module and a DNA-cleavage
module, as exemplified by the zinc finger nucleases
[ZFNs, (3–6)] and the TALE nucleases [TALENs,
(7–10)]. ZFNsand TALENs
cleavage domain of FokI as cleavage module and an
array of zinc fingers or the DNA-binding domain of
TAL effector proteins as DNA-binding module, respect-
ively. Recently, also fusion proteins with a catalytically
inactive I-SceI or a ZF array as DNA-binding modules
and the restriction endonuclease PvuII as sequence-
specific DNA-cleavage module were described (11,12), as
well as fusion proteins with a catalytically inactive I-OnuI
or a ZF array as DNA-binding modules and the I-TevI
nuclease domain as DNA-cleavage module (13).
double-strand breaks at a specific target site that can be
repaired by two different pathways: homology-directed
repair (HDR) which requires a donor DNA template, or
error-prone non-homologous end-joining (NHEJ), result-
ing in insertions, deletions or translocations. For the
purpose of genome engineering by introducing sequence
alterations or insertions at or near the site of the double-
strand break, NHEJ is an unwanted and possibly geno-
toxic side reaction. It can be largely circumvented by using
a DNA-nicking endonuclease [vulgo ‘nickase’, (14,15)]
which had been shown to stimulate HDR (16).
Nickases occur naturally or can be obtained by engin-
eering restriction or homing endonucleases [reviewed in
(17,18)]. Naturally occurring nickases are for example
the restriction endonuclease
subunits of heterodimeric restriction endonucleases, e.g.
Nt.BstD6I (21,22), Nb.BsrDI and Nb.BtsI (23). I-HmuI
and I-BasI represent naturally occurring nickases in the
homing endonuclease family (24–26). Restriction and
homing endonucleases that usually recognize palindromic
or quasi-palindromicDNA sequences
double-strand cuts making use of two catalytic centres
*To whom correspondence should be addressed. Tel: +49 641 35407; Fax: +49 641 35409; Email: email@example.com
Published online 13 February 2013Nucleic Acids Research, 2013, Vol. 41, No. 7e83
? The Author(s) 2013. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
can be engineered to become nickases by inactivating one
catalytic centre, as had been shown for EcoRV (27,28),
FokI (29), I-SceI (30) and I-AniI (31).
ZFNs that use the non-specific DNA-cleavage domain
of FokI to introduce a programmable DNA double-strand
cut can be converted to a ZF-nickase by inactivating the
catalytic centre of one FokI monomer in a ZFN
heterodimer. This has been shown in a few studies pub-
lished in 2012 (32–34), which demonstrated that the
ZF-nickases allow site-specific genome modifications at
the predetermined target site, while reducing unwanted
mutagenesis caused by error-prone NHEJ.
It had been argued by Halford (35) that ‘the reaction
mechanism of FokI excludes the possibility of targeting
ZFNs to unique DNA sites’. Although this risk has been
minimized by using obligate heterodimers (36,37), it was
shown in two recent studies that ZFNs that form obligate
heterodimers caused residual off-target cleavage (38,39).
What is a problem with ZFNs might be also one with
ZF-nickases, when the same FokI cleavage module is
used. The question arises, whether one can substitute the
catalytic domain of FokI in ZF-nickases with a different
DNA-cleavage module, as we have done in three different
fusion proteins, PvuII–I-SceI (12), ZF-PvuII (11) and
TALE–PvuII (M. Yanik, unpublished data) that intro-
duce double-strand cuts into DNA.
In this study, we engineered and tested two highly
specific nickases, MutH–I-SceI and TALE–MutH. The
DNA mismatch repair protein MutH is a naturally
occurring monomeric site-specific nickase that can nick
the DNA at GATC sites in un- or hemimethylated DNA
(40,41). It is part of the MutHLS system from Escherichia
coli and a few other bacteria; it serves to repair DNA
mismatches, e.g. caused by rare errors of the replicative
polymerases. The mismatch is recognized by MutS which
recruits MutL to the site of error to form a ternary complex
(42). This complex activates MutH to nick the erroneous
daughter strand at the 50-side of a hemimethylated GATC
site which can be >1000bp upstream or downstream from
the mismatch (43). The nick in the unmethylated strand is
the entrypointfor UvrD
single-stranded DNA that is digested by exonucleases
past the original mismatch. The resulting gap is filled by
DNA polymerase and the DNA is ligated (44). MutH by
itself hardly shows any cleavage activity on unmethylated
DNA under physiological ionic strength (41,45). Only
when guided by the fused DNA-binding module does
MutH exhibit nicking activity at the targeted GATC site.
We show here that MutH–I-SceI and TALE–MutH can be
considered as site- and strand-specific nickases, and
TALE–MutH, in addition, as a programmable nickase.
MATERIALS AND METHODS
Design and construction of the MutH–I-SceI and
TALE–MutH fusion proteins
For the construction of the MutH–I-SceI fusion protein, a
catalytically inactive variant of I-SceI (46), which had
been truncated at the C-terminus (?C9), was fused to
the C-terminal end of a cysteine-free variant of MutH
via a 10-amino-acid linker (ASENLYFQGG) harbouring
a TEV protease recognition site (underlined), or for
control a linker without the TEV site (TKQLVKSE).
The gene for MutH (C96S), which contains the coding
sequence for an N-terminal His6-tag, and the gene for
I-SceI (?C9 D44S D145A) were connected by the
coding sequence of the linker and inserted into the expres-
sion vector pASK-IBA63b-plus
Germany) coding for a C-terminal Strep-tag. Thus, the
For the TALE–MutH fusion protein, a truncated
variant of the TALE protein AvrBs4, corresponding to
the previously described AvrBs3 DNA-binding module
(9), was fused directly to the N-terminal end of MutH.
The gene for the TALE–MutH fusion contains two
parts: (i) the gene of the truncated TALE variant,
missing the coding sequence of the first 152 amino acids
at the N-terminal end and the last 250 amino acids at the
C-terminal end [28 amino acids remain after the last
half-repeat (M. Yanik, unpublished data)]. (ii) The gene
of MutH, which contains the coding sequence for a
C-terminal His6-tag. The two parts were connected and
introduced into the expression vector pQE30 (Qiagen),
coding for an N-terminal Strep-tag. Thus, the expected
fusion protein would be: Strep-TALE–MutH-His6. The
sequence of both fusion constructs was confirmed by
DNA sequencing of the entire coding region. We had
varied the linker length between AvrBs3 and PvuII
which is structurally very similar to MutH (47) and
found the 28-amino-acid linker superior to a 63- and 16-
amino-acid linker (M. Yanik, unpublished data). There-
fore, we have chosen the 28-amino-acid linker for the
TALE–MutH fusion protein.
(IBA, Go ¨ ttingen,
Protein expression and purification
The expression vectors for the recombinant fusion
XL1-Blue (Stratagene). The cells were grown at 37?C to
OD600nmca. 0.7 in LB-medium containing 75mg/ml ampi-
cillin. Protein expression was induced by adding 200mg/l
anhydrotetracycline or 1mM isopropyl-b-D-thiogalacto-
pyranoside for MutH–I-SceI or TALE–MutH, respect-
ively, followed by further growth at 20?C overnight. The
cells were harvested by centrifugation and resuspended in
20mM Tris–HCl, pH 7.9, 1 M NaCl, 20mM imidazole,
1mM phenylmethanesulfonyl fluoride (PMSF), lysed by
sonification and centrifuged for 30min (>17000g) at 4?C
to remove cell debris. The His6- and Strep-tagged proteins
were first purified via Ni2+-NTA agarose (Qiagen) by a 1h
incubation of Ni2+-NTA agarose with the supernatant at
4?C and washing with resuspension buffer; the protein was
eluted with 20mM Tris–HCl, pH 7.9, 1 M NaCl and
200mM imidazole. The eluted fractions were then
transferred to a column with Strep-Tactin Sepharose
(IBA) for further purification. After washing with
100mM Tris–HCl, pH 7.9, 1M NaCl, the protein was
eluted with 100mM Tris–HCl, pH 7.9, 500mM NaCl,
2.5mM desthiobiotin, dialyzed overnight at 4?C against
10mM HEPES-KOH, pH 7.9, 500mM KCl, 1mM
e83Nucleic Acids Research, 2013,Vol. 41,No. 7PAGE 2 OF 11
dithiothreitol (DTT), 50% (v/v) glycerol and stored at
?20?C. The protein concentration was determined by
measuring the absorbance at 280nm using the molar ex-
tinction coefficient as determined according to Pace et al.
(48). Protein purification was monitored by sodium
Plasmid cleavage assay
To test the activity and specificity of the fusion proteins,
the substrate plasmid pAT153 was used, which was
modified by introducing a DNA cassette with the appro-
priate target site (Table 1). Three kinds of substrates were
generated: one addressed substrate and two unaddressed
substrates. The addressed substrate contains the recogni-
tion site for the binding module [I-SceI (S) or AvrBs4 (T)]
and a MutH (H) recognition site separated by spacers of
different lengths. For MutH–I-SceI, only an addressed
substrate with a 3-bp spacer was used (S-3-H) and for
TALE–MutH, several spacer lengths were tested (T-x-H;
x=1, 2, 3, 4, 5, 6, 7, 8 and 9bp). The two unaddressed
substrates contain either the recognition site for the
binding module (S or T) without a nearby MutH recogni-
tion site or with a MutH recognition site (H) and without
a recognition site for a binding module. For the analysis of
the cleavage reactions, 4nM substrate plasmid and 16nM
enzyme (i.e. a 4-fold excess of enzyme over substrate) were
incubated in 20mM Tris-acetate, 120mM K-acetate,
1mM MgCl2, 50mg/ml bovine serum albumin (BSA),
pH 7.5 at 37?C. Taking the enzyme dilution into
account, the ionic strength of the reaction mixture was
ca. 160mM (which corresponds to physiological condi-
tions). The reaction progress was measured after defined
time intervals (1, 3, 10, 30, 60 and 120 min) by agarose gel
electrophoresis; gels were stained with ethidium bromide,
and the fluorescence of DNA bands was visualized with a
BioDocAnalyze System (Biometra). The time course of
the reaction (product versus time) was fitted to a
monoexponential function to give the rate constants for
nicking. For the determination of the strand-specificity of
MutH–I-SceI and TALE–MutH, respectively, a pAT153-
derived plasmid harbouring the specific substrate cassettes
S-3-H and T-3-H/T-6-H, respectively (Table 1), was used.
The nicked addressed plasmid was gel-purified and sent
for sequencing with primers 50-AATAGGCGTATCACG
AGGCCCTTTC-30(binding to the bottom strand) and
50-ACCCAGAGCGCTGCCGGCAC-30(binding to the
TEV cleavage of the linker of MutH–I-SceI
The TEV protease cleavage of MutH–I-SceI to separate
the DNA-binding and DNA-cleavage modules was per-
formed in 50mM Tris–HCl, pH 8.0, 0.5mM EDTA,
1mMDTT, 10 units/150ml
(Invitrogen) and 0.44mM MutH–I-SceI in the presence
of 4.4mM of a 53-bp oligonucleotide harbouring a
GATC recognition site. The reaction mixture was
incubated for 90min at 30?C. Afterwards, an aliquot
was taken to test the activity of MutH–I-SceI by a
plasmid cleavage assay as mentioned above, using the ad-
dressed substrate. To exclude that the loss of the catalytic
activity of MutH–I-SceI after TEV proteolysis is due to
the procedure, the whole experiment was performed in
parallel using water instead of the TEV protease.
Cleavage assay with fluorescently labelled PCR products
for the determination of the strand-specificity of
To investigate whether TALE–MutH nicks a DNA sub-
strate in the top or the bottom strand (defined by the
asymmetric recognition sequence of the DNA-binding
module), PCR products were generated using a forward
and a reverse primer which were 50-labelled with the
fluorophores Atto 488 and Atto 647N, respectively.
These experiments were carried out for the T-x-H sub-
strates (x=1, 2, 3, 4, 5, 6, 7, 8 and 9bp). The assay was
performed with 20nM of a 211-bp polynucleotide sub-
strate and 120nM of TALE–MutH (i.e. a 6-fold excess
of enzymeover substrate)
120mM K-acetate, 1mM MgCl2, 50mg/ml BSA, pH 7.5
at 37?C. The ionic strength of the reaction mixture was ca.
Table 1. DNA cassettes harbouring appropriate target sites that were introduced into the plasmid substrates
Cassettes for addressed substrates
Cassettes for unaddressed control substrates
Notes: Target sites are underlined and abbreviated with S (I-SceI), T (AvrBs4) and H (MutH). Numbers indicate
spacer lengths in base pairs. Sequences are shown in the 50–30direction for the sense strand.
PAGE 3 OF 11 Nucleic AcidsResearch, 2013, Vol.41,No. 7e83
160mM (which corresponds to physiological conditions).
The reaction progress was analysed after defined time
intervals (3, 10, 30, 60, 120 and 180 min) by denaturing
PAGE; the fluorescence of DNA bands was visualized
with the VersaDoc Imaging System (Bio Rad).
Design of the fusion constructs
Two principally different types of approaches had been
recently used by us to produce highly specific nucleases
for the purpose of genome engineering: (i) fusion proteins
with a specificity defined by a catalytically inactive homing
endonuclease [I-SceI, (12)] and (ii) fusion proteins with a
programmable specificity defined by a ZF array (11) or a
the Type II restriction endonuclease PvuII was the DNA-
cleavage module. We have now used these two different
MutH–I-SceI and TALE–MutH. In analogy to our
MutH–I-SceI fusion protein with an N-terminal His6-tag
and a C-terminal Strep-tag. Likewise, in analogy to our
ZF- (11) and TALE-PvuII constructs (M. Yanik, unpub-
lished data), a TALE–MutH fusion protein with an
N-terminal Strep-tag and a C-terminal His6-tag was
produced (Figure 1).
highly specific nickases,
we have produceda
Determination of the cleavage preference of MutH–I-SceI
for the addressed substrate
The rates of DNA nicking were determined with
unmethylated plasmid substrates with three different rec-
ognition sites: (i) the addressed bipartite recognition site
composed of an I-SceI recognition site next to a MutH
recognitionsite (GATC), (ii) a stand-aloneI-SceI
recognition site and (iii) a stand-alone MutH recognition
site. All three plasmid substrates have 19 additional
GATC sites. As shown in Figure 2, only the plasmid sub-
strate with the addressed bipartite site is nicked. Even at a
4-fold excess of enzyme over unaddressed substrate and at
prolonged incubation time (2h), no non-specific nicking
or cleavage is observed. Given the sensitivity of the assay,
these results show that the plasmid substrate with the ad-
dressed site is nicked by a factor of 1000 faster than the
other plasmid substrates. Actually, the preference for the
addressed site over an unaddressed site might exceed the
factor of 1000, because (i) the plasmids used in the assay
contain 19 unaddressed sites, i.e. stand-alone GATC sites
and only one addressed site and (ii) in the determination
of the rate of nicking of the undressed substrate, the lower
limit of detection had been reached.
Inactivation of the specific nicking activity of
MutH–I-SceI by proteolytic separation of the
binding and the cleavage module
The activity of the MutH–I-SceI fusion construct can be
suppressed by separating the DNA-binding and DNA-
cleavage modules by pre-incubating MutH–I-SceI with
the TEV protease which cleaves the linker peptide that
connects the two modules. Figure 3 shows that the
covalent linkage of I-SceI and MutH is necessary for
nicking of the addressed site. There is no nicking or
cleavage observed when I-SceI and MutH are present in
the reaction mixture but not covalently linked to each
other. If the MutH–I-SceI fusion construct, in which the
linker does not contain a TEV protease cleavage site,
is pre-incubated with TEV protease, the addressed sub-
strate is nicked, demonstrating that TEV protease does
not cleave MutH or I-SceI at a cryptic TEV protease
Figure 1. (A) Scheme of the fusion constructs MutH–I-SceI and TALE-MutH binding to their respective recognition sites (50!30). The highly
specific interaction is mediated mainly by the binding module, I-SceI or the DNA-binding domain of the TALE protein AvrBs4, respectively. The
monomeric cleavage module MutH is recruited by the binding module to nick the GATC site in close proximity to the I-SceI or TALE site. MutH is
fused to I-SceI via a 10-amino-acid linker (ASENLYFQGG, shown as an orange line; the star indicates a TEV protease recognition site). In the case
of the TALE–MutH construct, no additional linker was introduced; the fusion was done 28 amino acids (shown as red line) after the last half-repeat.
(B) Model of the designed fusion constructs bound to specific DNA, but without the oligopeptide linking the DNA-binding and DNA-cleavage
module. The colour code can be deduced from the scheme, I-SceI in green, the TALE protein in red (only the 23 repeats of the 3UGM structure are
shown) and MutH in blue. Modelling was done using the crystal structures 1R7M (I-SceI), 3UGM (TALE) and 2AOQ (MutH). The model was
generated with PyMOL and 3D-DART (56).
e83 Nucleic Acids Research, 2013,Vol. 41,No. 7PAGE 4 OF 11
Determination of the strand specificity of MutH–I-SceI
and the cleavage module were fused with a flexible linker,
the question arose, whether both strands can be nicked
by MutH or whether one strand is preferred. Guided
by the molecular model shown in Figure 1, we expected
the bottom strand to be cleaved preferentially if not
exclusively. The strand specificity of MutH–I-SceI was
determined by sequencing the nicked product. As shown
in Figure 4, the top strand remains intact and the bottom
strand is cleaved 50of the GATC site.
Determination of the cleavage preference of TALE–MutH
for the addressed substrate
Similar experiments as carried out to determine the pref-
erence of the MutH–I-SceI fusion protein for addressed
over unaddressed substrate cleavage were carried out for
the TALE–MutH fusion proteins. As shown in Figure 5,
the TALE–MutH fusion constructs are specific for the
addressed bipartite recognition site, consisting of the
TALE target site (here we used the AvrBs4 target site)
in a defined distance next to a MutH recognition site.
We tested spacer lengths of 1, 2, 3, 4, 5, 6, 7, 8 and 9bp
between the two sites on the addressed substrate and
found that a distance of 3bp (T-3-H) is optimal
(Figure 5C) for nicking. No double-strand breaks were
Investigation of the strand specificity of TALE–MutH
To determine the strand specificity of TALE–MutH, we
have used a 211-bp PCR product that carried different
fluorophores on the 50-ends of the top and bottom
strand, respectively. Depending on which strand is prefer-
entially nicked, the electrophoretic analysis of the nicking
reaction would yield a different characteristic fluorescence
image. As shown in Figure 6, TALE–MutH nicks the
bottom strand of the substrate T-3-H with more than
two orders of magnitude higher preference over the top
strand. Intriguingly, the substrate T-6-H is nicked prefer-
entially in the top strand, albeit by a factor of ca. 3 more
slowly than the bottom strand of T-3-H. Similar experi-
ments were performed also for the other substrates: T-1-H
to T-4-H are nicked preferentially in the bottom strand,
whereas T-5-H to T-9-H are nicked preferentially in the
top strand. This change in preference for bottom strand
nicking to top strand nicking could be correlated with the
finding shown in Figure 5 that there is a decrease in the
rate of nicking between T-3-H and T-6-H. Similarly, as for
MutH–I-SceI (Figure 4), determination of strand specifi-
city was also determined for the substrates T-3-H and
T-6-H by sequencing. The results show that the bottom
strand (T-3-H) and the top strand (T-6-H), respectively,
are nicked only at the #GATC site.
Figure 2. Kinetics of DNA nicking by the MutH–I-SceI fusion con-
struct of addressed and unaddressed substrates analysed with plasmid
cleavage assays using a 4-fold excess of enzyme over substrate (16nM
enzyme, 4nM substrate). The addressed substrate plasmid contains an
I-SceI and a MutH recognition site separated by 3bp (S-3-H). The
unaddressed substrate plasmids (controls) contain either an I-SceI
recognition site without a nearby MutH recognition site (S) or no
I-SceI recognition site (H). Each of the substrates has 19 additional
GATC sites distributed over the plasmid. The position of the bands
representing supercoiled (sc), open circular (oc) and linear (lin) forms of
the plasmid is indicated. The first lane shows the reaction without
MgCl2 (‘?’). ‘Nick’ and ‘lin’ represent the controls for nicking and
cleavage activity, respectively, and correspond to the open circular
and linear forms of the plasmid, respectively. The electrophoretic
analysis shows that the addressed substrate plasmid is nicked by the
fusion construct MutH–I-SceI, whereas the unaddressed substrate
Figure 2. Continued
plasmids are not nicked or cleaved. The calculated apparent cleavage
rate constant for the addressed plasmid substrate is 4.0?10?2min?1,
whereas the apparent cleavage rate constants for both unaddressed
PAGE 5 OF 11 Nucleic AcidsResearch, 2013, Vol.41,No. 7 e83
The efficiency of homologous recombination can be
increased by several orders of magnitude by a specific
double-strand break at the locus of interest (49). The
targeted insertion of DNA into a pre-defined locus by hom-
ologous recombination requires highly specific nucleases
(50,51). Such nucleases became available with the fusion
of zinc fingers to the FokI cleavage domain (52). It was
demonstrated, however, that engineered double-strand-spe-
cific nucleases could introduce mutations when the
double-strand break is repaired by the error-prone NHEJ
pathway (53) rather than by HDR and that nicking
enzymes suppress NHEJ (14,15), which means that DNA
nicks can initiate efficient gene correction, with less
genomic instability than a targeted DNA double-strand
break. Ramirez et al. (33), Wang et al. (34) and Kim
et al. (32) introduced Zinc finger nickases (ZFNs) for
targeted gene insertion and showed that they induce
HDR with reduced mutagenic effects. As there are other
architectures for highly specific double-strand-specific nu-
cleases [e.g. (11,12), M. Yanik, unpublished data], it should
be possible to generate highly site- and strand-specific
nickases other than the ZF-based nickases. In this article,
we have shown that specificity-determining DNA-binding
modules (catalytically inactive I-SceI and the DNA-binding
Figure 3. Effect of separating the I-SceI and the MutH module on addressed substrate nicking. The kinetics of DNA-nicking by the MutH–I-SceI
fusion construct of the addressed substrate was analysed with a plasmid cleavage assay (16nM enzyme, 4nM substrate). The addressed substrate
plasmid contains an I-SceI and a MutH recognition site separated by 3bp (S-3-H). The substrate has 19 additional GATC sites distributed over the
plasmid. (A) Assay performed with MutH–I-SceI that had not been pre-incubated with TEV protease. (B) Assay performed with MutH–I-SceI that
had been pre-incubated with TEV protease. (C) For control, we have also incubated the fusion protein, in which the linker did not contain a TEV
protease cleavage site, with the TEV protease. The position of the bands representing supercoiled (sc), open circular (oc) and linear (lin) forms of the
plasmid is indicated. The first lane shows the reaction without MgCl2(‘?’). ‘Nick’ and ‘lin’ are the controls for cleavage and nicking activity and
correspond to the open circular and linear forms of the plasmid, respectively. Whereas in (A) and (C), nicking is observed, in (B), it is not.
Figure 4. Determination of the strand specificity of MutH–I-SceI by
sequence analysis of the nicked addressed plasmid after cleavage with
gel-purified and sequenced (top strand and bottom strand) over the
specific site (S-3-H). For the purpose of illustration, the sequence is
shown in the reverse and complementary orientation. The recognition
sites of I-SceI and MutH are highlighted by green and blue bars,
respectively. The sequencing results indicate that the bottom strand is
cleaved 50of the addressed GATC site. *Taq polymerase artefact.
The cleavage productwas
e83 Nucleic Acids Research, 2013,Vol. 41,No. 7PAGE 6 OF 11
domain of a TALE protein, respectively) can be fused to a
specific nicking enzyme (MutH) to produce a highly
sequence- and strand-specific nickase. The fusion proteins
that we obtained, MutH–I-SceI and TALE–MutH, recog-
nize their respectivebipartite recognitionsequence,
consisting of the recognition site of the DNA-binding
module and the MutH recognition site (GATC). They
only nick one strand 50of the GATC site and do not
nick stand-alone GATC sites or any other sites, making
them potentially useful tools for site-specific nicking of
Figure 5. Kinetics of DNA-cleavage by TALE–MutH fusion constructs of addressed and unaddressed substrates analysed with plasmid cleavage
assays using a 4-fold excess of enzyme over substrate (16nM enzyme, 4nM substrate). (A) Catalytic activity of TALE–MutH with a plasmid
substrate containing an AvrBs4 and a MutH recognition site separated by?base pairs (T-x-H; x=1–9bp). (B) Catalytic activity on the unaddressed
substrate plasmids (controls) containing either an AvrBs4 recognition site without a nearby MutH recognition site (T) or no AvrBs4 recognition site
(H). Each of the substrates has 18 additional GATC sites distributed over the plasmid. The position of the bands representing supercoiled (sc), open
circular (oc) and linear (lin) forms of the plasmid is indicated. The first lane shows the reaction without MgCl2(‘?’). ‘Nick’ and ‘lin’ represent
controls for nicking and cleavage activity, respectively, and correspond to the open circular and linear forms of the plasmid, respectively.
(C) Calculated rate constants for TALE–MutH nicking the above-mentioned substrates. The electrophoretic analysis shows that the addressed
substrate plasmid is nicked by the fusion construct TALE–MutH, whereas the unaddressed substrate plasmids remain uncleaved. The graph
shows the quantitative evaluation of the experiments shown in (A) and (B). Note that the enzyme exhibits the best activity on the addressed
substrate with the 3bp spacer and no activity with the stand-alone TALE (T) or MutH (H) recognition sites. The quantitative analyses reveal the
following apparent cleavage rate constants: 1.3?10?4min?1(T-1-H), 1.2?10?2min?1(T-2-H), 3.6?10?2min?1(T-3-H), 1.1?10?2min?1(T-4-H),
0.3?10?2min?1(T-5-H), 1.8?10?2min?1(T-6-H), 0.6?10?2min?1(T-7-H), 0.8?10?2min?1(T-8-H) and 8.9?10?4min?1(T-9-H); the nicking
rate for T and H was below the detection limit of approximately 4?10?5min?1.
PAGE 7 OF 11 Nucleic AcidsResearch, 2013, Vol.41,No. 7e83
Figure 6. Determination of the strand specificity of TALE–MutH. (A) Scheme of the reaction: the 211bp polynucleotide substrate harbours the
AvrBs4 recognition site (T) and the MutH (H) recognition site separated by 3 (T-3-H; left) or 6 (T-6-H; right) bp. The substrates are 50-labelled at
the top strand with Atto 488 (green) and with Atto 467N (red) at the bottom strand. The expected nicking products have a length of 169 (172) and
38 (35) bp for top and bottom strand nicking of the T-3-H substrate (T-6-H substrate). Which strand is attacked preferentially can be determined in
the electrophoretic analysis by the characteristic fluorescence of the products. (B) DNA nicking kinetics of the addressed polynucleotide substrates
T-3-H (left) and T-6-H (right) by TALE-MutH. The nicking products for top and bottom strand of the T-3-H (T-6-H) substrate are indicated with
169 (172) and 38 (35) bp, respectively. The first lane shows the control reaction without MgCl2(‘?’). The lane designated as ‘ctrl’ shows the result of
the cleavage of the PCR substrate by BamHI which overlaps the MutH site and is shown here to indicate the size of the nicking products. (C) The
graphs show the quantitative evaluation of the experiments shown in (B). The calculated cleavage rate constants for T-3-H (T-6-H) top strand
nicking is 6.0?10?5min?1(7.8?10?3min?1) and for bottom strand nicking 2.2?10?2min?1(1.3?10?4min?1). The analysis of the strand specificity
shows that the addressed PCR substrate with the 3bp spacer is preferentially nicked in the bottom strand by the fusion construct TALE–MutH,
whereas the substrate with the 6bp spacer is preferentially nicked in the top strand. In (D), the rate constants for nicking are shown for all substrates
investigated: T-1-H to T-9-H.
e83 Nucleic Acids Research, 2013,Vol. 41,No. 7PAGE 8 OF 11
DNA in general and precision genome engineering in
MutH is a site-specific DNA nicking enzyme which in
its natural function requires complex formation with
activated MutL to be directed to its target site, a
hemimethylated GATC site that is nicked in the
unmethylated strand. By itself and at physiological ionic
strength and Mg2+concentration (which we had deliber-
ately chosen to be prepared for applications in vivo),
MutH does not attack unmethylated GATC sites, but is
strictly dependent on a covalent coupling to a DNA-
binding module, as was shown here for MutH–I-SceI.
Proteolytic separation of the DNA-binding and DNA-
cleavage module prevents DNA cleavage. This finding
suggests that MutH cannot bind in a productive manner
to the bipartite recognition site, unless it is positioned
properly by the I-SceI (or TALE) module as part of the
We had shown before (12) that catalytically inactive
I-SceI can serve as a specific DNA-binding module for
the restriction endonuclease PvuII. Our results with
MutH–I-SceI demonstrate that this is also possible with
other nucleases, here a nicking enzyme that is structurally
related to PvuII (47). MutH–I-SceI recognizes a unique
sequence. If such a sequence is introduced into a
complex genome, this sequence could be used as a target
site for genome engineering, as it was done with I-SceI
sites for in planta gene targeting (54).
Of particular interest is the TALE-MutH fusion
protein, which in contrast to MutH–I-SceI is program-
mable to recognize almost any DNA sequence. Similar
to the MutH–I-SceI, the TALE–MutH fusion protein
requires a GATC site next to the recognition sequence
of its DNA-binding module, the TALE recognition
sequence. This requirement should not limit the usefulness
of this programmable nickase, as GATC sites occur on
average every 256bp and are unmethylated in eukaryotic
genomes. In contrast to the ZF-nickases based on FokI
(32–34), TALE–MutH only needs one TALE protein for
targeting to a specific site, which reduces the size of a
MutH-based TALE-nickase by 50% compared with a
FokI-based TALE-nickase, because MutH functions as
a monomer whereas FokI is a functional dimer. As the
DNA-binding module of TALE proteins can be used to
program the restriction endonuclease PvuII to cleave a
bipartite recognition sequence consisting of the TALE rec-
ognition sequence and the PvuII recognition sequence (M.
Yanik, unpublished data), we believe that other single-
strand-specific nucleases can function as highly specific
nickases in fusion proteins consisting of a DNA-binding
module and a DNA-nicking module. Examples are restric-
tion enzymes such as Nt.CviPII (19,20) or subunits of
heterodimeric restriction endonucleases, e.g. Nt.BstD6I
(21,22), Nb.BsrDI and Nt.BtsI (23) (Nt or Nb indicate
the specificity for top or bottom strand nicking).
enzymes, the TALE–MutH fusion protein can nick the
upper or lower strand, depending on the distance
between the TALE recognition site and the GATC
sequence. For example, with a spacer length of 2–4bp
(optimally T-3-H), the bottom strand is nicked with
several hundred-fold preference over the other strand.
In contrast, with a spacer length of 5–8bp (optimally
T-6-H), the rate of nicking decreases but now the top
strand is preferred. Substrates with a spacer length of
1 and 9bp are hardly attacked at all. This suggests that
there is some flexibility in the junction of the DNA-
binding module and the DNA-cleavage module which
allows MutH to reach the scissile phosphodiester bond
either in the bottom strand or in the top strand, depending
on the distance between the MutH recognition site relative
to the TALE recognition site. We believe that decreasing
or increasing the length of the linker between TALE and
MutH could have a similar effect, i.e. changing the pref-
erence for bottom or top strand nicking. As we have not
determined the optimal linker between AvrBs4 and MutH
experimentally, but rather extrapolated the linker length
(28 aa) in our TALE–MutH fusion protein from a previ-
ously constructed TALE–PvuII fusion construct, it could
be that the specificity could be further increased by slight
modifications in the linker length. Likewise, we have not
optimized the DNA-binding module of TALE protein but
rather used the natural AvrBs4 protein sequence; it could
well be that exchanging some of the RVDs by ‘strong’
RVDs (55) could even further increase specificity.
The authors thank Mert Yanik, Marika Midon, Ines
Fonfara, Andreas Marx and Roger Heinze for fruitful
discussions and plasmids; Laura Waltl for assistance and
Anja Drescher for critical reading of the article. L.G. is
a member of the International Research Training Group
‘Enzymes and Multienzyme Complexes acting on Nucleic
Acids’ funded by the Deutsche Forschungsgemeinschaft
(DFG): B.S. has been a recipient of a grant (Just’us) by
the Justus-Liebig-University Giessen.
Research Training Group GRK 1384, and Excellence
Giessen [Just’us]. Funding for open access charge: DFG
and the Justus-Liebig-University Giessen.
Conflict of interest statement. None declared.
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