Photocaged variants of the MunI and PvuII restriction enzymes

Article abstract of DOI:10.1021/bi2000609

Regulation of proteins by light is a new and promising strategy for the external control of biological processes. In this study, we demonstrate the ability to regulate the catalytic activity of the MunI and PvuII restriction endonucleases with light. We used two different approaches to attach a photoremovable caging compound, 2-nitrobenzyl bromide (NBB), to functionally important regions of the two enzymes. First, we covalently attached a caging molecule at the dimer interface of MunI to generate an inactive monomer. Second, we attached NBB at the DNA binding site of the single-chain variant of PvuII (scPvuII) to prevent binding and cleavage of the DNA substrate. Upon removal of the caging group by UV irradiation, nearly 50% of the catalytic activity of MunI and 80% of the catalytic activity of PvuII could be restored.(Figure Presented)

Full text of DOI:10.1021/bi2000609

ARTICLE
pubs.acs.org/biochemistry
Photocaged Variants of the MunI and PvuII Restriction Enzymes
||
Arunas Silanskas,† Michael Foss,‡ Wolfgang Wende,‡ Claus Urbanke,§ Arunas Lagunavicius,†,
Alfred Pingoud,*,‡ and Virginijus Siksnys*,†
†Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania
‡Institut f€ur Biochemie, FB08, Justus-Liebig-Universitaet Giessen, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany
§Abteilung Strukturanalyse, Medizinische Hochschule, Carl Neuberg Strasse 1, D-30625 Hannover, Germany
S Supporting Information
b
ABSTRACT: Regulation of proteins by light is a new and
promising strategy for the external control of biological pro-
cesses. In this study, we demonstrate the ability to regulate the
catalytic activity of the MunI and PvuII restriction endonu-
cleases with light. We used two different approaches to attach a
photoremovable caging compound, 2-nitrobenzyl bromide
(NBB), to functionally important regions of the two enzymes.
First, we covalently attached a caging molecule at the dimer
interface of MunI to generate an inactive monomer. Second, we
attached NBB at the DNA binding site of the single-chain
variant of PvuII (scPvuII) to prevent binding and cleavage of the
DNA substrate. Upon removal of the caging group by UV
irradiation, nearly 50% of the catalytic activity of MunI and 80% of the catalytic activity of PvuII could be restored.
he ability to regulate biological processes by an external
signal (e.g., light) seems to be very attractive and useful for
by in vitro or in vivo translation. Furthermore, different functionally
important regions (active site, substrate binding site, and oligomer-
ization interface) of the enzymes can be selected for modification to
abolish their activity. Chemical caging of the active site cysteines of
the SssI DNA (cytosine-C5)-methyltransferase with 4,5-di-
methoxy-2-nitrobenzyl bromide (DMNBB) decreases the activity
by up to 95%, while irradiation of the caged methyltransferase
restored 60% of its activity.6 The Taq polymerase was inactivated
by sterically blocking the space reserved for incoming dNTPs.7 In
this case, a tyrosine residue containing the 2-nitrobenzyl group was
introduced at position 671 using an in vivo translation system.
Irradiation with UV light regenerated the wild-type (wt) Taq
polymerase and thus restored DNA polymerization activity. When
an enzyme is active in a particular oligomeric state, there is a possi-
bility of disrupting proteinꢀprotein interaction or damaging com-
munication between different monomers by introducing a bulky
photoremovable compound (see below as an example the restric-
tion endonuclease BamHI).
T
many in vivo and in vitro applications and/or experiments. This
could be achieved by caging of biomolecules with photoremo-
vable compounds. The principle of this strategy is simple: the
caged biomolecule is inactivated, while irradiation with light
removes the cage resulting in reactivation of the biomolecule.
When the biological process one wishes to regulate is an enzymatic
reaction, each participant in this reaction (substrate, inhibitor,
cofactor, and enzyme) could be targeted for caging.1 Introduction
of a photoremovable protecting group into a substrate could
prevent its binding by the enzyme. A successful example exploiting
this strategy made use of the incorporation of 6-nitropiperonylox-
ymethyl (NPOM)-caged thymine into DNA at a restriction enz-
yme recognition site, which dramatically decreased the level of
DNA cleavage by the restriction enzymes EcoRI, BglII, and
BamHI.2 DNA cleavage was completely restored upon removal
of the caging group by UV irradiation. In the case of the Src tyrosine
kinase, bivalent peptide-derived inhibitors containing a photoclea-
vable group were synthesized.3 Inhibitors associate with the active
site of the SH1 domain and the regulatory SH2 domain blocking
Src activity; photolysis splits the inhibitors and releases the active
enzyme. Cofactors such as NADPþ and NADþ, ATP, or Mg2þ have
been caged, allowing regulation of various enzymatic reactions.4,5
Several caging strategies are available when the target is
an enzyme or protein.1 Various functional groups of a protein
(carboxylates, amines, and thiols) can be caged by chemical
modification or introduction of unnatural (caged) amino acids
Photoremovable caging compounds could be used to regulate
the activity of DNA-cutting enzymes, which could have potential
applications in gene targeting and gene therapy.8 Homing
endonucleases that recognize and cleave large (>12 bp) DNA
sequences were successfully used as scaffolds for generating
molecular scissors for a variety of genomic applications.9ꢀ12
The type II restriction endonucleases that specifically act on
Received: January 14, 2011
Published: February 25, 2011
r
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short (4ꢀ8 bp) DNA sequences could be adapted for gene
targeting purposes by fusing them to a DNA binding module
recognizing long DNA sequences. The nonspecific DNA clea-
vage domain of the type IIS restriction endonuclease FokI was
extensively used for fusion with various DNA binding modules
such as Zn-finger motifs,13ꢀ17 TALE repeats,18ꢀ20 a noncleaving
mutant of the homing endonuclease I-SceI,21 and other DNA
binding domains.22ꢀ27 Triplex-forming oligonucleotides may
serve as DNA binding modules as demonstrated for the PvuII
restriction endonuclease.28 However, despite the advances made
in the construction of specific DNA scissors, cleavage of off-
targets remains a major limitation for their application in gene
therapy (in vivo),16,17,29 which could be minimized by control-
ling the activity of the nucleases. For example, formation of the
DNA triple helix is quite slow; therefore, the active enzyme in the
restriction enzymeꢀtriple-helix-forming oligonucleotide fusion
protein could start to cleave nonspecific DNA target sites that are
not addressed by triple helix formation. Caging with a photo-
removable compound could help to regulate activity of these
designed enzymes and prevent possible nonspecific activity.
To date, most of the work on caging of DNA-cleaving enzymes
with photosensitive compounds was conducted with the type IIP
restriction endonuclease BamHI. In the case of BamHI, which is
active as a dimer, the dimerization interface was chosen as the
target for caging to control the enzyme activity.30ꢀ32 Unnatural
amino acids, a lysine derivative with a photoremovable com-
pound (6-nitroveratryloxycarbonyl) and alanine derivatives such
as azophenylalanine or 40-carboxyphenylazophenylalanine,
which undergo cisꢀtrans isomerization upon illumination, were
incorporated at the dimer interface of BamHI via substitution of
amino acid Lys132 using an in vitro translation system with
special codons and the corresponding tRNA.30ꢀ32 These light
sensitive compounds destroy the intricate salt bridge network
at the dimer interface, thereby inactivating the enzyme. Irradia-
tion of the caged enzymes with UV light restores their catalytic
activity.
pRIZ0-scPvuII(S81C)2 was used to transform E. coli XL10-Gold
cells, which contain the pLGM plasmid (Cmr) encoding the
PvuII methyltransferase.
Protein Expression and Purification. ER2267 cells contain-
ing the pUHE25-2-MunIR plasmid were grown in LB medium
containing 50 mg/L Ap and 30 mg/L Cm at 37 °C. After being
induced with 0.2 mM IPTG for 4 h, cells were harvested by
centrifugation.
MunI was purified according to the method described in ref 36
and stored at ꢀ20 °C in storage buffer [10 mM Tris-HCl (pH 7.4
at 25 °C), 100 mM KCl, 1 mM EDTA, 1 mM DTT, and 50%
(v/v) glycerol]. The enzyme was >95% homogeneous as shown
by SDSꢀPAGE analysis. The concentration of protein was
determined spectrophotometrically at 280 nm using an extinc-
tion coefficient of 45720 Mꢀ1 cmꢀ1
.
scPvuII(S81C)2 was expressed in E. coli XL10-Gold cells,
purified as described previously,37 and finally stored at ꢀ20 °C
in 20 mM sodium phosphate (pH 7.4), 300 mM NaCl, 0.5 mM
EDTA, 1 mM DTT, 0.01% (w/v) Lubrol, and 50% (v/v)
glycerol. The enzyme was >95% homogeneous as shown by
SDSꢀPAGE analysis. The concentration of protein was deter-
mined spectrophotometrically at 280 nm using an extinction
coefficient of 71240 Mꢀ1 cmꢀ1
.
GdmCl-Induced Protein Unfolding of MunI. We conducted
protein denaturation experiments by mixing 1 μM (for fluores-
cence and AUC analysis) or 8 μM (for CD analysis) MunI with
various concentrations of GdmCl in 10 mM Tris-HCl (pH 7.5 at
25 °C), 200 mM KCl, and 1 mM DTT. Samples were incubated
overnight at 25 °C.
Fluorescence spectra were recorded at 25 °C in the thermo-
statically controlled cell holder of a Perkin-Elmer LS 50 lumines-
cence spectrophotometer. The excitation wavelength was
295 nm, and the emission was recorded from 310 to 380 nm
using a 5 nm bandwidth. Differences in fluorescence intensity
between native and denatured protein were measured at a fixed
emission wavelength of 335 nm. Data reported here represent
the average of three spectra.
CD spectra were recorded at 25 °C in the thermostatically
controlled cell holder of a Jasco J-710 spectropolarimeter, using a
cylindrical cuvette with a light path of 0.05 cm. CD spectra were
recorded between 185 and 250 nm. To monitor the denaturation
of MunI, 10 readings of the CD signal at 220 nm were taken (with
a 20 s averaging time) and the data averaged.
Analytical Ultracentrifugation of MunI. GdmCl-induced
protein denaturation was monitored with an analytical ultracen-
trifuge by determining apparent molar masses from sedimenta-
tion equilibrium experiments. Samples contained 5.4 μM MunI,
10 mM Tris-HCl (pH 7.4 at 25 °C), 200 mM KCl, and different
concentrations of GdmCl. A Beckman XLA analytical ultracen-
trifuge equipped with UV absorption optics scanning the samples
at 280 nm, an eight-hole analytical rotor, and six-channel charcoal
filled Epon centerpieces was used. Samples were spun at
16000 rpm and 20 °C and scanned continuously until no change
in the concentration profile could be detected over a period of
12 h. Scans collected over these 12 h were averaged and evaluated
assuming a single sedimenting species, yielding apparent molar
masses.38
Here we demonstrate activity regulation of two restriction
endonucleases, MunI and PvuII, using near-UV light. We used
two different strategies. We chemically introduced a photoremo-
vable cage compound, 2-nitrobenzyl bromide (NBB), at the
dimer interface of MunI, converting the nuclease into an inactive
monomer form, or at the DNA binding site of PvuII, preventing
substrate binding and cleavage. Irradiation of the caged restric-
tion endonucleases with near-UV light restores up to 50% of
MunI's cleavage ability and 80% of PvuII's cleavage ability as
compared with those of the uncaged enzymes.
’ MATERIALS AND METHODS
Strains and Plasmids. MunI. Plasmid pUHE25-2-MunIR
(Apr) containing the restriction endonuclease gene munIR
was used for protein expression in Escherichia coli ER2267 cells
containing a compatible plasmid pMunIM 6.2 (Tcr, Cmr)
harboring the methyltransferase gene munIM.33
PvuII. We have chosen the single-chain variant of the naturally
homodimeric restriction enzyme PvuII (scPvuII) for our study.34
The plasmid encoding the C-terminal His6-tagged variant
scPvuII(S81C)2, harboring a cysteine residue at position 81 in
both “halves” of the pseudodimer, was generated using plasmid
pRIZ0-scPvuII (Apr)34 as a template and employing a PCR-based
site-directed mutagenesis method.35 The sequence of the mu-
tated gene was confirmed by sequencing. For protein expression,
Caging of MunI and PvuII. Before being caged, the MunI
protein was dialyzed against 10 mM sodium phosphate (pH 7.4
at 25 °C) and 0.1 M NaCl. Caging of MunI was performed
by overnight incubation of 60 μM protein (monomer con-
centration) with 500 μM 2-nitrobenzyl bromide (NBB) in the
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presence of 1 M GdmCl at 4 °C. After the reaction, unreacted
NBB was quenched with 1 mM DTT. The reaction mixture was
dialyzed against 10 mM sodium phosphate (pH 7.4 at 25 °C) and
0.1 M NaCl. The caged MunI was purified by gel filtration using a
Superdex 200 HR column (Amersham Biosciences) equilibrated
with 20 mM Tris-HCl (pH 7.5 at 25 °C) and 0.2 M KCl.
Fractions containing caged MunI were pooled, dialyzed against
10 mM Tris-HCl (pH 7.4 at 25 °C), 100 mM KCl, 1 mM EDTA,
and 50% (v/v) glycerol, and stored at ꢀ20 °C.
Decaging of MunI and PvuII. Decaging of caged MunI (4 μM
monomer) was performed by irradiation with UV light of
different wavelengths for 1 h in 100 mM sodium acetate (pH
5.8 at 25 °C), 0.2 M KCl, and 20 mM DTT at 4 °C. The
Fluoromax-3 (Jobin Yvon, Stanmore, U.K.) spectrofluorimeter
equipped with a Xe lamp was used as the UV source. After
irradiation, the protein sample was analyzed by gel filtration with
a Superdex 200 HR column (Amersham Biosciences) equili-
brated with 20 mM Tris-HCl (pH 7.5 at 25 °C) and 0.2 M KCl.
The best efficiency of decaging was obtained by irradiation with a
wavelength of 330 nm (29 nm bandwidth).
Decaging of caged scPvuII(S81C)2 (1 μM) was performed in
20 mM sodium phosphate (pH 7.4 at 25 °C), 100 mM NaCl,
0.5 mM EDTA, and 9 mM DTT by irradiation at 325 nm using a
40 mW heliumꢀcadmium laser (Laser 2000). After different
time intervals, aliquots were withdrawn and tested for PvuII
activity.
DNA Cleavage Activity. DNA cleavage activity of MunI
was tested using bacteriophage λ DNA as a substrate. Protein
(400 nM dimer concentration) was incubated in 50 μL with 1 μg of
λ DNA in 10 mM Tris-HCl (pH 7.0 at 25 °C), 10 mM MgCl2,
50 mM NaCl, and 0.1 mg/mL BSA for 15 min at 37 °C. The
reaction was quenched with a loading dye solution containing
50 mM EDTA (pH 8.0 at 25 °C), 0.1% SDS, 50% (v/v) glycerol,
and 0.01% bromophenol blue and analyzed by agarose gel
electrophoresis.
The scPvuII(S81C)2 protein was dialyzed against 20 mM
sodium phosphate (pH 7.4 at 25 °C), 100 mM NaCl, and
0.5 mM EDTA to remove DTT from the storage buffer. Caging
of scPvuII(S81C)2 was performed by incubation of 10 μM
protein with 900 μM NBB for 2 h at 25 °C in 20 mM Tris-
HCl (pH 8.5 at 25 °C), 100 mM NaCl, and 0.5 mM EDTA. The
caging reaction was stopped by the addition of 9 mM DTT.
Finally, a Vivaspin 500 spin column (Satorius, Goettingen,
Germany) was used to remove unreacted NBB and to change
the buffer to 20 mM sodium phosphate (pH 7.4 at 25 °C),
100 mM NaCl, 0.5 mM EDTA, and 1 mM DTT.
The pUCGK-4 plasmid (provided by G. Kruckas) containing
a single copy of the MunI recognition sequence was used in DNA
cleavage experiments. DNA cleavage was performed by incuba-
tion of 0.75 nM wt MunI and 1.5 nM caged and decaged MunI
with 1.5 nM plasmid DNA in reaction buffer at 25 °C.
To analyze the PvuII cleavage activity, we used plasmid pAT-
PEB, a derivative of pAT153 with one PvuII site, as a substrate.
The efficiency of the caging reaction was tested by determining
the DNA cleavage activity using an excess of caged scPvuII-
(S81C)2 (20 nM) over plasmid DNA (9 nM) in 10 mM Tris-
HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl, and 0.1 mg/mL
Figure 1. Dimer interface of MunI. (A) Crystal structure of the MunI
restriction endonuclease. Ribbon representation of the two monomers
colored blue and red, respectively. Helices involved in protein dimeriza-
tion are shown in more intense colors. The region magnified in panel B is
circled. (B) Close-up of a region of the MunI dimerization interface
showing the cysteines residues (C123) subjected to caging.
Figure 2. Generation of photoswitchable restriction endonucleases. (A) Caging of the enzyme with 2-nitrobenzyl bromide (NBB) and reactivation by
irradiation with UV light. (B) Generation of photoswitchable MunI. A cysteine residue located at the dimer interface of wt MunI (residue 123) is
modified using NBB. The resulting caged enzyme is an inactive monomer that is unable to bind to DNA. Irradiation at 330 nm leads to the release of the
caging group and formation of the active MunI dimer. (C) Generation of the photoswitchable scPvuII. A single cysteine residue in each “half” of scPvuII
located at the enzymeꢀDNA interface (residue 81) is modified using NBB. The resulting caged enzyme is not able to bind to scPvuII recognition sites.
Illumination at 325 nm leads to the release of the caging group and reactivation of scPvuII.
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Figure 3. Unfolding of MunI induced by GdmCl. Unfolding was monitored by (A) fluorescence emission at 335 nm (λexc = 295 nm), (B) far-UV CD at
222 nm, and (C) analytical ultracentrifugation. (D) Schematic representation of the MunI denaturation pathway in the presence of GdmCl. The
monomeric state of MunI at 1 M GdmCl allows caging of native cysteines at the dimer interface.
BSA at 37 °C. The recovery of activity during the decaging of
caged scPvuII(S81C)2 was analyzed with 2 nM enzyme and 6 nM
plasmid substrate.
Samples were collected at given time intervals, and reactions
were quenched with the loading dye solution and analyzed by
agarose gel electrophoresis. The amounts of supercoiled (SC),
open-circular (OC), and linear (FLL) DNA forms were evalu-
ated by densitometric analysis of ethidium bromide-stained gels.
the protein after irradiation leaving unmodified cysteine residues
(Figure 2A).
A different strategy was used to cage the PvuII restriction
endonuclease. On the basis of the structure of PvuII bound to its
specific substrate,41 we selected different positions in the DNA
binding site of the protein, which presumably prevent DNA
binding if modified with a bulky photoremovable caging group
(Figure 2C). To modify the protein at the selected positions,
we produced single-cysteine variants using the single-chain
variant of the PvuII enzyme (scPvuII).34 scPvuII was chosen
because this variant had been used before for the production
of the scPvuIIꢀtriple-helix-forming oligonucleotide fusion
protein.28 For the modification studies, we selected the S81C
scPvuII variant with the Ser f Cys substitution in both halves of
the pseudodimer; scPvuII(S81C)2 shows only slightly reduced
DNA cleavage activity compared to that of scPvuII (data not
shown), which in turn has a 3-fold lower activity (kcat/Km) than wt
PvuII.34
Accessibility of Cys123 at the Dimer Interface of MunI.
Titration of MunI with 5,50-dithiobis(2-nitrobenzoic acid)
(DTNB)42 showed that the native cysteines are not accessible
at the dimer interface (Figure 1 of the Supporting Information).
One way to make cysteines accessible for chemical modification
is to induce dissociation of the MunI dimer into monomers using
denaturing agents. Therefore, we investigated unfolding of MunI
by guanidinium chloride (GdmCl).
’ RESULTS AND DISCUSSION
Strategy. To generate photoswitchable restriction enzymes,
we selected the MunI and PvuII restriction endonucleases as
model systems. Both enzymes are well-characterized both bio-
chemically and structurally. MunI recognizes the sequence 50-C/
AATTG-30 and cuts the phosphodiester bond after the C (as
indicated by the slash) to generate a double-strand break.39 Two
MunI monomers dimerize via a bundle of R-helices forming a
functional dimer (Figure 1A) that symmetrically binds and
cleaves the C/AATTG target site in both DNA strands.40 The
MunI dimer contains two cysteine residues at position 123 (one
per monomer) at the dimer interface (Figure 1B). These
cysteines are located in the dimerization helices close to each
other (Figure 1B). One can assume that chemical modification of
the Cys123 residues with a bulky photoremovable cage would
disrupt the functionally important dimer via formation of inactive
monomers (Figure 2A,B). Reactivation of the caged protein
could be performed with near-UV light. Irradiation of the caged
MunI removes the cage from the dimerization interface, allowing
association of the monomers into the active dimer. For caging,
we used 2-nitrobenzyl bromide (NBB), which dissociates from
MunI unfolding induced with increasing GdmCl concentra-
tions was monitored by the intrinsic protein fluorescence and
circular dichroism (CD) and by performing analytical ultracen-
trifugation (AUC) experiments (Figure 3). Intrinsic MunI
fluorescence was measured at a wavelength of 335 nm, where
the difference is largest between the native and fully denatured
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Figure 5. (A) Analysis of the cleavage activity of NBB-treated and
untreated scPvuII. Experiments were performed using an excess of
enzyme (20 nM) over plasmid DNA (9 nM). Samples after 5, 15, and 45
min were analyzed. (B) Decaging reaction of caged scPvuII. The plasmid
cleavage activity of the caged scPvuII was analyzed after irradiation with
with 325 nm light for different times (0ꢀ1280 s). The cleavage reaction
was performed for 30 min with 2 nM enzyme and 6 nM plasmid
substrate: uncleaved plasmid (C), untreated PvuII (PvuII), supercoiled
DNA (SC), open-circular DNA (OC), and full-length linear DNA
(FLL).
fluorescence between 1.5 and 2.5 M GdmCl corresponds to MunI
unfolding (Figure 3A,B). AUC data showed that the molecular mass
of MunI decreased from 51 to 26 kDa between 0 and 1 M GdmCl,
indicating dissociation of the dimer into monomers (Figure 3C).
Thus, the first stage of the fluorescence decrease in Figure 2A
correlates with dissociation of the MunI dimer. Taken together, our
data indicate that the MunI dimer first dissociates into native
monomers followed by monomer unfolding with increasing GdmCl
concentrations (Figure 3D). Additional studies revealed that the
GdmCl-induced denaturation of MunI is completely reversible and
that the refolded MunI has the same catalytic and DNA binding
properties as the native protein (data not shown).
In the case of an oligomeric protein, the dissociation into
monomers might occur prior to unfolding of the polypeptide
chain,43 or both processes can occur concertedly.44 GdmCl-
induced denaturation studies show that MunI first dissociates into
monomers followed by monomer unfolding at increased GdmCl
concentrations. A similar denaturation pathway has been reported
for the tetrameric restriction endonuclease Bse634I.45 On the other
hand, dissociation and unfolding of the dimeric restriction endo-
nucleases BfiI and PvuII occur simultaneously.46,47
According to the denaturation pathway of MunI (Figure 3D),
the Cys123 residues located at the dimer interface should be
accessible for chemical modification in the presence of 1.0ꢀ1.5 M
GdmCl when the MunI dimer is dissociated into monomers.
Indeed, titration of MunI with DTNB in the presence of 1.5 M
GdmCl showed that the Cys123 residues are accessible and
reactive (Figure 1 of the Supporting Information), allowing their
caging with photochemical reagents.
Figure 4. Caging of MunI with 2-nitrobenzyl bromide (NBB) (A) and
decaging with UV light (B) monitored by gel filtration. (A) Caging of
MunI with NBB at the dimer interface generates a monomer in solution.
The solid line indicates reaction products after caging of MunI; the
dashed line indicates the profile of wt MunI. (B) Irradiation of caged
monomeric MunI with UV light promotes formation of the dimer. The
solid line indicates reaction products after decaging; the dashed line
indicates the profile of wt MunI. (C) Cleavage of λ DNA with caged
and decaged MunI: lane 1, substrate DNA only; lane 2, cleavage with
wt MunI; lane 3, cleavage with caged MunI; lane 4, cleavage with
decaged MunI.
forms of the protein. The fluorescence of MunI decreases in two
discrete stages: the first between 0 and 1 M GdmCl and the
second between 1.5 and 2.5 M GdmCl (Figure 3A). Far-UV CD
spectra at 222 nm showed that the molar ellipticity of MunI
changed in the range from 1.5 to 3 M GdmCl (Figure 3B). The
changes in the CD signal indicate loss of secondary structures by
the protein accompanied by its unfolding. Therefore, the decrease in
Caging of MunI and PvuII with NBB. Caging of MunI was
performed by overnight incubation with 2-nitrobenzyl bromide
(NBB) in the presence of 1 M GdmCl at 4 °C. Reaction products
were dialyzed to remove GdmCl and analyzed by gel filtration
(Figure 4A). In contrast to wt MunI, which is a dimer in solution
(∼46 kDa), the caged MunI mainly elutes as a monomer with
a molecular mass of ∼26 kDa (Figure 4A and Figure 2 of the
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Supporting Information). After caging, only a small fraction
(∼10%) of MunI remains dimeric, presumably because of
incomplete chemical modification (Figure 4A). Altogether,
gel filtration analysis showed that the caging efficiency of MunI
with NBB is high, yielding ∼90% of the caged monomer.
Monomeric MunI showed no cleavage activity with phage λ
DNA as the substrate (Figure 4C).
Caging of scPvuII(S81C)2 was performed under native con-
ditions using a 90-fold excess of 2-nitrobenzyl bromide over
protein at 25 °C for 2 h. The reaction was stopped by the addition
of DTT. After the unreacted NBB had been removed, the
efficiency of the caging reaction was verified by a DNA cleavage
assay (Figure 5A). The inactivity of the caged scPvuII prepara-
tion suggests that the yield of caged protein was close to 100%.
Decaging of Caged MunI and PvuII. Decaging of caged
MunI was performed by irradiation with near-UV light using
different wavelengths (312ꢀ350 nm) for 1 h at 4 °C. The deca-
ging efficiency was determined by gel filtration by calculating the
fraction of the MunI dimer formed after irradiation (Figure 4B).
Irradiation at 330 nm yielded the largest amount (∼70%) of
MunI dimer (Figure 3 of the Supporting Information). Decaged
MunI showed the same λ DNA cleavage pattern (Figure 4C) as
the wt enzyme.
The photoreactivation of caged scPvuII was performed by
irradiation at 325 nm using a heliumꢀcadmium laser at 4 °C. The
efficiency of the decaging reaction was analyzed after defined
illumination times by a DNA cleavage assay (Figure 5B).
DNA Cleavage Studies. To determine more precisely the
catalytic activity of caged and decaged MunI, we performed
DNA cleavage experiments using supercoiled plasmid DNA that
contains a single copy of the MunI recognition sequence. At
equimolar concentrations of protein and DNA, caged MunI
exhibited no catalytic activity, not even nicking (Figure 6B).
However, in the case of decaged MunI, the profile of plasmid
DNA cleavage was similar to that of wt MunI with the exception
that a ∼2-fold higher concentration of the decaged protein had to
be used (Figure 6A,C). This result indicates that photoreactiva-
tion of MunI reaches ∼50%.
Kinetic experiments together with gel filtration data show that
the chemical modification of the cysteine residues in MunI by
NBB at the dimer interface inactivates the enzyme, prevent-
ing association of the caged monomers into the active dimer
(Figures 4A and 6B). Electrophoretic mobility shift assay
(EMSA) experiments demonstrated that in contrast to the wt
enzyme the caged monomeric MunI is unable to bind a cognate
DNA (Figure 4 of the Supporting Information).
The analysis of the caging reaction of scPvuII(S81C)2 was
performed using an excess of caged enzyme over supercoiled
plasmid DNA to detect traces of remaining activity. No DNA
cleavage activity was detected for the caged scPvuII(S81C)2
preparation, whereas the enzyme incubated without 2-nitroben-
zyl bromide exhibited full activity (Figure 5A). To test if the
inactivity of caged scPvuII is due to a weakened DNA binding
ability, the DNA binding of caged scPvuII(S81C)2 and scPvuII
was analyzed by fluorescence anisotropy. The results (Figure 5 of
the Supporting Information) indicate that introducing the bulky
NBB moiety at the DNA binding site at position 81 prevents specific
DNA binding. The time course of the photoreactivation of caged
scPvuII(S81C)2 showed that after of irradiation at 325 nm for 1 min
50% and after 20 min 80% of the scPvuII activity is obtained as
compared to that of the uncaged enzyme (Figure 5B).
Figure 6. Photoactivation of MunI. (AꢀC) Cleavage of the plasmid
DNA containing a single MunI recognition site by wt, caged, and
decaged MunI, respectively. Kinetic experiments were performed by
using 0.75 nM wt MunI and 1.5 nM caged and decaged MunI with 1.5
nM plasmid DNA. The following forms of DNA were observed during
the reaction: supercoiled DNA (SC), open-circular DNA (OC), and full-
length linear DNA (FLL).
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ARTICLE
’ CONCLUSIONS
Funding Sources
This work was supported by European Union Grant FP6-2006-
15509-NEST.
Two strategies for controlling the activity of restriction
enzymes by caging were presented: (1) preventing formation
of the active dimer in the case of MunI and (II) preventing DNA
binding in the case of PvuII.
(1) Selective caging of cysteine residues with NBB at the
dimer interface of MunI generated inactive monomers. The yield
of the caged protein obtained was ∼90%. Activation of the caged
enzyme was performed with near-UV light irradiation, resulting
in removal of the cage compound and formation of the active
MunI dimer. A decaging efficiency of up to 70% and regeneration
of cleavage activity of up to 50% were achieved under our experi-
mental conditions.
(2) Similar results were obtained by blocking the DNA binding
site of PvuII. Two cysteine residues were introduced at the DNA
binding site, which did not affect the DNA cleavage activity of PvuII.
Modification of these cysteine residues with NBB resulted in a DNA
binding deficient endonuclease. Photoreactivation of the caged
PvuII by near-UV light restored the DNA binding and consequently
the DNA cleavage activity up to 80%.
’ ACKNOWLEDGMENT
We thank Dr. Mindaugas Zaremba for suggestions and
discussions.
’ ABBREVIATIONS
AUC, analytical ultracentrifugation; CD, circular dichroism;
DMNBB, 4,5-dimethoxy-2-nitrobenzyl bromide; DTT, 1,4-
dithiothreitol; EDTA, ethylenedinitrilotetraacetic acid; EMSA,
electrophoretic mobility shift assay; FLL, linear (DNA); GdmCl,
guanidinium monochloride; λexc, excitation wavelength; NBB,
2-nitrobenzyl bromide;OC, open-circular (DNA); PCR, polymer-
ase chain reaction; PAGE, polyacrylamide gel electrophoresis; SC,
supercoiled (DNA); scPvuII, single-chain PvuII; SDS, sodium do-
decyl sulfate; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol;
wt, wild type.
The strategies used in this work could be used to regulate
activity of other enzymes. Native cysteine residues or cysteine
residues introduced by mutagenesis at the oligomerization inter-
face or at the substrate binding site could be selectively caged
with NBB, resulting in an inactive enzyme, whose catalytic
activity could be restored by irradiation with near-UV light.
The procedures that we have developed are sufficiently general
that they can be used for many other enzymes, for which it is
desirable to turn them “on” by light. This is the case, for example,
for so-called meganucleases that in addition to a cleavage module
have a specific DNA binding module. After delivery to the cell,
they can be turned “on” after the DNA binding module has
located the target site, thus preventing premature DNA cleavage
by the cleavage module.
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S
Supporting Information. Graph representing accessibil-
b
ity of cysteine residues at the MunI dimer interface to DTNB
reagent (Figure S1), calibration curve for determination of the
molecular mass of wt and caged MunI (Figure S2), bar diagram
showing the efficiency of MunI decaging at different wavelengths
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’ AUTHOR INFORMATION
Corresponding Author
*A.P.: Justus-Liebig-Universitaet Giessen, Institut f€ur Biochemie,
FB08, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany; tele-
phone, þ49-641-9935400; fax, þ49-641-9935400; e-mail, alfred.
m.pingoud@chemie.bio.uni-giessen.de. V.S.: Institute of Bio-
technology, Vilnius University, Graiciuno 8, Vilnius LT-02241,
Lithuania; telephone, þ370-5-2602108; fax, þ370-5-2602116;
e-mail, siksnys@ibt.lt.
Present Addresses
Thermo Fisher Scientific (formerly Fermentas), Graiciuno 8,
LT-02241 Vilnius, Lithuania.
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