 |
INTRODUCTION |
DNA methyltransferases
(Mtases)1 are a biologically
important class of enzymes that catalyze the transfer of the activated methyl group from the cofactor
S-adenosyl-L-methionine (AdoMet) to the N-6
nitrogen of adenine and the N-4 nitrogen of cytosine or the C-5 carbon
of cytosine within their DNA recognition sequences (1). As DNA
methylation is a postreplicative process that depends on the presence
or regulation of DNA Mtases, a particular DNA sequence may exist in its
fully methylated, its unmethylated, or transiently in its
hemimethylated form. Thus, DNA methylation can be regarded as an
increase of the information content of DNA (2), which serves a wide
variety of biological functions, including protection of the host
genome from endogenous restriction endonucleases, DNA mismatch repair
after replication, regulation of gene expression and DNA replication,
embryonic development, genomic imprinting, and X-chromosome
inactivation (3-5). Furthermore, it plays a role in carcinogenesis
(6).
Three-dimensional structures are available for the
C5-cytosine DNA Mtases M.HhaI (7) and
M.HaeIII (8) in complex with DNA. Both enzymes consist of
two domains forming a positively charged cleft, which accommodates the
DNA. However, the most striking feature of these protein-DNA complexes
is that the target cytosines are completely rotated out of the DNA
double helix and placed in a pocket within the cofactor binding
domains, where catalysis takes place. In addition, the structures of
the N6-adenine DNA Mtases M.TaqI (9)
and the N4-cytosine DNA Mtase M.PvuII
(10) in the absence of DNA were reported. These
N6-adenine and
N4-cytosine DNA Mtases (N-DNA Mtases) also have
a bilobal structure forming a positively charged cleft. Modeling B-DNA
in this cleft showed that the distance between the target base and the
bound AdoMet is too large for a direct methyl group transfer, and a base flipping mechanism as observed for the
C5-cytosine DNA Mtases was postulated.
Biochemical evidence for a base flipping mechanism of the
N6-adenine DNA Mtases M.EcoRI (11)
and M.TaqI (12) was obtained using duplex
oligodeoxyribonucleotides (ODNs) containing the fluorescent base
analogue 2-aminopurine at the target positions. In addition, tighter
binding of the N6-adenine DNA Mtases
M.EcoRV (13) and M.EcoRI (14) to duplex ODNs
carrying base analogues with reduced Watson-Crick hydrogen bonding
potential at the target positions was observed and attributed to a
reduced energetic cost to flip out the target base. Furthermore, a
thymine residue placed at the target position within a duplex ODN
showed an enhanced reactivity toward potassium permanganate oxidation
in the presence of M.TaqI, which was interpreted by a higher
accessibility of the thymine residue in the binary complex due to base
flipping (15). A structural comparison of M.TaqI with
M.HhaI showed that the cofactor binding domains of both
enzymes have a very similar fold (16). In addition, the cofactors and the motifs IV ((N/D/S)PP(Y/F/W)-motif of N-DNA Mtases and PCQ-motif of
C5-cytosine DNA Mtases) as well as the motifs
VIII (F/Y/W-motif in N-DNA Mtases and QXRXR-motif
in C5-cytosine DNA Mtases) overlay very well.
Thus, it was suggested that the extrahelical target adenine in
M.TaqI is located in a similar position as the extrahelical
cytosine in M.HhaI and that Tyr-108 (motif IV) and Phe-196
(motif VIII) in M.TaqI are responsible for proper
orientation of the extrahelical adenine.
To identify the binding site of the extrahelical target base in
N6-adenine DNA Mtases, we used ultraviolet
light-induced photochemical cross-linking, which is a powerful
technique to define specific contact points in nucleoprotein complexes,
where direct structural information is not available (for a review, see
Ref. 17). Non-substituted nucleic acids or nucleic acids containing
4-thiouracil, azido-substituted nucleobases, and halogenated
pyrimidines like 5-bromouracil, 5-iodouracil, or 5-iodocytosine are
typically used in the photo-cross-linking reaction. In contrast to
chemical cross-linking, which interposes spacers of varying length
between the nucleic acid and the protein, photo-cross-linking produces
zero-length cross-links. This allows the identification of contact
points in nucleoprotein complexes at high resolution. Although thymine
in non-substituted nucleic acids can be photo-cross-linked directly at
253 nm, the yields are generally low, which is most likely due to
photodegradation of the proteins and nucleic acids (18). This problem
can be minimized by incorporating photo-activable analogues into
nucleic acids, which can be excited above 300 nm. An additional
advantage of using substituted nucleic acids is the fact that the site
of modification within the nucleic acid sequence is already defined. The highest photo-cross-linking yields for different nucleoprotein complexes were obtained with nucleic acids containing 5-iodouracil, and
yields of up to 95% were reported (19-22).
In this publication we show that the N6-adenine
DNA Mtases M.TaqI from Thermus aquaticus (23, 24)
and M.CviBIII from the Chlorella virus NC-1A
(25), which both methylate adenine within the double-stranded
5'-TCGA-3' DNA sequence, give high photo-cross-linking yields with
duplex ODNs containing 5-iodouracil at the target position (Scheme
1). Using a combination of Edman
degradation and electrospray ionization mass spectrometry (ESI-MS) of a
nucleopeptide obtained by proteolytic fragmentation of the covalent
M.CviBIII-DNA complex we demonstrate that Tyr-122 (motif IV)
is modified in the photo-cross-linking reaction. In addition, results
from photo-cross-linking experiments with the mutant Mtases
M.TaqI/Y108A (Tyr in motif IV replaced by Ala) and
M.TaqI/F196A (Phe in motif VIII replaced by Ala) are
discussed.

View larger version (12K):
[in this window]
[in a new window]
|
Scheme 1.
Photo-cross-linking reaction of the
N6-adenine DNA Mtase
M.TaqI and its isoschizomer
M.CviBIII with duplex ODNs containing
5-iodouracil at the target position.
|
|
 |
EXPERIMENTAL PROCEDURES |
Oligodeoxyribonucleotides--
ODNs were synthesized on an
Applied Biosystems 392 DNA/RNA synthesizer or purchased from MWG
Biotech (Ebersfeld, Germany). The phosphoramidite for
5-iodo-2'-deoxyuridine was purchased from Glen Research. Due to the
base lability of 5-iodo-2'-deoxyuridine (26), the syntheses were
performed with Expedite phosphoramidites (Perseptive Biosystems)
containing the more labile tert-butylphenoxyacetyl protection group and allowing deprotection with concentrated ammonia at
room temperature for 2 h. Purification, evaluation, and
hybridization of ODNs were performed as described before (12).
Sequences of synthesized ODNs are listed in Table I.
DNA Methyltransferases--
M.TaqI and the mutant
Mtases M.TaqI/Y108A and M.TaqI/F196A were
prepared as described earlier (12, 27). M.CviBIII was expressed in DS1312 Escherichia coli cells harboring
pNC1A.14-lac (25), which carries the gene for M.CviBIII
under the inducible lac promotor. The expression system was
kindly provided by Dr. Waltraud Ankenbauer, Roche Molecular
Biochemicals. Cells were grown at 37 °C to an optical density of 0.6 at 600 nm and induced with
isopropyl-1-thio-
-D-galactopyranoside (0.1 mM final concentration) at 30 °C for 4 h. Cells
were harvested by centrifugation (15 min at 4,000 × g), resuspended in buffer A (20 mM Tris
hydrochloride, pH 7.6, 10 mM
-mercaptoethanol, 20 mg/liter phenylmethylsulfonyl fluoride, and 5% glycerol) supplemented
with potassium chloride (500 mM) and lysed by sonification.
After centrifugation (1 h at 30,000 × g), the
supernatant was diluted with buffer A to give a potassium chloride
concentration of 170 mM and loaded onto an anion-exchange
column (DEAE-Sepharose FF, Amersham Pharmacia Biotech). M.CviBIII was eluted with buffer A containing potassium
chloride (170 mM). The resulting protein solution was
diluted with buffer A to yield a potassium chloride concentration of
100 mM and loaded onto a heparin column (heparin-Sepharose
CL 6B, Amersham Pharmacia Biotech). A potassium chloride gradient (0.1 to 1.0 M) in buffer A was applied and M.CviBIII
eluted at a potassium chloride concentration of 200-250
mM. Fractions containing M.CviBIII were combined
and concentrated by ultrafiltration (Centriprep 30, Amicon). The
concentrated protein solution was loaded onto a gel filtration column
(Superdex 200, Amersham Pharmacia Biotech) and eluted with a Tris
hydrochloride buffer (20 mM, pH 7.4) containing potassium
chloride (600 mM), EDTA (0.2 mM), and DTT (2 mM). Fractions containing M.CviBIII were pooled,
2-fold diluted with glycerol, and stored at
20 °C. M.CviBIII was at least 90% pure, as judged by
polyacrylamide gel electrophoresis in the presence of sodium dodecyl
sulfate and staining with Coomassie Blue. Protein concentrations were
estimated by the method of Bradford (28) (Coomassie Protein Assay
Reagent, Pierce) using bovine serum albumin as standard.
Ultraviolet Light-induced Photochemical
Cross-linking--
Typically, photo-cross-linking reactions were
performed with a mixture (0.1 ml for analytical and 1 ml for
preparative scale) of duplex ODNs (1.2-1.5 µM) and DNA
Mtases (2.0-3.0 µM) in Tris acetate buffer (20 mM, pH 7.9) containing potassium acetate (50 mM), magnesium acetate (10 mM) and DTT (1 mM). Photo-cross-linking was induced with a HeCd laser
(Liconox, Santa Clara, CA) operating at 325 nm with an intensity of 25 miliwatts/mm2. Samples were irradiated in QS quartz
cuvettes (Helma, Mülheim, Germany) at 4 °C for 2 h or
indicated times. Photo-cross-linking reactions were either analyzed by
SDS-polyacrylamide gel electrophoresis or anion-exchange
chromatography. The long ODNs (36 nucleotides) were
32P-labeled by treatment with T4 polynucleotide kinase (New
England Biolabs) and [
-32P]ATP (110 TBq/mmol, 0.4 MBq/µl, Hartmann Analytik, Braunschweig, Germany) in Tris
hydrochloride buffer (70 mM, pH 7.6) containing magnesium
chloride (10 mM) and DTT (5 mM) prior to the
hybridization and photo-cross-linking reactions. Photo-cross-linked and
uncross-linked duplex ODNs were separated on 16% SDS-polyacrylamide
gels (29), and radioactive bands were quantified using a phosphorimager
(Molecular Imager System GS 525, Bio-Rad). Reaction mixtures with the
short duplex ODN (14 base pairs) were loaded onto an anion-exchange column (Poros HQ10, 4.6 × 100 mm, Perseptive Biosystems), and compounds were eluted with a Tris hydrochloride buffer (10 mM, pH 7.4) containing potassium chloride (0 mM
for 5 min followed by a linear gradient to 1 M within 15 min and 1 M for 10 min) at a flow rate of 2 ml/min.
Absorption was detected at 260 nm.
Determination of the Photo-cross-linked Amino Acid
Residue--
Proteolytic fragmentation was investigated using a crude
reaction mixture obtained with M.CviBIII and
32P-labeled TCGI/TCGA(S). The buffer of the reaction
mixture was changed to Tris hydrochloride (10 mM, pH 8.0)
and calcium chloride (20 mM) by repeated ultrafiltration
(Centricon 10, Amicon), chymotrypsin (50% of total protein weight) was
added, and the mixture incubated at 37 °C. Samples were taken after
different incubation times and analyzed on a 16% SDS-polyacrylamide
gel. For the isolation of the nucleopeptide, a preparative amount of
the crude reaction mixture containing approximately 700 pmol of the
covalent M.CviBIII-TCGI/TCGA(S) complex was enzymatically
fragmented for 16 h, as described above. The nucleopeptide was
purified on an anion-exchange column (Poros HQ10, 4.6 × 100 mm,
Perseptive Biosystems) using a linear gradient of potassium chloride
(0-1.0 M) in Tris hydrochloride buffer (10 mM,
pH 7.4). Fractions containing the nucleopeptide (detection at 260 nm)
were pooled and desalted by repeated ultrafiltration (Microsep 3K,
Pall-Gelman Sciences). Peptide sequencing of the isolated nucleopeptide
was performed with an Applied Biosystems 473A protein sequencer using
standard conditions. The electrospray ionization mass spectrum of the
isolated nucleopeptide was acquired using a double focusing sector
field mass spectrometer MAT 90 (Finnigan MAT) equipped with an ESI II
electrospray ion source.
 |
RESULTS |
Formation of a Photo-cross-link with M.TaqI--
Initial
photo-cross-linking experiments were performed with a hemimethylated
duplex ODN (36 base pairs) in which the target adenine within the
recognition sequence of M.TaqI is replaced by 5-iodouracil
(TCGI/TCGAMe(L); Table I).
32P-Labeled TCGI/TCGAMe(L) was irradiated with
a HeCd laser at 325 nm in the presence of an excess of
M.TaqI. Aliquots were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE) after different irradiation times, and a
time-dependent formation of a band with lower
electrophoretic mobility compared with the free duplex ODN was observed
(Fig. 1A). As the mass of the
duplex ODN in the DNA-protein cross-link is increased, such a lower
mobility is expected for the complex. The photo-cross-linking yields
after different irradiation times were determined using a
phosphorimager, and the obtained data were fitted to a single
exponential function (Fig. 1B). The photo-cross-linking reaction proceeded with a half-life of 20 ± 6 min and a maximum yield of 57 ± 3%. The unmethylated duplex ODN TCGI/TCGA(L) gave a photo-cross-linking yield similar to that for the hemimethylated duplex ODN TCGI/TCGAMe(L).
View this table:
[in this window]
[in a new window]
|
Table I
Sequences of duplex ODNs and their abbreviations used in this report
The recognition sequence of M.TaqI and M.CviBIII
is shown with larger spacing, and the following symbols are used:
I = 5-iodo-2'-deoxyuridine, AMc = N6-methyl-2'-deoxyadenosine, L = long (36 base
pairs), S = short (14 base pairs).
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Photo-cross-linking of a duplex ODN
containing 5-iodouracil at the target position and
M.TaqI as a function of time. A,
denaturing PAGE of the reaction mixture containing
32P-labeled duplex ODN TCGI/TCGAMe(L) (1.2 µM) and M.TaqI (3.0 µM) obtained
after irradiation at 325 nm for the indicated times. B,
photo-cross-linking yields after different reaction times were
determined from A using a phosphorimager, and the data were
fitted to a single exponential function.
|
|
In order to verify that the newly formed band in Fig. 1A is
a specific covalent DNA-M.TaqI complex, the following
control experiments (data not shown) were performed. Irradiation of
TCGI/TCGAMe(L) in the absence of M.TaqI did not
produce a band with lower mobility, which eliminates the possibility
that the band with lower mobility represents a DNA-DNA cross-link.
Additionally, incubation of TCGI/TCGAMe(L) and
M.TaqI without irradiation did not result in a band with lower mobility after denaturing PAGE, which demonstrates the covalent nature of the product formed. Furthermore, irradiation of the duplex
ODN ICGA/TCGAMe(L), in which the thymine within the
recognition sequence is replaced by 5-iodouracil, in the presence of
M.TaqI did not lead to a significant formation of a
photo-cross-linked product. This result indicates that the
photo-cross-linking reaction is DNA sequence-specific. Additionally,
irradiation of the control duplex ODNs TCGA/TCGAMe(L) and
TCGA/TCGA(L), which do not contain 5-iodouracil, in the presence of
M.TaqI yielded no photo-cross-linked product. In addition, electrophoretic mobility shift experiments under native conditions were
performed and revealed that binding of M.TaqI to canonical duplex ODNs is in the low nanomolar range and not altered significantly upon 5-iodouracil substitution.
Isolation of a Covalent DNA-M.TaqI Complex--
For the isolation
of a covalent DNA-M.TaqI complex, the photo-cross-linking
reaction was performed with TCGI/TCGA(S), a 14-base pair duplex ODN
containing 5-iodouracil at the target position, and the reaction
mixture was separated by anion-exchange chromatography (Fig.
2). M.TaqI did not bind to the
anion-exchange column under the conditions used and eluted in the void
volume, whereas TCGI/TCGA(S) eluted after 18.6 min. In addition, a new
compound eluting after 12.0 min was observed. Such a lower retention
time compared with the free ODN is expected for a DNA-M.TaqI
complex, because binding of M.TaqI should lead to some
steric shielding of the negatively charged phosphodiester backbone.
From the peak areas of the compounds eluting after 12.0 and 18.6 min, a
photo-cross-linking yield of 53% with regard to the duplex ODN
TCGI/TCGA(S) was calculated. The material eluting after 12.0 min was
isolated, desalted, 32P-labeled by treatment with T4
polynucleotide kinase and [
-32P]ATP, and analyzed by
denaturing PAGE (Fig. 3). Again, a
strongly reduced electrophoretic mobility (Fig. 3, lane
2) compared with the free ODN TCGI/TCGA(S) (Fig. 3,
lane 1) was observed. In addition, a mixture of
M.TaqI and TCGI/TCGA(S) did not show a band with reduced
mobility under denaturing conditions (Fig. 3, lane
3), demonstrating that the material eluting after 12.0 min
during anion-exchange chromatography is a covalent
DNA-M.TaqI complex.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Analysis of the photo-cross-linked
DNA-M.TaqI complex by anion-exchange
chromatography. The complex was produced by irradiation of the
short duplex ODN TCGI/TCGA(S) (1.5 µM) and
M.TaqI (2.0 µM) at 325 nm for 2 h.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3.
Denaturing PAGE of the isolated
photo-cross-linked DNA-M.TaqI complex.
Lane 1, 32P-labeled duplex ODN
TCGI/TCGA(S) alone; lane 2, isolated (material
eluting after 12.0 min in Fig. 2) and 32P-labeled
photo-cross-link between M.TaqI and the duplex ODN
TCGI/TCGA(S); lane 3, non-covalent complex
between M.TaqI and 32P-labeled duplex ODN
TCGI/TCGA(S).
|
|
In order to identify the photo-cross-linked amino acid in
M.TaqI, the covalent DNA-M.TaqI complex was
subjected to proteolytic degradation after purification by
anion-exchange chromatography. However, enzymatic fragmentation of the
covalent DNA-M.TaqI complex with different specific
proteases (trypsin, chymotrypsin, elastase, and V8 endopeptidase) even
under partially denaturing conditions (1 M guanidinium
hydrochloride, 1% SDS, or 30% methanol) did not produce a small
nucleopeptide complex. Thus, the resistance of the covalent
DNA-M.TaqI complex toward proteases, presumably due to the
thermophilic nature of M.TaqI, precluded further
characterization of the photo-cross-linked amino acid in
M.TaqI.
Formation of Photo-cross-links with M.TaqI/Y108A and
M.TaqI/F196A--
In order to investigate, whether Tyr at position 108 or Phe at position 196 of M.TaqI is involved in the
photo-cross-linking reaction, the two mutant Mtases
M.TaqI/Y108A and M.TaqI/F196A were tested. In
these mutant Mtases, the aromatic amino acid residues Tyr at position
108 or Phe at position 196 of M.TaqI are replaced by the
non-aromatic amino acid residue Ala. The photo-cross-linking yields of
the duplex ODN TCGI/TCGA(L) with M.TaqI/Y108A and
M.TaqI/F196A compared with the wild-type enzyme were 6- and
2-fold reduced, respectively (data not shown).
Formation and Proteolytic Fragmentation of a Photo-cross-link with
M.CviBIII--
For further photo-cross-linking reactions, we used the
mesophilic N6-adenine DNA Mtase
M.CviBIII, which is an isoschizomer of M.TaqI. Analyses of the photo-cross-linking reaction between
M.CviBIII and the short duplex ODN TCGI/TCGA(S) by
anion-exchange chromatography revealed that almost all the duplex ODN
had reacted, and a new compound with a retention time of 7.9 min was
formed (Fig. 4). M.CviBIII
eluted in the void volume as observed for M.TaqI. As the
photo-cross-linking reaction with M.CviBIII was almost
quantitative with respect to the duplex ODN, a crude reaction mixture
of 32P-labeled TCGI/TCGA(S) and M.CviBIII was
subjected to proteolytic fragmentation with chymotrypsin, and the time
course of the protease reaction was analyzed by denaturing PAGE (Fig.
5). After 16 h, almost all reaction
intermediates had converted to a final product. The smaller
electrophoretic mobility of this proteolytic end product compared with
the free duplex ODN indicates that a peptide fragment remained bound to
the DNA.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Analysis of the photo-cross-linked
DNA-M.CviBIII complex by anion-exchange
chromatography. The complex was produced by irradiation of the
short duplex ODN TCGI/TCGA(S) (1.5 µM) and
M.CviBIII (2.0 µM) at 325 nm for 2 h.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Proteolytic fragmentation of the
photo-cross-linked DNA-M.CviBIII complex with
chymotrypsin. The crude reaction mixture of
32P-labeled TCGI/TCGA(S) and M.CviBIII (Fig. 4)
was treated with chymotrypsin after buffer exchange and the digest
analyzed by denaturing PAGE after indicated incubation times.
|
|
Identification of the Modified Amino Acid in the Covalent
DNA-M.CviBIII Complex--
A preparative amount of the
photo-cross-linking reaction mixture was treated with chymotrypsin, and
the resulting nucleopeptide was purified by anion-exchange
chromatography. Fractions containing the nucleopeptide were
concentrated and desalted by ultrafiltration, and the isolated
nucleopeptide was subjected to automated peptide sequencing by Edman
degradation. The peptide sequence of the first 13 residues was found to
be DFIVGNPPXVVRP, and at position 9 (X) none of
the 20 natural amino acids was observed in appreciable amounts. This
peptide sequence corresponds to the amino acid residues 114-126 of
M.CviBIII and contains a Tyr residue at position 122. The
absence of Tyr at position 9 in the sequenced peptide suggests that
Tyr-122 in M.CviBIII was modified in the photo-cross-linking reaction. The observed peptide sequence is preceded by a Phe residue in
M.CviBIII, providing a cleavage site for chymotrypsin, which cleaves after aromatic amino acid residues. However, the non-aromatic residue Pro was found at the C terminus, which indicates that some
C-terminal amino acid residues were missing. The C-terminal part of the
nucleopeptide and its overall structure were verified by ESI-MS. The
DNA strands of the nucleopeptide underwent dissociation in the
transport region of the electrospray interface and the modified strand
was detected as 5-9-fold negatively charged ions. A deconvoluted mass
spectrum of the modified strand is shown in Fig.
6. The mass of the nucleopeptide was
found to be 5900.5, and the higher observed masses in Fig. 6 represent
sodium and potassium adducts. The observed mass corresponds well to the
calculated mass of 5900.6 for a nucleopeptide containing the amino acid
residues 114-129 of M.CviBIII, which has a chymotrypsin
cleavage site at its C-terminal end (Fig.
7).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
Deconvoluted electrospray ionization mass
spectrum of the nucleopeptide derived from the photo-cross-linked
DNA-M.CviBIII complex. The nucleopeptide was
obtained by treatment of the crude reaction mixture of TCGI/TCGA(S) and
M.CviBIII (Fig. 4) with chymotrypsin for 16 h and
purification by anion-exchange chromatography, followed by desalting.
I = relative intensity.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 7.
Structure of the nucleopeptide derived from
the photo-cross-linked DNA-M.CviBIII complex. The
calculated mass of 5900.6 of the shown nucleopeptide is in good
agreement with the observed mass of 5900.5 (Fig. 6). The structure
contains the amino acid sequence from position 114 to 129 of
M.CviBIII and the nucleotide sequence of the short
5-iodouracil containing single-stranded ODN. Photo-cross-linking of
5-iodouracil to Tyr-122 is suggested from the peptide sequence
DFIVGNPPXVVRP obtained by Edman degradation, where
X at position 122 represents an unidentified amino acid
residue. The peptide sequence NPPY of M.CviBIII belongs to
the conserved motif IV found in all N6-adenine
and N4-cytosine DNA as well as in
N6-adenine RNA Mtases.
|
|
 |
DISCUSSION |
DNA Binding and Base Flipping by M.TaqI--
The crystal structure
of M.TaqI in complex with the cofactor shows that the enzyme
consists of two domains forming a positively charged cleft with a
diameter of 21 Å, which closely matches the diameter of B-DNA (9).
However, modeling B-DNA into the structure showed that the distance
between the amino group of the target adenine within the DNA helix and
the methyl group of AdoMet is 15 Å, which is too far for a direct
methyl group transfer (30). By rotating the adenine out of the DNA
helix toward the cofactor, the distance between the methyl group donor
and acceptor can be reduced significantly, and hence a base flipping
mechanism, as observed in the crystal structures of the
C5-cytosine DNA Mtases M.HhaI (7) and
M.HaeIII (8) in complex with DNA, was proposed for
M.TaqI. Biochemical evidence for a base flipping mechanism
of M.TaqI was obtained using a duplex ODN, in which the
target adenine is replaced with the fluorescent base analogue
2-aminopurine (12). The 2-aminopurine fluorescence is highly quenched
in polynucleotides as a result of interactions with neighboring bases
and is strongly enhanced upon binding of M.TaqI. In
addition, a thymine residue placed at the target position within a
duplex ODN showed an enhanced reactivity toward potassium permanganate
oxidation in the presence of M.TaqI (15). This result can
readily be explained by a higher accessibility of the thymine residue
toward permanganate in the DNA-M.TaqI complex because of
base flipping. As the Van der Waals radius of iodine (2.15 Å) is only
slightly larger than that of a methyl group (2.0 Å), base flipping is
also expected for the 5-iodouracil residue in a complex between
M.TaqI and a duplex ODN containing 5-iodouracil at the
target position.
Binding of M.TaqI to canonical and 5-iodouracil modified
duplex ODNs was analyzed in a electrophoretic mobility shift assay under native conditions, and revealed that substitution of the target
adenine with 5-iodouracil exerted only little effect on the binding
strength. This result is consistent with earlier binding studies, which
showed that a duplex ODN carrying a mismatched thymine at the target
position binds with a similar affinity as the canonical substrate (15).
Recently, tighter binding of the C5-cytosine DNA Mtases M.HhaI (31,
32) and M.HpaII (31) as well as the
N6-adenine DNA Mtases M.EcoRV (13)
and M.EcoRI (14) to duplex ODNs carrying mismatched bases or
base analogues with reduced Watson-Crick hydrogen bonding potential at
the target positions was observed and attributed to less energy
required to disrupt a mismatched base pair and flip out the target
base. As the energetic contribution of base flipping to DNA binding
should at least involve the energy needed to flip out the target base
and the energy gained by binding the extrahelical base, replacement of
the matched target base by a mismatched base or a base analogue can
lead to tighter, unchanged, or even worse binding, depending on the sum
of positive and negative energetic terms. Thus, the similar affinities
of M.TaqI for duplex ODNs containing adenine, 5-iodouracil,
or thymine at the target position are compatible with a base flipping mechanism.
Photo-cross-linking Experiments with M.TaqI--
M.TaqI
was photo-cross-linked to the hemimethylated TCGI/TCGAMe(L)
and the unmethylated TCGI/TCGA(L) duplex ODNs carrying the photo-activable 5-iodouracil at the target position in 50-60% yield.
The DNA sequence specificity of the cross-linking reaction is readily
demonstrated by our observation, that the hemimethylated duplex ODN
ICGA/TCGAMe(L), in which thymine within the recognition
sequence of M.TaqI is replaced by 5-iodouracil, yielded no
significant amounts of a photo-cross-linked product. In order to
facilitate purification of the photo-cross-linked complex by
anion-exchange chromatography, the short duplex ODN TCGI/TCGA(S) was
photo-cross-linked with M.TaqI and the covalent complex
isolated. However, all attempts to digest the covalent
DNA-M.TaqI complex with various specific proteases did not
result in short nucleopeptide fragments which precluded further
analysis. This resistance toward proteases is presumably due to the
thermophilic nature of M.TaqI. A relatively high resistance
toward proteolytic cleavage was also reported for a photo-cross-link
between the thermostable MutS protein from T. aquaticus and
a duplex ODN (33).
Photo-cross-linking Experiments with the Mutant Mtases M.TaqI/Y108A
and M.TaqI/F196A--
In order to test whether Tyr-108 or Phe-196 of
M.TaqI are involved in the photo-cross-linking reaction, two
mutants of M.TaqI were investigated. In
M.TaqI/Y108A Tyr at position 108 and in M.TaqI/F196A Phe at position 196 are replaced by Ala. The
photo-cross-linking yield of TCGI/TCGA(L) and M.TaqI/Y108A
was found to be 6-fold and that of M.TaqI/F196A 2-fold
reduced compared with the wild-type enzyme. In an earlier study, we
have already investigated DNA binding of these mutant Mtases using a
fluorescence-based assay (27). Titrations of a duplex ODN, in which the
target adenine was replaced by the fluorescent base analogue
2-aminopurine, yielded a slightly increased DNA binding affinity of
M.TaqI/Y108A, whereas that of M.TaqI/F196A was
unchanged compared with the wild-type enzyme. This demonstrates that
the reduced photo-cross-linking yield with the mutant Mtases is not
caused by a lower DNA binding affinity. However, the magnitude of the
2-aminopurine fluorescence increase observed in the titrations, which
correlates with the ability of the Mtases to flip out the target base,
was different. It was found that M.TaqI/F196A is impaired in
base flipping, which offers a convenient explanation for its somewhat
reduced ability to form a photo-cross-linked complex. In contrast, the
mutant Mtase M.TaqI/Y108A is still able to flip the target
base. Thus, the strongly reduced photo-cross-linking yield observed
with M.TaqI/Y108A can directly be associated with the
missing aromatic side chain and suggests that Tyr at position 108 is
the primary site of modification in M.TaqI. A similar result
was obtained in photo-cross-linking experiments with RNA containing
5-iodouracil and a variant of the MS2 coat protein deficient in forming
phagelike capsid particles (21). The photo-cross-link was formed to a
Tyr residue at position 85 in high yield, and a mutant containing Ala
at this position failed to form a photo-cross-linked complex.
Photo-cross-linking Experiments with M.CviBIII--
For further
photo-cross-linking experiments, we used the
N6-adenine DNA Mtase M.CviBIII, a
mesophilic isoschizomer of M.TaqI. Photo-cross-linking of
the short duplex ODN TCGI/TCGA(S) with M.CviBIII proceeded
with an almost quantitative yield (Fig. 4). This represents the highest
photo-cross-linking yield reported for double-stranded DNA and a
protein. Furthermore, it was possible to fragment the DNA-protein
complex enzymatically (Fig. 5), isolate the formed nucleopeptide, and
determine its amino acid sequence by Edman degradation and its mass by
ESI-MS (Fig. 6). We found that Tyr at position 122 in
M.CviBIII is the site of modification in the
photo-cross-linking reaction. Very high photo-cross-linking yields were
also reported for the RNA-binding domain of the U1A spliceosomal
protein (20) and the bacteriophage MS2 coat protein (21), where
three-dimensional structures of the protein-RNA complexes are known
(34, 35). In these complexes a Tyr residue is found in a
-stacking
arrangement with a cytosine residue, and replacement of these cytosine
residues by 5-iodouracil resulted in 67-90% photo-cross-linking
yields. In fact, high photo-cross-link efficiencies with 5-iodouracil
were attributed to such a
-stacking arrangement, in which an initial
photoelectron transfer could take place before the resulting radical
ion pair collapses to form a covalent bond (17, 36). Thus, the high
yield of the photo-cross-linking reaction with M.CviBIII
strongly indicates that Tyr-122 is in close proximity and most likely
-stacked with 5-iodouracil in the protein-DNA complex.
A Structural Model of M.TaqI in Complex with DNA--
In an amino
acid sequence alignment, Tyr-122 of M.CviBIII and Tyr-108 of
M.TaqI were found in corresponding positions within a
20-amino acid fragment with 70% sequence identity (37). This supports
our conclusion from the photo-cross-linking experiments with the mutant
Mtases M.TaqI/Y108A and M.TaqI/F196A that Tyr at
position 108 is the primary site of modification in M.TaqI. A model of M.TaqI in complex with TCGI/TCGA(S) is presented
in Fig. 8. Docking B-DNA into the
proposed DNA binding cleft of the M.TaqI-AdoMet structure
(38) showed that the distance between an inner helical 5-iodouracil and
Tyr-108 is too large for an efficient photo-cross-linking reaction. The
5-iodo-2'-deoxyuridine residue was then rotated out of the DNA helix
toward the cofactor, as observed in the cocrystal structure of the
C5-cytosine DNA Mtase M.HhaI (7).
Although this movement clearly reduces the distance to Tyr-108, it also
leads to a steric overlap between the 5-iodouracil residue and Tyr-108.
Thus, Tyr-108 was rotated toward the outside of M.TaqI in a
location, where it can form a
-stacking interaction with the
extrahelical 5-iodouracil residue. Phe-196, which was shown to be
important for stabilizing the extrahelical target base (27), is also
included in Fig. 8. For the extrahelical target adenine in the
M.TaqI-DNA complex, a similar position as that of
5-iodouracil is expected.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 8.
Model of M.TaqI in complex
with TCGI/TCGA(S). The model was produced by docking B-DNA
(green) containing an extrahelical 5-iodo-2'-deoxyuridine
residue (red) into the structure of M.TaqI
(gray) in complex with the cofactor AdoMet
(yellow) (38). In addition, Tyr-108 (motif IV,
purple) was rotated toward the outside of M.TaqI,
where it can form -stacking interactions with the extrahelical
5-iodouracil residue. The conserved Phe residue at position 196 (motif
VIII, blue) is also included in the figure. Modeling was
performed with the graphic program O (42), and the figure was created
using the computer program ICM (43).
|
|
Our results with the related N6-adenine DNA
Mtases M.TaqI and M.CviBIII are also relevant for
other DNA and RNA Mtases. Tyr-122 in M.CviBIII and Tyr-108
in M.TaqI belong to the conserved motif IV
((N/D/S)(P/I)P(Y/F/W)) found in all
N6-adenine and
N4-cytosine DNA Mtases as well as in
N6-adenine RNA Mtases (N-DNA/RNA Mtases) (39).
In addition, the structures of several other N-DNA/RNA Mtases in the
absence of DNA or RNA were determined and it was found that their
cofactor binding domain structures are very similar (10, 40, 41). Thus,
the spatial relationship between the target base and the aromatic amino
acid residue within motif IV is expected to be similar for all
N-DNA/RNA Mtases.