(Received for publication, July 9, 1996, and in revised form, September 18, 1996)
From the Department of Biochemistry and Genetics, The University of Newcastle, Newcastle upon Tyne, NE2 4HH, United Kingdom
The EcoRV DNA methyltransferase
introduces a CH3 group at the 6-amino position of the first
dA in the duplex sequence d(GATATC). It has previously been reported
that the methylase contacts the four phosphates (pNpNpGpA) at, and
preceding, the 5-end of the recognition sequence as well as the single
dG in this sequence (Szczelkun, M. D., Jones, H., and Connolly, B. A. (1995) Biochemistry 34, 10734-10743). To study the
possible role of the dA and T bases within the ATAT sequence,
interference studies have been carried out using diethylpyrocarbonate
and osmium tetroxide. The methylase bound very strongly to
hemimethylated oligonucleotides modified at the second AT, of the
AT
sequence, in the unmethylated strand of the duplex.
This probably arises because these modifications facilitate DNA
distortion that follows the binding of the nucleic acid to the protein.
Oligonucleotides containing modified bases at both the target dA base
and its complementary T were used to determine whether this dA
methylase flips out its target base in a similar manner to that
observed for dC DNA methylases. In binary EcoRV
methylase-DNA complexes, analogues that weakened the base pair caused
an increase in affinity between the protein and the nucleic acid. In
contrast, in ternary EcoRV methylase-DNA-sinefungin (an
analogue of the natural co-factor,
S-adenosyl-L-methionine (AdoMet)) complexes,
only small differences in affinity were observed between the normal
dA-T base pair and the analogues. These results are almost identical to
those seen with DNA dC methylases (Klimasauskas, S., and Roberts R. J. (1995) Nucleic Acid Res. 23, 1388-1395; Yang, S. A.,
Jiang-Cheng, S., Zingg, J. M., Mi, S., and Jones, P. A. (1995)
Nucleic Acids Res. 23, 1380-1387) and support a
base-flipping mechanism for DNA dA methylases.
The EcoRV methylase adds CH3 groups to the first dA in GATATC1 targets (1) and forms a stable complex with this sequence in the presence of its co-factor, S-adenosyl-L-methionine (AdoMet),2 or co-factor analogues such as sinefungin (2, 3, 4, 5). Tightest binding is observed with hemimethylated GATATC sequences (Kd = 13 nM), and both unmethylated (Kd = 46 nM) and dimethylated (Kd = 143 nM) sequences bind less strongly. Weak binding is observed in the absence of co-factor. DNA footprinting showed that the methylase interacted with four phosphates (NpNpNpGpA), symmetrically disposed on both DNA strands of the duplex, and also with the dG in the recognition sequence (3). The protein also caused a 60° bending of DNA in the direction of the major groove (4). These footprinting studies gave no indication of the function that the central ATAT bases played in DNA recognition by the methylase. Experiments using modified bases suggested a critical role for the first AT pair and a much less important function for the second (2, 5). However, it is not known whether these bases contact the protein directly or facilitate the bending of the DNA, particularly at the easily deformed, central, TA step (6, 7, 8).
To further define the role of the central ATAT bases, we have used additional interference methods. The best reagent for interference studies is dimethylsulfate, which reacts predominantly with the N7 position of dG and has been widely used (9, 10, 11). This reagent also reacts more weakly with the N3 of dA, but in our hands, gave no useful information with the EcoRV methylase (3). Other base-specific chemicals have received much less attention. One potential reagent for dA is diethylpyrocarbonate (DEPC), which causes carbethoxylation at N7 and N6 of dA and N7 of dG (10, 12, 13, 14). However, this reagent shows low reactivity with B-DNA although it is more reactive with the Z-form (15, 16). Binding of small ligands such as echinomycin and quinoxoline antibiotics, which change the conformation of B-DNA, can also cause increased reactivity (17, 18, 19). DEPC has also been used as a footprinting agent to study anti-Z-DNA antibodies (20), the tet repressor (21) and HMG box proteins (22). Osmium tetroxide reacts preferentially with T residues although slower reactions with dC and dG have been observed (23, 24). OsO4 oxidizes the 5-6 double bond of T via the formation of an addition compound and shows a strong preference for single-stranded DNA. Using this reagent, it has been possible to probe DNA structural features such as cruciforms, which show hyper-reactivity (25). In this paper, both DEPC and OsO4 have been used to study the interaction of EcoRV methylase with the ATAT bases within its recognition sequence.
A key question with EcoRV methylase, and DNA dA methylases generally, is whether they "flip out" the dA base that is the target for methylation. Base flipping was first observed, using x-ray crystallography, with the DNA dC methylase HhaI (26). The target dC is flipped out of the DNA helix by 180° and placed at the catalytic site of the methylase. The structure of a second dC methylase, HaeIII, also indicated base flipping (27). The high sequence homologies seen between dC methylases (28, 29) suggests that base flipping is universal for these enzymes. It has been postulated that base flipping could be a common mechanistic feature shared by several enzymes that act on DNA, particularly methylases and DNA repair enzymes (30). However, base flipping can only be unequivocally confirmed by the solution of a structure of an enzyme complexed with double-stranded DNA. Apart from the two dC methylases mentioned above, this has only been done with the repair enzyme endonuclease V (31). Endonuclease V repairs pyrimidine dimers in DNA and flips out one of the dA bases opposite the lesion. Other repair enzymes including uracil-DNA glycosylase (32, 33), the Ada enzyme (34), and Escherichia coli photolyase (35) have had their structures solved, either as apo-enzymes or with short single-stranded oligonucleotides. In these cases, the putative distance between the catalytic apparatus and the inferred position of the target base in double-stranded DNA suggested that base flipping might occur. A similar situation is found with DNA dA methylases. Only one structure (Tag I methylase) is available but lacks bound DNA (36). However, the dA target (obtained by modelling B-DNA into the structure) is so far from the AdoMet that these authors suggest that base flipping will be necessary for catalysis. Recently, it has been postulated that all dA methylases have a common structure, related to dC methylases, and so are likely to share a common mechanism (37).
Although structures of enzyme-DNA complexes provide the ultimate proof for base flipping, other methods have been used. Tighter binding was observed for both HhaI methylase and HpaII methylase using oligonucleotides in which the dG/dC base pair (containing the target dC) was replaced with mismatched bases (38, 39). Furthermore, an inverse correlation was observed between the binding affinity and the strength of the target base pair. It was argued that tighter binding occurs with mismatches because less energy needs to be expended by the methylase to flip out the base. The "saved" energy manifests itself as a lower Kd. Tighter binding is only observed in enzyme-DNA binary complexes, and in ternary (enzyme-DNA-AdoMet) complexes, the differences in Kd values between the correct dC/dG base pair and mismatches largely disappear. This is due to the complete stabilization of the flipped out base in ternary complexes by interactions between the protein and both the flipped dC and orphan dG. In this publication, the mismatch base approach has been used with the EcoRV methylase to evaluate base flipping with a DNA dA methylase.
The
purification of the EcoRV methyltransferase was as reported
(1, 2, 4). Oligonucleotides were prepared using the phosphoramidite
method with standard DNA synthesis reagents from Cruachem Ltd.
(Glasgow, Scotland). The phosphoramidites of the modified bases were
obtained from Cruachem (deoxyinosine), Pharmacia Biotech Inc. (St.
Albans, UK) (6-methyldeoxyadenosine), and Cambio Ltd. (Cambridge, UK)
(purine-1--D-2
-deoxyriboside,
2,6-diaminopurine-1-
-D-2
-deoxyriboside, spacer). All
oligonucleotides were purified by high pressure liquid chromatography
and their concentrations determined by absorbance at 260 nm using the
sum of the extinction coefficients of the individual bases (40, 41).
Oligonucleotides were 5
-labeled with polynucleotide kinase (Pharmacia)
and [
-32P]ATP (3000 µCi/mmol, Amersham) (42). The
non-incorporated radioactivity was removed with two rounds of ethanol
precipitation. The pellet was dried in a Savant Speed-Vac centrifuge
and resuspended in the appropriate volume of 10 mM
Tris-HCl, pH 8, containing 1 mM EDTA (TE buffer). Duplexes
were made by mixing equimolar amounts of the individual strands in TE
buffer and heating to 85 °C. Annealing was allowed to take place by
cooling to room temperature over 6-8 h.
OsO4
(Sigma) was dissolved in distilled water as a 20 mM stock solution (caution OsO4 is very toxic,
see Refs. 23 and 24 for safety precautions). Before adding to the DNA,
an aliquot was activated by the addition of an equal volume of a 6%
(v/v) aqueous solution of pyridine for 5 min. 10 pmol of the duplex oligonucleotide (with the strand under investigation, 5
-labeled with
32P) was incubated in 50 µl of 5 mM Tris-HCl,
pH 8.0, and 5 mM of the activated OsO4. After
15 min at 37 °C, the reaction was stopped by precipitating the
oligonucleotide with ethanol. After two further rounds of ethanol
precipitation, the oligonucleotide was suspended in 50 µl of TE
buffer. The binding of EcoRV methylase to
OsO4-modified DNA was carried out in 20 µl of 50 mM Hepes-KOH, pH 7.5, containing 10 mM EDTA,
100 mM NaCl, 1 µg/µl acetylated bovine serum albumin, and 1.5 mM sinefungin (Sigma, Poole, UK).
The DNA was titrated with between 50 and 400 nM of the
methylase to produce an enzyme concentration that gave approximately
50% bound and 50% free DNA. Samples were incubated at room
temperature for 10 min, and the bound and free DNA pools were separated
by non-denaturing polyacrylamide gel retardation as described
previously (2, 3, 4). The two DNA fractions were visualized by
autoradiography of the gels, and the appropriate slices were excised.
The oligonucleotides were eluted from the gel by shaking overnight in
TE buffer at 37 °C. After centrifugation, the supernatant was washed
three times with n-butanol, and the oligonucleotide was
precipitated using ethanol. The pellet was resuspended in 45 µl of
water and 5 µl of piperidine and incubated at 90 °C for 30 min in
order to cleave at the modified positions. The sample was dried, then dissolved in 300 µl of water, and again evaporated to dryness. This
step was repeated twice, and the sample was dissolved in 5 µl of
loading buffer prior to running on a denaturing DNA sequencing gel
(42). Both the free and bound DNA pools, together with a control
(consisting of oligonucleotide treated with OsO4 alone) and
a Maxam-Gilbert dG chemical sequencing track (43), were run on a 20%
denaturing gel and visualized by autoradiography.
About 10 pmol of the oligonucleotide (with the strand to be investigated labeled as above) was incubated with 5 µl of DEPC in 50 µl of 5 mM Tris-HCl, pH 8.0, at room temperature for 12 min. Due to the poor solubility of DEPC, the sample was vigorously shaken using a whirly mixer for 2 min at both the beginning and halfway through the reaction. Following the modification of the oligonucleotide, the interference experiment was carried out exactly as for the OsO4 experiment.
Determination of Kd Values for Oligonucleotide Binding to EcoRV MethylaseThe protocols used based on band-shift
gel retardation analysis have been described in detail (2). Briefly,
for the evaluation of Kd values in ternary
(enzyme-DNA-sinefungin) complexes, the 5-labeled oligonucleotide
(0.125 nM) was incubated with EcoRV methylase
(0.1-81 nM) in 20 µl of 50 mM Hepes-KOH, pH
7, 10 mM EDTA, 100 mM NaCl, 20 µg of
acetylated bovine serum albumin, and 1.5 mM sinefungin. For
binary (enzyme-DNA) complexes, the methylase was pre-incubated with an
equimolar amount of GACGATATCGTC to remove any traces of tightly bound
AdoMet (2). Kd values were then determined as for
the ternary complexes with the omission of sinefungin. The
methylase concentration in these experiments was varied between 13 and 856 µM, except when the target dA was replaced with
the spacer; here, 2.1 to 117 µM was used. After
incubation at room temperature for ten minutes, the free and
enzyme-bound DNA fractions were separated by non-denaturing gel
electrophoresis (2). The amounts present in each pool were determined
using a Fuji BAS-1500 phosphoimager. Kd values were
evaluated using "GraFit" (44) to fit the data to binding isotherms.
For the evaluation of the Kd values for ternary complexes, only the methylase concentrations above 2.7 nM
were used. This ensures that the enzyme concentration is at least 16 times greater than that of the DNA, allowing the approximation [Efree] = [Etotal]. Kd
determinations were based on the material present in the free DNA band.
This is more accurate than evaluations based on the bound band due to
protein-DNA dissociation during electrophoresis (45). Three to five
determinations were averaged to give the reported values.
The oligonucleotides used in these
studies are shown in Fig. 1. All are based on a parent
duplex, which contains an EcoRV GATATC site, formed by the
hybridization of a 30- and 33-mer (Fig. 1A). All the
experiments have been carried out with hemimethylated derivatives of
the parent, i.e. containing GATATC on one stand and GMTATC
(M = 6-methyldeoxyadenosine) on the other. This is because
hemimethylated oligonucleotides bind most tightly to the methylase (2,
3). For the OsO4 and DEPC interference studies, the two
possible hemimethylated oligonucleotides (having M in either the top or
bottom strand) have been used (Fig. 1B). To study base
flipping, a set of oligonucleotides containing M in the top strand was
utilized (Fig. 1C). The target deoxyadenosine base on the
bottom strand has been replaced with a variety of analogues (Fig.
1D), which vary the number of Watson-Crick hydrogen bonds to
the partner thymidine. Purine-1--D-2
-deoxyriboside (P)
reduces the number from two to one, whereas
2,6-diaminopurine-1-
-D-2
-deoxyriboside (D) increases
the value to three. With deoxyinosine (dI) and the spacer (S; a
derivative giving an abasic site), no hydrogen bonds can be formed to
the thymidine. Thus, these base analogues allow the base pair strength
to be correlated with binding affinity, as has been done with
HhaI and HpaI (38, 39). In one case, the target
deoxyadenosine has been retained and the base pair altered by replacing
the partner thymidine with the spacer.
Interference Studies
DEPC and OsO4 mainly react with DNA at purines and thymidines, respectively. Nevertheless, their use as DNA footprinting agents has not been extensive, probably due to the low reactivity that B-DNA shows toward the two chemicals. Initially, we attempted protection footprinting by adding the reagent to the pre-formed methylase-DNA complex without success. No reaction was seen between OsO4 and DNA in the buffer used to study the methylase-DNA complex. It is perhaps not surprising that this reagent fails to work under these conditions as the methylase requires double-stranded DNA, which reacts poorly with OsO4 (23, 24). Additionally, OsO4 shows a low reactivity in the presence of NaCl (46), and 100 mM levels of this salt are necessary to keep the methylase active. Addition of DEPC to methylase-DNA complexes caused a rapid, irreversible inactivation of the protein, as assessed by gel shifts (not shown). As DEPC has been used as a protein modification reagent, reacting with lysine and histidine (47), this effect is easily rationalized.
Interference footprinting provided more success. Here, the DNA was
modified with the reagent, and the reagent was removed before the
addition of the enzyme. This means the reaction conditions can be
manipulated to allow, for example, reaction between the DNA and
OsO4. The protocols outlined under "Experimental
Procedures" gave a reasonable degree of modification of the DNA by
both OsO4 and DEPC, as can be seen by examination of the
control lanes in Fig. 2. Both reagents also exhibited
the expected specificities, i.e. OsO4 reacting
most strongly with T and DEPC with dA followed by dG. However, the
selectivities are not absolute. In interference footprinting, the
reagent used to modify the DNA never comes into contact with the
protein. This prevents the irreversible inactivation of the methylase,
previously seen using DEPC.
The use of DEPC (Fig. 2) produced a clear cut result with
the hemimethylated (GATATC/GMTATC) oligonucleotides. Namely the binding of the EcoRV methylase was strongly enhanced by
modification to the second dA in the unmethylated strand
(GATTC). Better binding occurs for both orientations of
the oligonucleotide (i.e. with 6-methyldeoxyadenosine on the
top or the bottom strand) and is clearly visible as very pronounced
bands at the second dA position for the bound lanes in gels A(I) and
B(I) in Fig. 2. No other strong effects involving either reduced or
enhanced binding were observed, and in view of the untested nature of
DEPC as a footprinting agent, we feel it is unwarranted to draw more
conclusions. The results with OsO4 are similar to those
observed with DEPC (Fig. 2). The most prominent effect is a very strong
enhancement of methylase binding due to modification of the second
T in the unmethylated strand (GATA
C) of the duplex
(Fig. 2, gels A(I) and B(I)). OsO4 is not completely
specific and also reacts with dA bases. Reaction of the second dA in
the unmethylated strand (GAT
TC) with this reagent also
improves methylase binding, as seen with DEPC. The interference results
can be summarized as follows. 1) Modifications to the second AT
(GAT
C) in the unmethylated strand strongly enhance
methylase binding; 2) no strong effects are seen for reaction at the
methylated strand (Fig. 2, gels A(II) and B(II)); and 3) in no case
does reaction with either OsO4 or DEPC diminish
binding.
We used the mismatch base approach
previously applied to DNA dC methylases (38, 39) with the
EcoRV methylase to determine whether or not base flipping
takes place. The oligonucleotides shown in Fig. 1, C and
D, were used to manipulate the dA/dT base pair by varying
the target dA base while keeping the partner T constant. The binding of
these oligonucleotides to the methylase, in the absence of sinefungin,
i.e. binary complexes, has been investigated by gel
retardation analysis as shown in Fig. 3. Quantification of these gel retardation experiments (Fig. 4 and Table
I) demonstrated, as expected, rather poor binding of the
parent oligonucleotide. The Kd of 181 nM
is in good agreement with that of 200 nM found in our
earlier study. This is characteristic of the EcoRV methylase, which requires the presence of AdoMet or sinefungin for
tight binding to GATATC sequences (2). When the target dA was replaced
with 2,6-diaminopurine, the Kd value (177 nM) was unaffected (Fig. 4 and Table I). The presence of the other three analogues (deoxyinosine, purine, and the spacer) increased affinity (Fig. 4 and Table I). With deoxyinosine, the lowering of the Kd was small (about 2.4-fold).
However, the oligonucleotides containing purine and the spacer bound
particularly tightly with Kd values of 33 and 28 nM, respectively. These values represent about a six-fold
improvement in affinity over the wild type.
|
When the Kd values for the binding of DNA to the methylase in the presence of sinefungin, i.e. ternary complexes, were evaluated, a different pattern was seen in the gel shifts (not shown). The results are given graphically in Fig. 4 and summarized in Table I. The Kd of 20 nM obtained for the hemimethylated parent is in reasonable agreement with the value of 11 nM reported earlier for the same oligonucleotide (2). Although all the oligonucleotides in which the target dA has been replaced bind to the methylase more tightly than the wild type, differences are small. The substitution of dA with deoxyinosine, 2,6-diaminopurine, and the abasic spacer gives a less than 2-fold increase in affinity. Even the variant with the lowest Kd, where purine replaces dA, binds only 2.5 times more strongly. Given the inherent difficulties in the evaluation of Kd values using gel shift assays (38, 39, 45) and the errors we observe of ± 20%, it is unlikely that these small effects are significant.
Binding of EcoRV Methylase to Oligonucleotides Having the T, Partnering the Target dA, Replaced by an Abasic SiteThis substrate manipulates the target T/dA base pair by the alteration of the T base. Only an abasic site was used as it gave the most dramatic results in the above set of experiments. Fig. 4 and Table I show that, in binary complexes, the abasic site only causes a slight increase in binding affinity. Thus, a Kd of 153 nM is seen for the spacer, similar to the value of 227 nM for the control. The small decrease (1.5-fold) in Kd when the partner T is replaced by the spacer contrasts with the larger effect (about 6-fold) observed when the target dA is substituted with the spacer. In the presence of sinefungin, both oligonucleotides bound to the methylase with similar affinities (Fig. 4 and Table I). Thus, Kd values of 14 and 18 nM were observed for the modified and control oligonucleotides, respectively.
The results obtained in this paper are consistent with the
EcoRV methylase causing distortions to its GATATC cognate
sequence. Previously, we showed that the methylase caused a 60° bend
toward the major groove on binding to GATATC sequences (4), and this paper extends this observation. The roles of the T and dA bases have
been probed by interference studies using OsO4 and DEPC. We
found that the reaction of either of the second two AT bases (GATC) in the non-methylated strand caused a considerable
increase in affinity. OsO4 adds across the 5-6 double bond
of T (23, 24), and DEPC reacts with the 7-N and 6-NH2
positions of dA (10, 12, 13, 14), giving bulky adducts. These modifications would be expected to decrease both base stacking and Watson-Crick hydrogen bonding and so weaken the double helix and facilitate DNA
distortion. The increased binding is most likely due to the DNA being
able to easily adopt the distorted, bound conformation. The distortion
will include DNA bending (4) and base flipping. Earlier results showed
that the presence of base analogues at this second AT base step often
increased the turnover number of the methylase (5). These experiments
were carried out with self-complementary unmethylated dodecamers, and
the reason for the rate enhancement was obscure. Later, we suggested
that product release could be rate-limiting for unmethylated DNA (3).
However, many of the modified bases disrupt Watson-Crick base pairs and may, therefore, favor DNA distortion by a general weakening of the
double helix. Thus the increase in catalysis, when these AT bases are
modified, may be due to facilitation of DNA distortion in an analogous
manner to the tighter binding seen on their chemical modification.
The results seen on substituting the target dA and its partner T are
quantitatively similar and qualitatively identical to those seen with
the DNA dC methylases, HhaI and HpaII (38, 39). With the EcoRV methylase in enzyme-DNA binary complexes,
manipulations of the T/dA base pair that weaken Watson-Crick or
stacking interactions result in an increased binding affinity. In the
series T/X (replacement of target dA), the Kd values
followed the order of X = dA D > dI > P
S. The purine and spacer containing oligonucleotides bound about
six times more strongly than the control. We did not carry out
extensive studies for the X/dA series (replacement of the partner T).
However, although the S/dA combination bound more tightly than T/dA,
the difference was less pronounced than when S replaced dA. With the dC
methylases, a good inverse correlation was observed between the base
pair stability and the strength of binding to the enzyme in binary
complexes. This trend is also seen with the RV methylase although the
correlation is not perfect. Extensive studies on base pair stabilities
with modified bases have not been carried out, as has been done with
natural base mismatches. Base pair stability depends on several factors
including the number of Watson-Crick hydrogen bonds, incorrect
alignment of H-bond donor/acceptors, and base stacking. One might
expect base pair stability to decline in the following order for the T/X series; X = D (three Watson-Crick hydrogen bonds) > dA (two bonds) > P (one bond) > dI (no bonds, misalignment of H-bond
donor/acceptors) > S (no bonds, no base stacking). Thus, although the
two series representing binding affinities and base pair stabilities do
not exactly match, they are a reasonable approximation of each other. It is noteworthy that 2,6-diaminopurine is the only analogue that does
not decrease the Kd significantly, and this is the only modified base expected to stabilize, rather than weaken, the base
pair. All three changes (T/P, T/dI, and T/S) that destabilize the base
pair increase binding to the methylase. The change is small for
T/dI but a factor of about six for T/P and T/S, similar to the 10-fold
increase in binding seen with dC methylases using mismatches (38, 39).
With the dC methylases, the results were explained in terms of the
base flipping mechanism. Here, as clearly seen by crystallography (26,
27), the target dC is completely extruded from the double helix and
placed in the vicinity of the AdoMet co-factor. Base flipping is
facilitated by weakening the base pair, and this results in tighter
binding. The similarity of the results seen with EcoRV
methylase, supports the proposition that this dA methylase will also
flip its target dA out of the double helix.
Unfortunately, as previously mentioned (38), it is not possible to fully interpret Kd changes consequent to base pair alteration as modifications to the target dA and its partner T are likely to have multiple consequences. On the one hand, destabilizing the base pair facilitates base flipping and leads to a lower Kd. However, as observed with HhaI methylase (26) and HaeIII methylase (27), both the flipped base and the orphan base, which remains in the helix, make additional stabilizing interactions with the protein. Perturbing these interactions is most likely going to weaken binding and so lead to an increase in Kd. The general improvement in binding, seen with HhaI methylase, HpaII methylase, and EcoRV methylase using base mismatches, suggests that the ease of base flipping is the predominant thermodynamic force in binary complexes. However, these complexities are probably responsible for the less than perfect correlation between Kd values and base pair strength, seen with EcoRV methylase, and features such as the T/S base pair binding much more tightly than the S/dA. With EcoRV methylase, the preference for mismatches all but disappears in ternary (enzyme-DNA-sinefungin) complexes, and a similar effect is observed with both HhaI methylase and HpaII methylase (38, 39). In the case of EcoRV methylase, only the T/P base pair binds significantly better than the T/dA, and even here, the effect is not large. The base P may stabilize conformational features other than base flipping, as has previously been with the Trp repressor (48) and the EcoRI endonuclease (49). The increased binding affinity seen with the correct sequence on ternary complex formation occurs because only in the ternary complex are the full complement of energetically favorable interactions to both the flipped and the orphan base made by the protein. This leads to the stabilization of what would normally be a highly unstable and difficult to achieve DNA structure, containing a base fully extruded from the double helix. Further stabilization of mismatches is not possible on ternary complex formation as the bases are either absent (i.e. contain the abasic spacer, S) or of the incorrect structure and so do not interact correctly with the protein.
In conclusion, this paper supports the hypothesis of DNA distortion, including a flipped dA base, on nucleic acid binding to the EcoRV methylase. It has been proposed that methylases may have arisen from base-mismatch binding DNA repair enzymes (30, 38) by the fusion of three protein domains with the ability to recognize a mismatch, recognize a specific sequence, and carry out methylation. In this regard, it is interesting that the RV methylase appears to share some features with the repair enzyme endonuclease V. This enzyme recognizes thymine photodimers and flips out one of the dA bases opposite to the lesion. It also kinks the DNA by 60° (31). Likewise, the EcoRV methylase also flips out a dA base and bends the DNA by 60° (4).