(Received for publication, August 7, 1995; and in revised form, October 30, 1995)
From the
The EcoRV methyltransferase modifies DNA by the
introduction of a methyl group at the 6-NH position of the
first deoxyadenosine in GATATC sequences. The enzyme forms a stable and
specific complex with GATATC sequences in the presence of a nonreactive
analogue, such as sinefungin, of its natural cofactor S-adenosyl-L-methionine. Using circular permutation
band mobility shift analysis (in which the distance between the GATATC
sequence and the end of the DNA is varied) of protein-DNA-cofactor
complexes we have shown the methylase induces a bend of just over
60° in the bound DNA. This was confirmed by phasing analysis, in
which the spacing between the GATATC site and a poly(dA) tract is
varied through a helical turn, which showed that the orientation of the
induced curve was toward the major groove. There was no significant
difference in the bend angle measured using unmethylated GATATC
sequences and hemimethylated sequences which contain
G
ATATC in one strand only. These are the natural
substrates for the enzyme. The EcoRV endonuclease, a very well
characterized protein, served as a positive control. DNA bending by
this protein has been previously determined both by crystallographic
and solution methods. The two proteins bend DNA toward the major groove
but the bend angle produced by the methylase, slightly greater than
60°, is a little larger than that observed with the endonuclease,
which is approximately 44°.
Bending of DNA is fairly common and can be due to: 1)
sequence-specific intrinsic curvature (Crothers et al., 1990;
Hagerman, 1990, 1992; Trivinov, 1991), for example with
poly(dA)poly(dT), which induces curvature toward the minor
groove; 2) the binding of small ligands (Feuerstein et al.,
1990); and 3) the binding of proteins. One of the earliest,
structurally characterized, complexes in which proteins induce bending
of DNA was the nucleosome (Richmond et al., 1984). However,
DNA curvature does not only occur in these macromolecular structures. A
large number of proteins involved in important cellular processes such
as replication, recombination, or transcription are known to produce
bending of the double helix (Travers, 1990). An excellent example is
the prokaryotic catabolite gene activator protein (CAP protein), which
produces a 90° bend, as measured both in crystals (Schultz et
al., 1991) and solution (Wu and Crothers, 1984; Kim et
al., 1989; Zinkel and Crothers 1990). The importance of the bend
produced by this protein has been demonstrated by its replacement with
intrinsically bent DNA; this is enough to activate transcription in
vitro (Bracco et al., 1989). Inducing the bending of DNA
in order to activate transcription has been reported for other
transcriptional proteins (Perez-Martin and Espinosa, 1991), and this
seems to be a very common feature for this class of protein. The bend
angles obtained using solution methods range from 30°, for Cro
protein binding to O
1 operator, to
112° for the GalR protein binding to O
operator (Kim et al., 1989).
The interactions of type II restriction enzymes with their target sites can also result in DNA bending. Crystal structures of the EcoRI (Kim et al., 1990; Rosenberg, 1991) and EcoRV (Winkler et al., 1993; Kostrewa and Winkler, 1995) endonucleases with their cognates sequences (GAATTC and GATATC, respectively) show that the bound DNA is distorted and both enzymes induce considerable bends. In both cases studies in solution have confirmed the bending of the DNA observed in the crystalline state (Thompson and Landy, 1988; Douc-Rasy et al., 1989; Stöver et al., 1993; Vipond and Halford, 1995). Bend angles of 50° and 44° have been reported for RI and RV, respectively. However, the crystal structure of the cognate DNA(CAGCTG)-PvuII complex (Cheng et al., 1994) shows no significant DNA curvature, even though this nuclease shares structural similarities with EcoRV. Other data, obtained using only solution methods, showed that RsrI bent DNA in a very similar manner to EcoRI (Aiken et al., 1991). These two enzymes are perfect isoschizomers. SmaI and XmaI (imperfect isoschizomers that recognize the sequence CCCGGG) induce bending of 32° and 40°, respectively. Interestingly the DNA is bent in opposite directions, with SmaI toward the major groove and with XmaI in the direction of the minor groove (Withers and Dunbar, 1993).
There
is much less structural data available for type II DNA
methyltransferases. Two deoxycytidine methylases, HhaI (Cheng et al., 1993; Klimasauskas et al., 1994) and HaeIII (Reinisch et al., 1995), have been
crystallized as complexes with DNA. Both structures show that the
target deoxycytidine base is completely flipped out of the helix, but
there is little or no bending to the nucleic acid. One deoxyadenosine
methylase, TaqI (Labahn et al., 1994), has also had
its structure determined albeit in the absence of an oligonucleotide.
We are unaware of experiments describing solution studies in which any
DNA curvature, induced by complexation with a DNA methyltransferase,
has been measured. In this study we investigate DNA bending induced by
the EcoRV methyltransferase. This is a type II deoxyadenosine
methylase which adds CH groups to the first deoxyadenosine
in GATATC sequences (Nwosu et al., 1988). We have recently
shown that this methylase binds specifically to GATATC sequences with
the following order of affinity; hemimethylated DNA > unmethylated
DNA > dimethylated DNA. Sinefungin, an analogue of the cofactor S-adenosyl-L-methionine (AdoMet) (
)increased binding by a factor of 20, and ternary
enzyme/cofactor/substrate (un- or hemimethylated DNA) migrated slightly
more rapidly than the equivalent product complexes containing
dimethylated DNA. The enzyme copurified with traces of AdoMet and so
was capable of methylating small quantities of DNA without the addition
of this cofactor (Szczelkun and Connolly, 1995). Several of the
methylase-DNA contacts were determined using ethylation and methylation
interference analysis (Szczelkun et al., 1995). The formation
of a specific protein-DNA complex means that solution methods, such as
the circular permutation band mobility shift assay (Wu and Crothers,
1984; Liu-Johnson et al., 1986; Kim et al., 1989;
Crothers et al., 1991; Zwieb and Adhya, 1994) and helical
phasing analysis (Zinkel and Crothers, 1987; Crothers et al.,
1991; Gartenberg et al., 1990; Withers and Dunbar, 1993), can
be used to see if the methylase bends DNA. The two approaches have been
used with both unmethylated and hemimethylated GATATC sites to reveal
details of DNA bending by the EcoRV methylase and to elucidate
the means by which this protein recognizes its target sequence.
Figure 1: Oligonucleotide fragments containing a permutated EcoRV site. These were produced by PCR amplification and subsequent restriction endonuclease digestion (enzymes used shown) of a pBEND3 derivative. The position of the EcoRV site is shown, relative to the ends of the fragments. These fragments were used to study DNA bending by the EcoRV methylase and gave the results shown in Fig. 4.
Figure 4: Gel retardation of DNA fragments containing a permutated GATATC site by the EcoRV methylase and endonuclease. A, autoradiographs of the gels obtained. The lanes 1-6 correspond exactly to the fragments 1-6 shown in Fig. 1. B, plots of relative mobility (migration of protein-DNA complex/migration of free DNA) versus distance of EcoRV site from the 5`-end of the DNA fragment.
A second set of four
different 150-bp DNA fragments, containing a permutated EcoRV
site, were generated by the PCR amplification of pAD10; a plasmid that
contains a chemically synthesized DNase I gene which has a unique EcoRV site within its sequence (Worrall and Connolly, 1990;
Doherty et al., 1993). In the first instance four pairs of
primers, 15 bp long, were used to generate the four 150-bp fragments.
The positions at which the primers bound to the template, and hence the
products produced, are shown in Fig. 2. The second four pairs of
primers were used to give products containing a hemimethylated,
permutated, EcoRV site (i.e. GATATC
and GATATC on the two strands). One of each of the pairs of the primers
was of varying length and contained a G
ATATC sequence.
The second was 15 bp long and was selected to maintain the product
length at 150 bp (see Fig. 2).
Figure 2:
The production of 150-bp fragments
containing either an un- or a hemimethylated EcoRV site at
various distances along the oligonucleotide. These were prepared by PCR
amplification of part of a DNase I gene (shown in bold type)
containing a single GATATC site (highlighted). Pairing of the
short forward () primers (containing only normal bases) 1, 2, 3,
and 4 with the reverse (
) primers 1, 2, 3 and 4, respectively,
gave the fragments 1, 2, 3, and 4 which contain an unmethylated EcoRV site at the locations shown. Pairing of the longer
forward (
) primers (containing a single 6-methyldeoxyadenosine,
shown by an asterisk (*)) 1, 2, 3, and 4 with the reverse (
)
primers 1, 2, 3, and 4 gave the fragments 1, 2, 3, and 4, containing a
hemimethylated EcoRV site at the positions illustrated. All
primers are shown in italic type. The results found with these
oligonucleotides are shown in Fig. 5.
Figure 5: Gel retardation of DNA fragments containing a GATATC site at various positions along a 150-bp oligonucleotide by the EcoRV methylase and endonuclease. A, autoradiographs of the gels obtained. The lanes 1-4 correspond to the fragments 1-4 shown in Fig. 2. The experiments were performed with the methylase and both un- and hemimethylated (indicated by an asterisk (*)) fragments. With the endonuclease only the unmethylated fragment was used. The hemimethylated one did not form a complex, and hence did not show a gel shift, with this protein. B, plots of the relative mobility (migration of protein-DNA fragment/migration of free DNA) versus distance of the EcoRV site from the 5`-end of the fragment. With the methylase, the triangles refer to hemimethylated DNA and the circles to unmethylated.
For the phasing analysis, five
different M13 derivatives which contain poly(dA) tracts (Withers and
Dunbar, 1993) were used. These have the poly(dA) tracts phased with a
polylinker and were a generous gift of J. C. Dunbar (Wayne State
University, Detroit, MI). Unfortunately the polylinker does not contain
an EcoRV site. Therefore a partially complementary pair of
oligonucleotides (AATTCCGATGATATCC and GGCTACTATAGGGGCC, both written
in the 5` 3` direction), containing an EcoRV site and EcoRI/XmaI cohesive ends, were cloned between the EcoRI and XmaI sites in the M13 derivatives, as
illustrated in Fig. 3. The plasmids were transfected into Escherichia coli TG1-competent cells, and the resulting
reisolated plasmids had their sequences confirmed by automated DNA
sequencing. These plasmids were then used as templates for PCR
reactions with a common forward primer (CACTTTATGCTTCCGGCT) and five
different reverse primers (phasing oligonucleotide 1,
AGTCACGACGTTGTAAAAA; 2, TCACGACGTTGTAAAACG; 3, CGACGTTGTAAAACGACG; 4,
ACGTTGTAAAACGACGGC; 5, GTTGTAAAACGACGGCCA). This produces five
fragments of the same length (206 bp) with a GATATC EcoRV site
phased with the poly(dA) tract (see Fig. 3).
Figure 3: The DNA fragments used for phasing analysis. These were prepared by inserting an EcoRV site between the EcoRI and XmaI sites of the five phasing plasmids described by Withers and Dunbar(1993). This gives the five plasmids shown, each of which contains a GATATC site at a defined distance from a poly(dA) tract. These distances, measured from the center of the EcoRV site (between the thymidine and deoxyadenosine bases) to the middle deoxyadenosine of the poly(dA) tract are given. The five plasmids were then amplified by PCR to give the 206-bp fragments illustrated which were used to confirm DNA bending by the EcoRV methylase and gave the results shown in Fig. 6.
Figure 6: Gel retardation of DNA fragments containing a GATATC site at varying separations from a poly(dA) tract. A, autoradiographs of the gels obtained. The lanes 1-5 correspond to the fragments 1-5 shown in Fig. 3. Two bands are once again visible for the methylase that correspond to un- and hemimethylated DNA (fast band) or dimethylated DNA (slow band). B, plots of the relative mobility (migration of protein-DNA complex/migration of free DNA versus spacing between the center of the GATATC site and the poly(dA) tract.
Initially we evaluated any DNA bending produced by the binding of the methylase to GATATC sequences using the circular permutation band mobility shift assay (Wu and Crothers, 1984; Liu-Johnson et al., 1986; Kim et al., 1989; Crothers et al., 1991). The six fragments shown in Fig. 1were bound to the methylase, in the presence of N-methyl-AdoMet, and the complexes formed subjected to nondenaturing gel electrophoresis. The results obtained are illustrated in Fig. 4A. This figure clearly shows that fragments containing a centrally located GATATC site give complexes with lower electrophoretic mobility than do those with the EcoRV site at the end of the DNA. Furthermore, there is a good correlation between the degree of retardation and the nearness of the GATATC site to the center of the fragment as shown in Fig. 4B, a graphical representation of the gel data. Close inspection of Fig. 4A shows that the retarded band is actually composed of two just-resolved species (this is most apparent in lane 5). However, the separation of the two bands is much less obvious here than in our earlier publication (Szczelkun and Connolly, 1995), possibly because of the much longer oligonucleotides used in this study. The two bands arise because of the presence of AdoMet that copurifies with the methylase and adds methyl groups to the first deoxyadenosine in the GATATC sequence to generate hemi- and dimethylated species. For Fig. 4B the mobility has been calculated using the leading edges of all the bands. We take this to represent the faster bands, which, as described earlier (Szczelkun and Connolly, 1995) represent un- and hemimethylated oligonucleotides, i.e. substrates for the methylase. However, very similar results were seen if the trailing edges were used for mobility determination. This is a measure of the slower bands, which represent dimethylated oligonucleotide products. The mobility of a DNA fragment through a gel is lowered as the distance between its two ends decreases and this most commonly arises because of DNA bending. Furthermore the more centrally located the bend the smaller the end to end distance and, in consequence, the greater the retardation on gels (Wu and Crothers, 1984; Hagerman, 1990; Crothers et al., 1991). The results shown in Fig. 4, A and B, are therefore consistent with the methylase bending of the DNA.
It is possible to calculate the degree of bending using the empirical equation described by Thompson and Landy(1988), in which the mobility of fragments containing the bend at the center and at the end on the molecules are compared. The fragments we have used do not contain an exactly centered GATATC site but nevertheless comparing the two most central sites (Fig. 1, fragments 3 and 4) with the two most peripheral (fragments 1 and 6) gave a bend angle of 62 ± 3° (number of determinations = 4). This angle was produced regardless of which combination of the two central and peripheral sites were used to evaluate µM and µE. An angle of 62° was found for both the faster, substrate, complexes and the slower, product, complexes. This demonstrates that the differences in mobility between enzyme/un- and hemimethylated DNA complexes and those that comprise enzyme/dimethylated DNA cannot be due to a change in DNA bending.
As
a positive control we have used the binding of the very well
characterized EcoRV endonuclease to the DNA fragments, in the
presence of Ca. The results are also shown in Fig. 4, A and B, and quantitative analysis,
exactly as above, gave a bending angle of 47 ± 3° (number of
determinations = 4). The slower migration of the
endonuclease-DNA complexes compared to those of the methylase-DNA are
consistent with the different molecular weights of the two proteins:
endonuclease 58 kDa (comprised of a dimer of subunit molecular weight
29 kDa), methylase a monomer of 32 kDa. Using the same
oligonucleotides, Vipond and Halford(1995) reported a bend angle of
53° ± 4° and with a different set of oligonucleotides,
Stöver et al. (1993) found an angle of 44
± 4°. A figure of 55° has been seen by crystallography
(Winkler et al., 1993; Kostrewa and Winkler, 1995). The
similarity of the results we obtain with the endonuclease, to those
seen by several investigators, validates the data and conclusions drawn
with the relatively uncharacterized methylase.
We have confirmed
that the EcoRV methylase bends DNA by using a second set of
oligonucleotides, shown in Fig. 2. Once again in this set of
experiments N-methyl-AdoMet was used as cofactor. One of the
four 150-bp oligonucleotides (fragment 1) contains an exactly
center GATATC sequence, and in the others this is moved toward the end
of the DNA. Using GATATC containing PCR primers an
analogous set, containing a hemimethylated EcoRV site, was
also prepared. The results found with these oligonucleotides are shown
in Fig. 5. With these fragments the resolution of
enzyme/substrate (un- and hemimethylated DNA) and enzyme/product
(dimethylated DNA) was poor, and the leading edges have been used to
evaluate fragment mobility. By analogy with the above experiment we
assume that the bands seen in Fig. 5, using the unmethylated
oligonucleotides, are actually a mixture of un-, hemi-, and
dimethylated species. Once again we observe that the more central the
location of the EcoRV site, and hence any bend induced on
methylase binding, the more retarded the complex runs on gels (Fig. 5, A and B). Quantitation using the
Thompson-Landy equation and fragments 1 (center) and 4 (end) gives a
bend angle of 60 ± 3° (four determinations) in excellent
agreement with the values produced above. When samples of these
unmethylated fragments were reisolated, following a normal pre-gel
shift incubation with the methylase, they could be gel-shifted by the
endonuclease (not shown). This confirms that at least some of the DNA
remains fully unmethylated, because hemimethylated species are not
gel-shifted by the endonuclease (see later). The EcoRV
endonuclease was again used as a positive control, and the data for the
unmethylated oligonucleotides shown in Fig. 5, A and B, give a bend angle of 46 ± 3°. Almost identical
results were seen when gel shifts were carried out with the methylase
and hemimethylated oligonucleotides (Fig. 5, A and B), and the bend angle found was 61 ± 3°. In this
instance the material in the shifted band will consist of hemi- and
dimethylated DNA, and unmethylated material cannot be present. These
experiments confirm the bending angle determined above but also show
that methylase/un- and hemimethylated DNA complexes have the nucleic
acid bent to the same degree. Thus all three possible methylase-nucleic
acid complexes (unmethylated, hemimethylated, and dimethylated) bend
DNA to the same extent, just over 60°. It is interesting that the
hemimethylated oligonucleotides shown in Fig. 2were not
gel-shifted by the endonuclease (not shown). Under the conditions used, i.e. in the presence of Ca
, an analogue of
the normal cofactor Mg
, the endonuclease binds
specifically to GATATC sequences and rejects all others (Vipond and
Halford, 1995). The function of methylation is to protect the host DNA
from endonuclease action, and the lack of binding of hemimethylated
(and presumably dimethylated) DNA, in the presence of cations,
presumably facilitates this.
When the EcoRV methylase binds to its GATATC target
site, the DNA is bent toward the major groove by just over 60°. The
degree of bending is identical, irrespective of whether the GATATC site
is un-, hemi-, or dimethylated. The hemimethylated species is the true
physiological substrate for the enzyme although the protein is also
capable of de novo CH group addition to
unmethylated sequences. Dimethylated oligonucleotides represent
enzyme-product complexes. The different gel mobility of the
enzyme-substrate and enzyme-product complexes, observed here and
previously (Szczelkun and Connolly, 1995), are most probably due to a
protein conformational change, given that the bending of the DNA does
not alter. The parallel, positive control experiments, carried out with EcoRV endonuclease, gave a bend angle of about 47°, in
excellent agreement with earlier crystallographic (Winkler et
al., 1993; Kostrewa and Winkler, 1995) and solution studies
(Stöver et al., 1993; Vipond and Halford,
1995). It is also apparent that the two proteins, which recognize the
same GATATC sequence, both bend DNA toward the major groove, although
the methylase induces more curvature than does the endonuclease.
It
has been extensively reported that the binding to DNA of a great number
of proteins introduces curvature into the double helix. This is often
observed with repressor/activator proteins that must bind specifically
to particular DNA sequences (Wu and Crothers, 1984; Kim et
al., 1989; Schultz et al., 1991; Aggarwal et
al., 1988; Kerrpola and Curran 1991a, 1991b) or enzymes,
especially restriction endonucleases, that must catalyze a chemical
transformation with high fidelity at their target sites (Thompsom and
Landy, 1988; Douc-Rasy et al., 1899; Rosenberg, 1991; Aiken et al., 1991; Stöver et al.,
1993; Winkler et al., 1993; Vipond and Halford, 1995; Kostrewa
and Winkler, 1995; Withers and Dunbar, 1993). In all of these cases the
bending of the DNA serves to increase the specificity of the proteins
and to aid in the selection of target sequences and in the
discrimination against noncognate ones. Proteins achieve selectivity
for particular DNA sequences by a variety of mechanisms that include:
1) direct readout, where proteins recognize the bases (Seeman et
al., 1976) and 2) indirect readout, where a particular,
nontypical, DNA structure is recognized (Otwinowski et al.,
1988; Matthews, 1988; Brennan and Matthews, 1989). It is known that
many DNA sequences have intrinsic curvature or other distortions, and
it is thought that this can used in the recognition process (Heitman,
1992). Thus it has been suggested that the GAATTC EcoRI
recognition sequence is intrinsically distorted and that this is
enhanced on binding to the EcoRI restriction endonuclease
(Thomas et al., 1989). A variant on this theme is the concept
of a DNA sequence that is easy to bend or distort. Here a bendable
cognate sequence can easily adapt to a complementary protein binding
site, whereas stiff noncognate sequences cannot. This seems to occur
with DNase I, where rigid poly(dA)poly(dT) tracts are poorly cut,
whereas more flexible sequences are able to adopt the bound, bent,
conformation and so are better substrates (Drew and Travers, 1984; Suck et al., 1988; Weston et al., 1992). It should be
recognized that bending of DNA also serves to correctly line up the
protein and nucleic acid partners and so enhance protein-DNA
interaction. Thus direct and indirect readout are intimately linked.
Formation of interactions between the bases and the protein may produce
DNA curvature, but equally the ability to adopt a bent conformation may
enhance favorable contacts between the two macromolecules. Finally,
although DNA bending is common with proteins that act on specific DNA
sequences, it is not universal. Thus certain repressors, e.g. the
repressor (Jordan and Pabo, 1988), the 434 Cro protein
(Wolberger et al., 1988) and the PvuII restriction
endonuclease (Cheng et al., 1994) have a bound DNA that is
essentially straight. These proteins are able to achieve
discriminations as good as those that utilize DNA bending.
It is
likely that DNA methylases, which must add CH groups
accurately to specific sequences, would utilize the mechanisms outlined
above to generate specificity. However, there is little structural data
for these proteins. Two protein-DNA structures, for the type II
DNA-deoxycytidine methylases HhaI (Cheng et al.,
1993; Klimasauskas et al., 1994) and HaeIII (Reinisch et al., 1995), have been solved by crystallography. With HhaI the target deoxycytidine is flipped out of the double
helix and into the active site but hardly any other distortion, and no
bending, takes place. Base flipping is also seen with HaeIII,
and in addition the other bases in the recognition site are also
severely distorted. However, once again DNA bending is not observed.
Both these methylases have similar recognition sites (HhaI,
GCGC; HaeIII, GGCC), containing only (G/C) base pairs. A
structure of the type II deoxyadenosine methylase TaqI (Labahn et al., 1994), has been solved but this is for the apoenzyme
lacking bound DNA. The distance from the putative DNA binding site to
the AdoMet cofactor suggested base flipping. However, in the absence of
bound DNA it was not possible to draw any conclusions about DNA
bending. Based on comparisons of several AdoMet-dependent enzymes, it
has been proposed that all DNA methylases have a common, two domain,
topology (Schluckebier et al., 1995). The AdoMet and catalytic
functions are present in the N-terminal domain, which is extremely
similar for the enzymes studied. A more variable C-terminal domain, the
target recognition domain, is involved in making specific interactions
with the cognate DNA sequence. Support for this proposal has come from
the recent successful modelling of the structure of a type I methylase EcoKI (Dryden et al., 1995). Again flipping of the
target deoxyadenosine base was required to bring it close to AdoMet.
With so little data it is difficult to generalize about methylase-DNA
structures. It does seem likely that all of the proteins will have the
two-domain structure, with a common catalytic domain, and will flip out
the target base, although this has still to be unequivocally
demonstrated for deoxyadenosine methylases. However, within the
framework of these common elements the rest of the DNA cognate sequence
could be handled very differently. Thus some methylases, such as HhaI and HaeIII, may be able to achieve selectivity
without inducing DNA curvature. Others, like EcoRV, build DNA
bending into the recognition mechanism. In this regard most of the
repressors mentioned above use a common DNA recognition element, the
helix-turn-helix motif. Some, but not all of them, cause DNA bending.
Support for the bendability of the GATATC, and related, sequences
has come from the study of several protein-DNA structures. With the EcoRV restriction endonuclease the 55° bend, toward the
major groove, is centered on the TpA step (Winkler et al.,
1993; Kostrewa and Winkler, 1995), which has a sharp kink. It has long
been recognized that the two base pairs in TpA steps, in particular,
and YpR steps, in general, are poorly stacked (Klug et al.,
1979; Calladine, 1982; Dickerson, 1983). This gives these steps high
flexibility and deformability and, in particular, the ability to adopt
high positive roles and become untwisted (Suzuki and Yagi, 1995).
Deformations to protein-bound DNA structures are thus very often
associated with TpA steps, e.g. Trp repressor (Otwinowski et al., 1988; Shakked et al., 1994), met repressor (Somers and Phillips, 1992), TATA-binding protein (Kim et al., 1993a, 1993b) and the resolvase (Yang and
Steitz, 1995). The latter two proteins produce DNA bending very similar
to the EcoRV endonuclease, despite the three proteins having
absolutely no structural similarities. In all of these cases the key
seems to be the instability of the TpA step, which provides a low
energy barrier to any DNA structural perturbations that must take place
on binding the protein. We would thus propose that the bending seen
with the EcoRV methylase is centered on the TpA step of the
GATATC sequence. A kink, at this step, directed toward the major
groove, will cause this groove to become deeper and narrower and is
also likely to cause unwinding of the helix. This is exactly what is
seen with the EcoRV endonuclease. We have recently shown that
the methylase makes many of its contacts to the GATATC bases via the
major groove (Szczelkun et al., 1995). The consequence of the
motion we have described is actually to make the major groove
determinants of the bases less accessible and with the endonuclease the
middle TpA step is not contacted by the protein directly (Winkler et al., 1993; Kostrewa and Winkler, 1995). However, these
bases can be efficiently recognized, indirectly, by facilitating the
distortion and, also by the formation of new non-Watson-Crick hydrogen
bonds or dipolar interactions between them (Winkler et al.,
1993; Kim et al., 1993a). The problem of accessibility of the
target deoxyadenosine base, which must be placed near the AdoMet, can
also be solved by swinging it out of the helix, as for HhaI.
This means that the cofactor does not need to find its way into the
smaller major groove. Finally, we have also shown that important
contacts are made between the methylase and the phosphates pNpNpGpA on
each DNA strand. These lie to the 5`-ends of the symmetrical GATATC
site (Szczelkun et al., 1995). As pointed out for the
TATA-binding protein (Kim et al., 1993a), the bending we
describe tends to bring bordering phosphates from each strand closer to
each other and provide strong polar anchoring interactions which will
help to stabilize the complex.