(Received for publication, August 22, 1994; and in revised form, January 11, 1995)
From the
SmaI endonuclease recognizes and cleaves the sequence
CCCGGG. The enzyme requires magnesium for catalysis; however,
equilibrium binding assays revealed that the enzyme binds specifically
to DNA in the absence of magnesium. A specific association constant of
0.9
10
M
was determined
for SmaI binding to a 22-base duplex oligonucleotide.
Furthermore, the K
was a function of the
length of the DNA substrate and the enzyme exhibited an affinity of 1.2
10
M
for a 195-base
pair fragment and which represented a 10
-fold increase in
affinity over binding to nonspecific sequences. A K
of 17.5 nM was estimated from kinetic assays based
on cleavage of the 22-base oligonucleotide and is not significantly
different from the K
estimated from the
thermodynamic analyses. Footprinting (dimethyl sulfate and missing
nucleoside) analyses revealed that SmaI interacts with each of
the base pairs within the recognition sequence. Ethylation interference
assays suggested that the protein contacts three adjacent phosphates on
each strand of the recognition sequence. Significantly, a predicted
protein contact with the phosphate 3` of the scissile bond may have
implications in the mechanism of catalysis by SmaI.
The molecular mechanisms of sequence-specific recognition by DNA
binding proteins is a complex phenomena of which the details are still
emerging. In recent years, resolution of the structures of a number of
DNA-binding proteins and of proteinDNA cocomplexes has provided
insight into the variety of structures of the DNA-binding domains, the
network of bonding between the macromolecules, and also the
significance of both protein and DNA conformational changes in the
specific recognition
process(1, 2, 3, 4) .
The
endonucleases of the bacterial type II restriction-modification systems
provide excellent models by which to study mechanisms of sequence
specificity of DNA-binding
proteins(5, 6, 7) . Two of the endonucleases, EcoRI and EcoRV, have been extensively studied by
both biochemical and x-ray crystal structural analyses and have
revealed distinct mechanisms by which they achieve their high degree of
sequence specificity: the cleavage selectivity of EcoRI is
derived from both binding and catalytic specificity (8, 9) whereas the EcoRV endonuclease
exhibits the same intrinsic affinity for all DNA sequences but achieves
catalytic specificity in the presence of
magnesium(10, 11, 12) . The crystal
structures also revealed that the enzymes utilize novel protein
structures for DNA recognition and that there are significant
differences in the topology of the respective proteinDNA
complexes. In the EcoRI
DNA complex, the protein
approaches the DNA predominantly from the major groove and makes
specific interactions in the major groove with each of the purines and
pyrimidines of the recognition site. In contrast, the EcoRV
endonuclease approaches the DNA predominantly from the minor groove
although the sequence specific contacts are made in the major groove.
Protein-induced unwinding and bending of the DNA, including unstacking
of the central base pairs, are features that characterize both
endonucleases. However, whereas the net effect in the EcoRI
DNA complex is widening of the major
groove(9) , the DNA in the EcoRV
DNA complex is
reminiscent of A-form DNA with a more narrow and deeper major
groove(10) . Furthermore, the overall kink and curvature
appears to preclude the formation of hydrogen bonds between EcoRV and the central base pairs of the recognition sequence
such that direct interactions only occur at the outer 2 base pairs of
each half site of the recognition palindrome (10) .
The
general lack of sequence similarity between the type II endonucleases (13) has restricted the use of sequence comparisons to probe
the structure-function relationship of these enzymes. Furthermore,
detailed biochemical and structural analyses of endonucleases has in
the past been limited. Nonetheless, recent reports suggest that EcoRI and EcoRV may be paradigms for other
endonucleases. 1) It has now been shown (14) that TaqI
is similar to EcoRV in that it achieves specificity only in
the presence of magnesium. 2) The recent determination of the structure
of the BamHI endonuclease (15) has revealed an overall
conformation very similar to that of EcoRI. The structural
similarity occurs in the absence of any obvious sequence similarity
between these enzymes. Furthermore, it has been suggested that the
structure of the ``common core motif'' which, in EcoRI and BamHI, provides an ideal scaffold for
positioning the active sites of the enzyme near the scissile bond, may
also be conserved in other enzymes that similarly cleave their
hexanucleotide recognition sequences to yield a 5`, 4 bp ()stagger (15) . 3) Similarities have been detected
in the architecture of the active site of all four endonucleases (EcoRI(9) , EcoRV(10) , BamHI(15) , and PvuII(16, 17) ) for which crystal structures
are currently available.
Analyses of additional endonucleases should therefore enable potential trends to be discerned in the mechanism of recognition by these DNA-binding proteins. Furthermore, the existence of endonuclease isoschizomers makes it possible to analyze and compare the mechanism by which different enzymes interact with the same DNA sequence and how the requirements for recognition and catalysis are satisfied. We have initiated a comparative study of the SmaI and XmaI endonucleases. The enzymes recognize the sequence CCCGGG but cleave at different positions within the sequence such that SmaI cleaves at the internal CpG to yield a blunt-end scission whereas XmaI cleaves between the external cytosines to yield a 4 bp stagger. In the present study, an initial examination has been undertaken of the mechanism of sequence-specific recognition by the SmaI endonuclease and compared to that of EcoRV and PvuII which also produce blunt-end scissions.
The DNA in the
proteinDNA complexes and the free DNA was electrotransferred onto
NA45 membranes, eluted with 1 M NaCl, extracted with
phenol-chloroform, and recovered by ethanol precipitation. Strand
cleavage at the sites of modification was carried out by incubation of
the sample in 100 µl of 10% piperidine at 90 °C for 30 min.
Samples were three times lyophilized to remove the piperidine and
subsequently analyzed on denaturing (20%) polyacrylamide gels.
Pyrimidine modification of the 5`-end-labeled oligonucleotides (10 µg) was carried out essentially as described by Brunelle and Schlief (22) . The reaction was stopped by the addition of 200 µl of hydrazine stop buffer (0.3 M sodium acetate, pH 7.0, containing 1 mM EDTA). The DNA was ethanol precipitated in the presence of 5 µg of tRNA, washed with 80% ethanol, and dried under vacuum. Depurination reactions were carried out using formic acid as described previously(22) .
Figure 1:
Gel retardation
assays of DNA binding by SmaI endonuclease. A,
proteinDNA complexes formed after incubation of the specific
22-bp duplex oligonucleotide (0.3 nM) with SmaI
endonuclease. Lanes 1-7 correspond to 0, 0.2, 0.38,
0.75, 1.5, 3.0, and 6.0 nMSmaI, respectively. B, electrophoresis of binding reactions in which SmaI
was incubated with a 22-bp nonspecific DNA fragment (0.3 nM). Lanes 1 and 2 correspond to endonuclease
concentrations of 0.75 and 3 nM, respectively. C,
0.3-nm specific oligonucleotide (lane 1) and nonspecific
oligonucleotide (lane 2) were incubated with SmaI
endonuclease for 1 h at room temperature in the presence of 10 mm of
MgCl
.
Binding of the endonuclease to the recognition fragment occurs over a narrow range of KCl concentrations with maximum binding occurring at approximately 25 mM KCl. The apparent salt dependence of the protein-DNA interaction was anion-specific. At higher (>30 mM) salt concentrations, the inhibition by potassium glutamate was considerably less than that of potassium chloride at an equivalent concentration (Fig. 2). A similar effect of glutamate has been observed with RsrI (5) as well as other DNA-binding proteins including DNA polymerase III holoenzyme (23) and the lac repressor(24) . In the latter study, glutamate was concluded to be an inert anion in the relative competition between anions and DNA phosphate groups for binding to the protein. It is significant that, as also shown in Fig. 2, binding of SmaI to the specific substrate also occurred in the absence of KCl or potassium glutamate. SmaI has an absolute requirement for potassium, as well as magnesium, for catalytic activity. This requirement, therefore, appears to reflect a property of the cleavage reaction rather than a role for potassium in substrate recognition.
Figure 2:
Salt dependence of the formation of the SmaIDNA complexes. The endonuclease (2 nM) was
incubated with the specific oligonucleotide (0.3 nM) in 20
mM HEPES, pH 8.0, containing 0.5 mM EDTA and 0.1
mM dithiothreitol and various concentrations of KCl (C) or potassium glutamate (G) as shown. In the last
lane (0 mM), the reactions were carried out in 20 mM HEPES, pH 7.8, containing 0.5 mM EDTA and 0.1 mM dithiothreitol. Binding reactions were carried out at room
temperature for 1 h.
Preliminary studies revealed that the SmaIDNA
complexes are not efficiently retained on nitrocellulose filters under
standard binding reaction conditions. Quantitation of the
protein
DNA complexes in gel retardation assays was therefore used
to estimate the equilibrium association constant for the protein-DNA
interaction. The binding isotherm for the interaction of SmaI
with the 22-bp oligonucleotide is shown in Fig. 3A.
Incubation of a fixed concentration of the enzyme with an increasing
concentration of the recognition fragment resulted in a saturatable and
hyperbolic binding curve. The site-specific association constant was
calculated from the derived Scatchard plot (Fig. 3B)
and yielded a K
of 0.91 (±0.32)
10
M
. Competition assays,
similar to those described by Terry et al.(20) and
Aiken and Gumport (5) were used to determine the relative
affinity of SmaI for specific and nonspecific DNA sequences.
The K
for a 22-bp GC-rich fragment, lacking the
recognition site, was estimated to be 1.09
10
M
. It can be estimated from the
minimum size of the oligonucleotide required for maximum activity of
the enzyme that the stable interaction of SmaI with DNA
appears to require at least 12 bp. The competitor oligonucleotide
therefore contains at least 10 possible binding sites for the
endonuclease. Consequently, a K
of approximately 1
10
M
/site can be
estimated. This value suggests that there is at least a 1,000-fold
difference in the affinity of the enzyme for its recognition sequence
over that for non-cognate sequences.
Figure 3: A, binding isotherm of SmaI endonuclease to a 22-bp specific recognition oligonucleotide. 4.0 nM endonuclease was incubated with 0-80 nM DNA as shown. B, Scatchard analysis of the binding data.
The specificity of the
endonuclease was further examined using a 195-bp recognition fragment.
Titration of the DNA substrate with increasing concentrations of the
enzyme again resulted in the appearance of a single retarded complex (Fig. 4A). There was no evidence of multiple
proteinDNA complexes indicative of nonspecific binding of the
endonuclease to the DNA fragment. At the higher concentrations of
enzyme there was smearing of the band corresponding to the
protein
DNA complex. This effect arises from adding a larger
volume of the enzyme (and hence increasing the percentage of glycerol
(in which the enzyme is stored)) in the samples. Decreasing the
concentration of glycerol in the reactions eliminated the smearing.
Titration of the enzyme with the 195-bp fragment and the resulting
binding isotherm is shown in Fig. 4, B and C.
The corresponding specific association constant was calculated to be
1.23 (± 0.1)
10
M
and represents an affinity which is an order of magnitude greater
than that observed for the short oligonucleotide substrate. Binding
assays carried out in the presence of the 185-bp competitor fragment
yielded a value of 3.7
10
M
for the K
as determined directly from the
Dixon plot (Fig. 4D). The site-specific association
constant for non-cognate sites (K
/number of
potential binding sites(25) ) is approximately 2.08
10
M
. The SmaI
endonuclease therefore exhibited an affinity for its recognition site
approximately 10
times greater than that of random DNA
sequences and which is indicative of sequence-specific binding by the
enzyme in the absence of magnesium.
Figure 4:
Analysis of binding of SmaI
endonuclease to a specific 195-bp substrate. A, titration of
0.3 nM specific DNA with SmaI endonuclease. Lanes
1-7 correspond to 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4
nM endonuclease, respectively. Binding reactions were carried
out in 20 mm HEPES, pH 7.8, containing 20 mM potassium
glutamate for 1 h at room termperature. The proteinDNA complexes
were visualized by autoradiography after electrophoresis of the samples
on 7.5% non-denaturing gels. B, titration of 1 nMSmaI with increasing concentrations of 195-bp substrate. Lanes 1-9 correspond to 0, 0.01, 0.025, 0.05, 0.10,
0.25, 0.5, 1.0, 2.5, and 5.0 nM DNA, respectively. C,
binding isotherm (inset) and derived Scatchard analysis of the
binding data shown in B. D, Dixon plot representing
competition for SmaI binding by a 185-bp competitor lacking
the CCCGGG recognition sequence.
Steady-state kinetic analysis of SmaI cleavage of the specific oligonucleotide substrate is
shown in Fig. 5. The reactions were carried out at room
temperature, and no dissociation of the double-stranded substrate
during the course of the reaction was evident in the gel
electrophoresis assays. The endonuclease obeys Michealis-Menten
kinetics and can be saturated with substrate. The kinetic analyses
yielded a K of 17.3 nM and a k
of 23.8 min
(average of
three determinations).
Figure 5: Steady-state kinetic analysis of SmaI endonuclease cleavage of the 22- base duplex oligonucleotide substrate. SmaI was incubated with 0.005-0.085 µM DNA as described under ``Materials and Methods'' and the initial rates of hydrolysis determined. A, initial velocity of cleavage as a function of the substrate concentration. B, Eadie-Scatchard analysis of the initial velocity data. The line represents a linear least-squares fit to the data.
Figure 6:
A, DMS interference footprinting assays of
the SmaIDNA complexes for the top and bottom strand of
the 22-bp recognition fragment. Lane G corresponds to the
G+A specific (Maxam-Gilbert) sequencing products. B (bound) and F (free) correspond to the fragments
generated from the DNA isolated from the protein
DNA complexes and
free DNA, respectively. B, missing nucleoside analysis of SmaI binding to the 30-bp recognition oligonculeotide. Lanes G+A correspond to the fragments generated from the
acid depurination of the substrate; T+C represents the
depyrimidation of the substrate DNA. B and F correspond to the protein-bound and free DNA as described in A. C, histogram summary of the missing nucleoside
analysis for SmaI binding to the top and bottom strands of the
30-bp recognition oligonucleotide. Relative intensity represents the
ratio of the intensities of the free/bound sample for each
fragment.
The importance of the guanine bases in sequence specific binding was further indicated by missing nucleoside analyses(22) . This latter approach, in which the purine or pyrimidine bases can be selectivley removed from the DNA, provides a method for analyzing the contribution of both purines and pyrimidines to the specific recognition of the DNA by the protein. Furthermore, while methylation effects may arise from steric hindrance, the effect of base removal, as used in these experiments, more accurately reflects potential H-bond interactions.
The oligonucleotide substrate used in the missing base analysis was a 30-bp oligomer GCATGCACATGACTGGCCCGGGATCCAGT, ACGTGTACTGACCGGGCCCTAGGTCATCGT. The internal sequence of the oligonucleotide was identical to that of the 22-mer, used in the methylation interference assays, but the fragment contained additional bases at the 5`-end to facilitate the recovery of more fragments flanking the recognition site after cleavage of the DNA. Maxam-Gilbert sequencing of each strand of the control oligonucleotide revealed that the upper strand was heterogeneous in that it contained a dG in addition to the correct dC at the third position of the recognition site, i.e. CC(C/G)GGG (and is apparent in the control T+C lane in Fig. 6B). The presence of the mixed bases is presumed not to interfere with the footprinting experiments since the fragments with the incorrect recognition sequence will not be specifically bound by the endonuclease.
Fig. 6B is representative of the
autoradiograms obtained for the missing base analyses of the SmaI-DNA interaction. An autoradiogram corresponding to the
top strand only is shown for illustrative purposes and the results for
both top and bottom strands are summarized in Fig. 6C and are representative of three different experiments. Comparison
of the band intensities in the lanes corresponding to the protein-bound
and free DNA revealed that depurination of the guanines within the
recognition site significantly reduced protein binding. The absence of
guanines in the lanes corresponding to the proteinDNA complex was
evident for both the top and bottom strands of the recognition
fragment. Similarly, in the protein-bound lanes there was a marked
reduction in intensity of the fragments corresponding to the
modification of cytosines within the recognition sequence. However,
there appeared to be an increase in the band intensity for the cytosine
of the central, CpG dinucleotide relative to the adjacent cytosines of
the recognition site. Similar differences in the relative intensity
were detected in the free and control DNA samples and suggest that this
base may be generally more reactive toward the modification reagent
than the adjacent bases. Nonetheless, the free/bound ratio was
consistently less than that of the outer two cytosines of the
recognition sequence. There appeared to be no significant interaction
of the SmaI endonuclease with bases flanking the CCCGGG
recognition site.
The interference assays suggest full site
recognition by the SmaI endonuclease. At least part of the
recognition appears to occur in the major groove of the DNA, as
evidenced by the effect of guanine N methylation
on the formation of the protein
DNA complexes. Furthermore, the
concordance between the DMS and missing base analyses suggests the
effect of DMS methylation may be attributed to the loss of hydrogen
bond interactions rather than a steric effect of the methyl group.
Figure 7: A, ethylation interference footprints of SmaI binding to the top strand of the recognition fragment. Lanes G+A and T+C correspond to the Maxam-Gilbert sequencing products. B and F correspond to the fragments derived from the protein-bound and free DNA, respectively. C1 represents the ladder of sized DNA fragments generated by partial phosphodiesterase cleavage of the substrate DNA. C2 corresponds to fragments generated from XmaI cleavage of the duplex substrate and which yields a 17- and 12-base fragment resulting from cleavage of the top and bottom strands, respectively. B, histogram summary of the phosphate alkylation interference assays for the top and bottom strands of the duplex substrate.
The
interference pattern obtained for the SmaIDNA complex
revealed only three potential phosphate contacts/strand. The contacts
were symmetrical and corresponded to the GGG trinucleotide on each
strand. Inspection of the autoradiogram and resulting histogram summary (Fig. 7B) suggested there may be additional
protein-phosphate contacts beyond the recognition site on the upper
strand. However, the relative intensity (bound/free) is considerably
less than that of the proposed phosphate contacts within the
recognition site.
The SmaI endonuclease appears to readily
discriminate between specific and nonspecific sequences in the absence
of magnesium. The endonuclease formed a stable complex with a short (22
bp) recognition oligonucleotide but failed to bind to oligonucleotides
lacking the cognate sequence. Furthermore, titration of a 195-bp
fragment containing the recognition site revealed only a single
proteinDNA complex even in the presence of greater than a 20-fold
molar excess of the enzyme.
The specific association constants have
been determined for only a limited number of endonucleases. The
affinity of SmaI for short oligonucleotide substrates is lower
than that of EcoRI for which an association constant of
approximately 1 10
M
(for a 34-bp substrate) has previously been
reported(20) . Nonetheless, there are additional examples of
endonucleases which have relatively low specific association constants
yet bind specifically to DNA. Thielking and co-workers(11) ,
using a 20-mer as a substrate, have determined an affinity constant of
4
10
M
for an inactive
mutant of EcoRV that in the presence of magnesium binds
specifically to DNA but fails to cleave the substrate. Furthermore,
Jen-Jacobsen et al.(27) constructed N-terminal
deletion mutants of EcoRI of reduced (100-fold) binding
affinity but which retained the ability to discriminate between
specific and nonspecific sequences. Furthermore, the specificity index
for SmaI of 10
-10
is also consistent
with sequence-specific binding when compared to the less than a 40-fold
difference in the affinity of binding of EcoRV to specific and
nonspecific sequences (25) and the 4-fold difference reported
for TaqI(14) .
The affinity of the SmaI
endonuclease for specific (and nonspecific) sequences was also a
function of the length of the DNA substrate. Increasing the substrate
from 22 to 195 bp resulted in an apparent 10-fold increase in the
binding affinity. Similar trends have previously been observed for
other proteins including the lac repressor (28) and
the EcoRV endonuclease (29) . In addition, Taylor et al. (25) have reported that the effective equilibrium
constant for EcoRV binding to its recognition sequence
(calculated from preferential cleavage assays) ranged from 5
10
(55-mer) to 2.5
10
(381-mer) to 1
10
M
for a 3.9-kilobase
fragment. The dependence of the enzyme affinity (determined as K
for several endonucleases) on the length of the
DNA substrate is frequently interpreted in terms of long range effects
such as facilitated diffusion. It should be noted, however, that
although facilitated diffusion has been well documented for EcoRI (29, 30) there is no significant
difference in the K
of EcoRI for a 34-bp
and pBR322 substrate(20) . Furthermore, for SmaI the
apparent dependence of K
on the length of the
substrate may also the reflect the conformation or conformational
stability of the substrate: the 22-base duplex substrate is a GC-rich
oligonucleotide. The recognition sequence of a decamer containing the
CCCGGG recognition sequence has been shown to assume an A-form
conformation under cystallographic conditions(32) . The
substrate DNA may therefore be subject to local DNA distortions.
Furthermore, binding of SmaI appears to bend the DNA toward
the major groove(19) . It is possible that the intrinsic
sequence-specific conformation of the substrate or the SmaI-induced DNA conformational changes may not be stable
under the conditions (of low cation concentrations) used for the
binding assays. The longer substrate may help stabilize such
conformations.
It generally has been noted that K and K
for an enzyme are not necessarily
equivalent (33) . There is a considerable (greater than
100-fold) difference in the value of the affinity constant estimated
for EcoRI from the the kinetic and thermodynamic
assays(31, 34) . The discrepancy has been attributed
to differences between the dissociation rate constant and the cleavage
rate constant(34, 35) . A much smaller,
3-5-fold, difference is apparent between the K
and K
reported for RsrI using
pBR322 as a substrate(36) . The K
(17.5
nM) for SmaI for the 22-base oligonucleotide is lower
than that reported for several other endonucleases. However, many of
the previously described kinetic assays have utilized very short
(8-12 bp) oligonucleotide substrates (37, 38, 39) which may not be optimal for
endonuclease binding(35) . A K
near 30
nM reported for the PaeR7 endonuclease with a 30
nucleotide substrate (40) is comparable to that obtained for SmaI. Furthermore, the K
for SmaI is not significantly different from the K
(calculated from the inverse of the equilibrium association
constant). A mechanism in which strand scission is the rate-limiting
step in the SmaI cleavage reaction would be one interpretation
of the similarity between the kinetic and thermodynamic constants.
Alternatively, magnesium, which is present only in the kinetic assays,
may increase the DNA affinity of SmaI so that the value of K
more closely approaches the K
. Magnesium, in addition to conferring substrate
specificity, has been attributed with increasing the DNA affinity of EcoRV(29) , and it has been suggested that PaeR7 fails to bind DNA in the absence of magnesium (40) . For EcoRI it has been shown that magnesium does
not influence the equilibrium association constant(27) . There
appears, then, to be a variable role for magnesium in the activity of
the type II endonucleases. The isolation of catalytic defective mutants
that retain the ability to bind to DNA will be useful for examining the
role of magnesium in the sequence-specific binding of the SmaI
endonuclease.
Footprinting analyses of the SmaIDNA
complexes suggest a direct readout of each of the bases within the
recognition site by the endonuclease. DMS interference analyses
indicated that the protein contacted each of the guanines of the
recognition site within the major groove of the DNA. This conclusion
was supported by the missing base analyses and implies specific
hydrogen bond interactions between the protein and the donor and/or
acceptor groups of the purines. Missing base analyses also implicated
interactions between the protein and each of the cytosines within the
recognition sequence. Since both the DMS interference assays and the
missing base analyses implicated each of the guanine bases, the
selective removal of the cytosines in the latter assays may result in
subtle changes in the conformation of the guanines and, therefore,
indirectly influence binding of the enzyme. Nonetheless, N-4
methylation of the second cytosine of the recognition sequence by the
cognate methylase inhibits binding of the enzyme. C-5 methylation of
the external cytosine also markedly reduces the K
of the enzyme
suggesting a direct role for the
cytosines in the sequence specific recognition by SmaI.
Protein-DNA contacts at each of the base pairs within the recognition
site also characterizes the EcoRI (9) and PvuII (16) protein
DNA complexes.
Ethylation
interference assays revealed that SmaI interacts with the
phosphates of three adjacent bases on each strand of the recognition
hexanucleotide. Lesser and colleagues (35) examined the
protein-phosphate interactions in the EcoRIDNA complex
and have similarly determined that only six symmetry related phosphates
have a crucial role in recognition, although the pattern of phosphate
interactions is quite distinct for SmaI and EcoRI. SmaI also exhibited ``half-site'' recognition of the
phosphates by interacting only with those phosphates 5` of the
guanosines. The proposed phosphate contacts for SmaI therefore
differ from the other characterized blunt-end cutters, EcoRV (10) and PvuII(16) , both of which exhibit an
extensive network of phosphate interactions both within and flanking
the recognition sequence.
Identification of the potential
protein-phosphate contacts is important not only for the analysis of
sequence specific recognition but also for the potential mechanism of
catalysis. Substrate-assisted catalysis has recently been suggested for
the EcoRI and EcoRV endonucleases based on the
structural similarity of the PD(X) EXK catalytic
motif(41, 42) . It has been proposed that the
attacking water molecule in the cleavage reaction is activated by the
phosphoryl oxygen of the phosphate group on the 3`-side of the scissile
bond. Recent studies of EcoRI and EcoRV cleavage of
substrates containing phosphate substitutions 3` of the scissile bond
are consistent with the proposal of substrate assisted
catalysis(43) . In neither of the protein
DNA complexes
does the phosphate make a contact required for specific
binding(43) . Although the active site structure BamHI
is very similar to that of EcoRI and EcoRV, the
sequence of the catalytic motif is not well
conserved(15, 44) . Consequently, it has been
suggested that BamHI may utilize an alternative mechanism for
the activation of a water molecule for nucleophilic attack during
catalysis(15) . SmaI similarly lacks the consensus
PD(X)
EXK sequence motif(45) , and a
protein contact with the 3`-phosphate is inferred from the ethylation
interference studies. SmaI may, therefore, resemble BamHI in a reaction mechanism that differs from that proposed
for EcoRI and EcoRV. The requirement for KCl by SmaI also suggests some differences in the reaction mechanism.
Potential mechanisms of sequence discrimination and catalysis by the type II endonucleases have begun to emerge from recent biochemical and structural analyses of these proteins. The architecture of the active site appears to be conserved although the functional amino acids may differ(15, 16) . Furthermore, the similarity in the overall structure of the EcoRI and BamHI endonucleases has prompted the suggestion of a relationship between the position of cleavage within the recognition site and the structure of the enzyme(15) . Anderson (6) has suggested a correlation between the position of the scissile bond (i.e. within the major or minor groove of the DNA) and the orientation of the DNA-binding domain. The SmaI and EcoRV endonucleases are similar in that they each cleave within a 6-bp recognition sequence to produce a blunt-end scission. However, they appear to differ significantly in the interaction with their specific sequences: SmaI belongs to the class of enzymes designated by Zebala et al.(14) as SEL (Specificity Early and Late) whereas EcoRV is the prototype of the SLO class at which specificity occurs predominantly at the cleavage reaction(25, 29) . SmaI induces bending of the DNA, and although the direction of the bend is similar to that of EcoRV the bend angle is significantly smaller(19) . Consequently, whereas the extensive EcoRV-induced DNA conformational changes preclude the formation of hydrogen bonds at the central base pairs of the binding site(10) , SmaI appears to interact with each of the base pairs within the recognition sequence. Furthermore, it appears that the amino acids within the active sites of SmaI and EcoRV differ, and SmaI may not utilize substrate assisted catalysis. The only other blunt-end cutter that has been examined to date is PvuII(16) . Although the role of magnesium in the specificity of the PvuII has not yet been determined, the enzyme displays certain similarities with SmaI including (i) interaction with each of the base pairs within the recognition site, (ii) a potential protein contact to the phosphate 3` to the scissile bond, (iii) does not significantly bend the DNA, and (iv) the active site residues differ from the consensus sequence motif. It will be of interest to determine whether there is any conservation of the structures of the PvuII and SmaI endonucleases.
In contrast to the general lack of sequence similarities between the type II restriction endonucleases, current studies are beginning to reveal some common themes in their mechanism of interaction with their DNA substrate. Determination of the structure and interactions of more endonucleases will provide insight into the extent of the diversity of mechanisms by which these enzymes achieve their binding and catalytic specificity.