Download Recognition and Cleavage of DNA by Type‐II Restriction

Eur. J. Biochem. 246, 1-22 (1997)
0 FEBS 1997
Review
Recognition and cleavage of DNA by type-I1 restriction endonucleases
Alfred PINGOUD and Albert JELTSCH
Institut fur Biochemie, Fachbereich Biologie, Justus-Liebig-Universitat,Giessen, Germany
(Received 5 November 1996/20 January 1997) - EJB 96 163YO
Restriction endonucleases are enzymes which recognize short DNA sequences and cleave the DNA
in both strands. Depending on the enzymological properties different types are distinguished. Type I1
restriction endonucleases are homodimers which recognize short palindromic sequences 4- 8 bp in length
and, in the presence of Mg”, cleave the DNA within or next to the recognition site. They are capable of
non-specific binding to DNA and make use of linear diffusion to locate their target site. Binding and
recognition of the specific site involves contacts to the bases of the recognition sequence and the phosphodiester backbone over approximately 10- 12 bp. In general, recognition is highly redundant which explains the extreme specificity of these enzymes. Specific binding is accompanied by conformational
changes over both the protein and the DNA. This mutual induced fit leads to the activation of the catalytic
centers. The precise mechanism of cleavage has not yet been established for any restriction endonuclease.
Currently two models are discussed: the substrate-assisted catalysis mechanism and the two-metal-ion
mechanism. Structural similarities identified between EcoRI, EcoRV, BarnHI, PvuII and Cfrl 01 suggest
that many type I1 restriciton endonucleases are not only functionally but also evolutionarily related.
Keywords: protein -nucleic-acid interaction ; restriction-modification system ; restriction endonuclease ;
DNA methyltransferase; non-specific DNA binding ; facilitated diffusion; specific DNA binding ; DNA
recognition; phosphodiester bond cleavage ; catalytic mechanism.
Overview of restriction endonucleases
Restriction endonucleases are parts of restriction-modification (RM) systems which occur ubiquitously among prokaryotic
organisms. RM systems comprise an endonuclease and a methyltransferase activity. They serve to protect bacterial cells against
bacteriophage infection, because incoining foreign DNA is
highly specifically cleaved by the restriction enzyme if it contains the recognition sequence of the endonuclease. The cellular
DNA is protected from cleavage by a specific methylation
within the recognition sequence, which is introduced by the
methyltransferase (reviews : Noyer-Weidner and Trautner, 1993;
Cheng, 1995). RM systems can be divided into different types
according to the subunit composition and the cofactor requirement (Table 1) (reviews: Wilson, 1991 ; Wilson and Murray,
1991; Heitman, 1993). Type I RM systems consist of three subunits: R for restriction, M for modification and S for specificity.
They usually form pentameric complexes comprising two R, two
M and one S subunit which recognizes a bipartite recognition
sequence. Modification requires the presence of S-adenosyl-methionine (AdoMet) and occurs within the recognition sequence,
while cleavage takes place at distant random sites and requires
AdoMet as well as ATP which is hydrolyzed in large amounts
Correspondence to A. Pingoud, Institut fur Biochemie, Fachbereich
Biologie, Justus-Liebig-Universitat, Heinrich-Buff-Ring 58, D-35392
Giessen, Germany
Fax: +49 641 99 35409.
Abbreviutions. RM, restriction modification; AdoMet, S-adenosylmethionine.
Nore. This Review will be reprinted in EJB Reviews I997 which will
be available in April 1998.
following DNA cleavage (review: Bickle, 1993). Type I1 RM
systems comprise two separate enzymes, a homodimeric restriction endonuclease which is not dependent on AdoMet or ATP,
and a monomeric methyltransferase which only requires AdoMet as cofactor. Type I1 enzymes typically recognize palindromic recognition sites, 4-8 bp in length. Modification occurs
within the recognition site, cleavage within or in the immediate
vicinity of the recognition site. Some RM systems, originally
defined as type 11, turned out to have unusual properties and,
therefore, form distinct subgroups. One group, type He, comprises systems where the endonuclease is allosterically activated
by binding of a second recognition sequence (effector), e.g. the
EcoRII system. Such enzymes require two recognition sites for
DNA cleavage (review: Kriiger et al., 1995). A second group,
type IIS, comprises systems where a monomeric endonuclease
cleaves the DNA at a defined distance outside of the non-palindromic recognition site, e.g. FokI (review: Szybalski et al.,
1991). Type 111 RM systems consist of two subunits: R and M.
While M alone functions as a methyltransferase in the presence
of AdoMet, restriction requires the cooperation of R and M, as
well as the presence of ATP which, however, is not hydrolyzed
during the reaction. Modification occurs within the recognition
site, cleavage approximately 25 bp downstream from this site
(review: Bickle, 1993). Recently, several new RM systems have
been characterized and some of them, like the E d 7 1 system,
have been classified as type IV, because DNA cleavage is stimulated by AdoMet, but not dependent on ATP as in type I11
systems (Janulaitis et al., 1992a,b; Janulaitis, A., personal communication). Unique among restriction endonucleases are the
Bcgl-like enzymes which are dependent on AdoMet and cleave
DNA on both sides of their recognition sequence, thereby excis-
2
Pingoud and Jeltsch (Em J. Biochem. 246)
Table 1. Overview over different types of restriction endonucleases and related enzymes. Cofactors that are not essential for DNA cleavage to
occur but stimulate the reaction are given in brackets.
Type
Cofactor(s)
Recognition sequence
Cleavage
ATP (hydrolysis)
Ado Met
Mgz+
asymmetric, interrupted
7 bP
-TGA ( N , ) TGCT-
statistically at great distance
Type I1
EcoRV
Mg2+
palindromic 4-8 bp
GATATC
within recognition sequence
-GAT~ATC
- CTA~TAG-
Type IIS
Mi' +
asymmetric
4-7 bp
-GGATG-
outside
at defined distance
-GGATG-N,1
-CCTAC-N,,T
Mg"
DNA
palindromic
4-8 bp
-CC
GG-
within recognition sequence
ATP (no hydrolysis)
Mg2+
(AdoMet)
(DNA)
asymmetric
not interrupted
5-6 bp
-AGACC-
outside
at defined distance
Mg2 +
(AdoMet)
asymmetric
6 bP
- CTGAAG-
outside
at defined distance
- CTGAAG-N,,1
-GACTTC- N ,
Mg2+
interrupted
outside, at defined distances on both
sides of the recognition sequences
~N,,ACN,GTA('lT) CN,,~
fN,,TGN,CAT ( G / A ) GN,,T
loosely defined
bipartite
at different positions
between the parts of the recognition
sequence
asymmetric
20-40 bp
within recognition sequence
TYPe I
EcoB
Structure
FokT
Type He
EcoRII
Type I11
EcoPl
Type 1V
(A/T)
Ec057 1
BcgI-like
AdoMet
Bael
Methylation dependent
McrBC
Homing endonuclease
GTP (hydrolysis)
MgZ+
( A / T ) GG-GG ( A / , r ) CCT-
-1CC
-AGACC- TCTGG -
(DNA)
Mg'
PI-SceJ
ing a short DNA fragment (Kong et al., 1994; Sears et al., 1996).
Related in function to restriction endonucleases but not part of
an RM system are methylation-dependent restriction enzymes
(review: Noyer-Weidner and Trautner, 1993) and homing endonucleases (review: Mueller et al., 1993). Examples of type I, 11,
I11 and 1V restriction enzymes as well as related endonucleases
are given in Table 1. As shown in Table 1, all restriction and
homing endonucleases require Mg" for DNA cleavage which,
depending on the system, can be substit.uted by other divalent
metal ions.
By far the best studied restriction enzymes are the type I1
enzymes of which over 2500 have been identified (Roberts and
Macelis, 1996), over 50 sequenced and in part biochemically
characterized. Many of the identified restriction endonucleases
are isoschizomers (i.e. enzymes which have an identical recognition sequence and cleave it at the same position) or neoschizomers (i.e. enzymes which have an identical recognition
. . . GTGC~.. .
. . .TCACG . . .
sequence but cleave it at different positions). Type I1 restriction
enzymes are indispensable tools for genetic engineering ; presumably, they are among the most often used enzymes in the
biochemical laboratory and have been exquisitely instrumental
for the enormous progress in our understanding of genome structure and gene expsession made in recent years. They have been
the subject of several recent reviews in which different aspects
are addressed : recognition sequences (Kessler and Manta, 1990 ;
McClelland et al., 1994; Roberts and Macelis, 1996), practical
use (Bhagwat, 1992; Pingoud et al., 1993), genetics (Wilson,
1991 ; Wilson and Murray, 1991), biology (Bickle and Kriiger,
1993 ; Heitman, 1993), biochemistry (Roberts and Halford,
1993; Eun, 1994), structure (Rosenberg, 1991 ; Winkler, 1992;
Anderson, 1993 ; Aggarwal, 1995) and protein engineering
(Jeltsch et al., 1996a). In the present review we will focus on
the enzymology of these enzymes, with special emphasis on the
mechanism of DNA recognition and cleavage. We will organize
Pingoud and Jeltsch ( E m J. Biochem. 246)
Fig. 1. Schematic illustration of the steps involved in DNA recognition and cleavage by restriction endonucleases.
this review by following the reaction cycle of a restriction endonuclease which in vitro as well as in vivo is initiated by nonspecific binding to the DNA, followed by a series of dissociation
and association steps and/or facilitated diffusion along the DNA
until the target site is located. There, conformational changes
take place which lead to the activation of the catalytic centers
and cleavage of the DNA. Subsequently, the products are released, allowing for a new reaction cycle to take place (Fig. 1).
After dealing with these separate aspects of the reaction catalyzed by restriction enzymes, we will finally discuss to what
extent results obtained for individual restriction endonucleases
can be generalized and extended to other highly specific endonucleases. This discussion will cover several aspects relevant to
protein-DNA interaction in general (review : von Hippel,
1994) ; it will become clear that restriction endonucleases can be
regarded as a paradigm for proteins that specifically interact
with nucleic acids.
Non-specific DNA binding and linear diffusion
The biological function of restriction endonucleases is based
on the fast cleavage of an invading phage DNA. Cleavage must
occur before the phage DNA is methylated by the corresponding
methyltransferase and before it initiates its deleterious action in
the bacterial cell. This requires very rapid target site location,
which is not an easy task given the large excess of non-specific
over specific sites on the DNA. To overcome this problem, restriction endonucleases seem to have developed a common strategy for facilitated target site location: the protein very quickly
binds non-specifically anywhere to the DNA and then scans the
DNA in search of its recognition site in a one-dimensional diffusion process. The following paragraph summarizes our knowledge about non-specific DNA binding of restriction endonucleases and on the mechanism of linear diffusion of restriction endonucleases along DNA.
Non-specific DNA binding. Non-specific binding to DNA
has been shown for all restriction endonucleases studied in this
respect so far (BcgI: Kong et al., 1994; CfrSI: Siksnys and
Pleckaityte, 1993 ; EcoRI: Woodhead and Malcolm, 1980; Goppelt et al., 1980; EcoRV: Taylor et al., 1991; Alves et al., 1995;
H i n f f : Frankel et al., 1985; MboII: Sektas et al., 1995; RsrI:
Aiken et al., 1991a; SmaI: Withers and Dunbar, 1995a; T'qI:
Zebala et al., 1992; XmaI: Withers and Dunbar, 1995b). However, there are only very few studies regarding the intenactions
involved in non-specific binding of restriction endonucleases to
DNA and, in particular, no mutagenesis studies. In general, nonspecific binding is weak under cleavage conditions, i.e. i j the.
3
presence of millimolar concentrations of Mg" (e.g. K,< lo2M-'/
mol bp for EcoRV, Jeltsch et al., 1995a). In the absence of Mg",
non-specific DNA binding is stronger (Ka =lo5 M-'/mol bp,
EcoRI: Terry et al., 1983; EcoRV: Taylor et al., 1991; Jeltsch
et al., 1995a). It is strongly dependent on the salt concentration
of the buffer, indicative of a large contribution of electrostatic
interactions between the protein and the phosphate groups of the
DNA to the overall binding energy. The kinetics of association
of restriction endonucleases to oligodeoxynucleotide substrates
have been analyzed by fluorescence stopped-flow experiments
(EcoRI: Alves et al., 1989a; EcoRV: Baldwin et al., 1995). In
agreement with the biological needs, the association rates in both
cases were found to be diffusion controlled with association rate
constants of the enzymes to oligodeoxynucleotides in the order
of 107-108 M-* s-1.
The only structure available of a restriction enzyme bound
non-specifically to DNA is that of EcoRV (Winkler et al., 1993).
The complex is held together by contacts of the protein to the
backbone of the DNA. Five contacts of amino acid residues
from each subunit are formed to the phosphate groups, but no
direct interactions with the bases of the DNA are observed. In
the non-specific complex the large central bend of the DNA
characteristic of the specific EcoRV-DNA complex is not observed (Fig. 2) and the catalytic center of the enzyme is not
properly assembled. Those parts of the protein that in the specific complex are responsible for base recognition are disordered
in the non-specific complex, presumably because the DNA and
the protein do not have chemically complementary surfaces.
Large parts of the disordered interface are probably filled with
loosely bound water molecules and ions to avoid buried, unsaturated hydrogen bond donors and acceptors. In agreement with
this model, it has been shown for EcoRI that approximately 110
water molecules are sequestered in the non-specific complex
suggesting that a full hydration layer of water separates the protein and the DNA (Sidorova and Rau, 1996). Most of the water
molecules and ions that are present at the protein-DNA interface
are released during formation of the specific complex (cf. specific binding) as more and more direct contacts between the enzyme and the DNA are being formed. Interference with the
water release reaction might be the cause of star activity (i.e.
cleavage at sites that differ in one base pair from the recognition
site) observed for many restriction enzymes under osmotic pressure (for example in the presence of glycerol, see below). The
large conformational changes of EcoRV upon formation of the
non-specific and specific complex, respectively, are illustrated
in Fig. 2.
Linear diffusion. Non-specific binding of restriction endonucleases to DNA is the prerequisite for facilitated diffusion. A
diffusional search, in general, is characterized by a random walk
until the target is found eventually. The speed of target site location depends on the rate and geometry of the movement, because
a three-dimensional random walk in principle requires much
more steps than a two-dimensional search in a plane or even a
one-dimensional search on a line. The concept of facilitated
target site location by reduction in dimensionality was developed
for molecules interacting with membranes or with DNA (Adam
and Delbriick, 1968; Richter and Eigen, 1974; reviews: Berg
and von Hippel, 1985; von Hippel and Berg, 1989). Experimentally, facilitated diffusion along DNA was first shown for the lac
repressor (Barkley, 1981 ; Winter and von Hippel, 1981 ; Winter
et al., 1981) and subsequently for several restriction endonucleases (EcoRI: Jack et al., 1982; Terry et al., 1985; Ehbrecht et
al., 1985; BamHI: Ehbrecht et al., 1985; Nardone et al., 1986;
HindIII: Ehbrecht et al., 1985; EcoRV: Taylor et al., 1991;
Jeltsch et al., 1996b; BssHJI: Berkhout and van Wamel, 1996).
4
Pingoud and Jeltsch ( E m J. Biochem. 246)
apoenzyme
non-specific
complex
specific
complex
Fig.2. Comparison of the structures of EcoRV in the apoenzyme form, as well as bound non-specifically to a pseudo-hexadecamer
d(CGAGCTCG)-d(CGAGCTCG)and specifically to d(GGGATATCCC) (Winkler et al., 1993). In addition, the DNA in the non-specific and
specific complex is shown viewed along the dyad axis.
Due to the reduction in dimensionality, a search by linear diffusion is much faster than a search by three-dimensional diffusion,
although the one-dimensional diffusion constant is smaller than
the three-dimensional diffusion constant of the enzyme in solution. For example, EcoRI scans the DNA with a rate of approximately 7X lo6 bp s-' corresponding to a diffusion coefficient of
approximately 5X1O-l4 mz s-' which is about two orders of
magnitude smaller than the three-dimensional diffusion coefficient in solution (Ehbrecht et al., 1985). Linear diffusion is dependent on electrostatic interactions between the enzyme and
the DNA. By a counterbalance of attractive and repulsive forces,
the enzyme forms an electrostatic trap in which the DNA can
move easily, if not too many directed contacts are formed. All
restriction-endonuclease- DNA complexes known so far have
an architecture in which the enzyme enwraps the DNA (Fig. 3),
suggesting that the concept of the DNA being electrostatically
trapped by the enzyme might be of general validity. The rate of
linear diffusion is limited by the friction experienced by the protein when moving on the DNA, presumably caused by unbalanced electrostatic interactions or hydrogen bonds between the
protein and the DNA, which have to be broken transiently in
order to move on (Jeltsch et al., 1996b).
Facilitated diffusion, in principle, could be mediated by distinct molecular mechanisms that differ in the degree of preservation of the contact of the protein to the DNA during the movement (Berg et al., 1981). If the protein slides along the DNA
(linear diffusion) an intimate contact is preserved during the process, whereas if facilitated diffusion occurs via fast microscopic
dissociation and reassociation reaction? (hopping) this contact is
transiently lost. It has been shown for EcoRI (Jeltsch et al.,
1994) and BssHII (Berkhout and van Wamel, 1996) that these
enzymes do not overlook any recognition site during linear diffusion under optimum conditions. Moreover, EcoRI follows the
helical pitch of the DNA during the movement (Jeltsch et al.,
1994) and the process is prevented by blocks on the DNA like
a DNA triple helix (Jeltsch et al., 1994) or proteins bound specifically (Jeltsch et al., 1994) or non-specifically (Ehbrecht et al.,
1985) to the DNA. It has also been shown that EcoRV is re-
flected at the ends of linear DNA (our unpublished results).
These results demonstrate that the movement is a linear diffusion
rather than a hopping process. During one single binding event,
EcoRI and EcoRV scan thousands of base pairs by linear diffusion (Fig. 4). As the movement can be described as a one-dimensional random walk, the average number ( P ) of diffusion steps
by one base pair is given by the square of the number of base
pairs scanned. P also represents the probability that the enzyme
moves on by linear diffusion instead of dissociating, i.e. the ratio
of the rate constants for diffusion and dissociation (kd,Jko,,). This
value is in the order of lo6 both for EcoRI (Ehbrecht et al.,
1985) and EcoRV (Jeltsch et al., 1996b). During linear diffusion
EcoRI is continuously scanning the DNA sequence in search for
its recognition site (Jeltsch et al., 1994). Star sites, i.e. sequences
that differ from the recognition sequence by one base pair, provide an almost matching groove geometry compared to the recognition sequence. These sites are bound more strongly by the
enzyme than non-specific DNA (Lesser et al., 1990; Thielking
et al., 1990) and cause a pause in linear diffusion of up to 20 s.
Pausing times measured for different star sites are roughly correlated to the affinity of the enzyme to these sites (Jeltsch et al.,
1994), indicating that it takes more time for the enzyme to
discriminate between closely related sequences (i.e. the canonical site and star sites) than between unrelated sequences (i.e. the
canonical site and non-specific sites).
As linear diffusion is disturbed by other proteins bound to
the DNA, it has always been debated whether this phenomenon
is just an in vitro artifact or whether it takes place in vivo. This
question was investigated with EcoRV mutants in vivo, where it
was found that phage restriction in Escherichia coli cells is
highly significantly correlated to the ability of EcoRV mutants
to diffuse along the DNA (Jeltsch et al., 1996b). This result
strongly suggests that linear diffusion of restriction endonucleases along DNA indeed takes place in vivo and is of fundamental
importance for the biological function of these enzymes. It may
be rationalized by considering that a phage DNA shortly after
infecting a cell probably does not carry many tightly bound proteins that would interfere with linear diffusion of the enzyme.
Pingoud and Jeltsch (Eur J . Biochem. 246)
5
Fig.3. Compilation of the structures of restriction endonuclease-DNAcomplexes. EcoRI: Kim et al., 1990; EcoRV: Winkler et al., 1993; PvuII:
Cheng et al., 1994; BamHI: Newman et al., 1995. All structures are shown viewed along the helix axis of the DNA.
Linear diffusion of EcoRl
Linear diffusion of EcoRV
krel
klongsubstrate /k26 bp substrate
0.6
0.4
I
,
'
Y
l
0'
slowly than the canonical site. This was systematically analyzed
by cleavage experiments using oligonucleotide substrates for
EcoRI (Lesser et al., 1990; Thielking et al., 1990) and EcoRV
(Alves et al., 1995), but is also true for other restriction endonucleases, as every-day experience of using these enzymes tells us.
These experiments, as well as experiments in which cleavage of
methylated DNA by EcoRI was studied (Jen-Jacobson et al.,
1996), furthermore, showed that at non-canonical or methylated
sites very slow cleavage occurs mainly in one strand of the DNA
leading to nicked products that in vivo can be rejoined by the
cellular DNA ligase (Taylor et al., 1990). Thus, if erroneous
nicking occurs, it is likely to be repaired in the cell. The high
specificity of restriction endonucleases is the result of preferential interactions between enzyme and substrate which differ
qualitatively and quantitatively from non-specific contacts to
DNA. The formation of these preferential interactions collectively defines the recognition process, i.e. the transition from a
non-specific to a specific complex that results in the activation
of the catalytic centers. As recognition is operationally defined
as the process which leads to cleavage at the recognition site,
only the transition state of the cleavage reaction can be considered as a bona fide recognition complex. However, it can be
anticipated that by deleting or replacing a catalytically important
element either by altering the substrate or the enzyme, or by
leaving out the essential cofactor Mg", complexes are formed
that are not cleaved, but nevertheless have a high stability constant and are formed with high specificity and, thus, are likely
to mimic a recognition complex. This expectation has been
proven to be correct in most cases.
Metal ions play an essential role in the recognition process,
as only in the presence of Mg2' or Mn2+,and to a certain extent
also in the presence of other divalent metal ions, does a productive interaction result, i.e. an interaction that leads to cleavage of the DNA. Nevertheless, most restriction enzymes analyzed so far do not require divalent metal ions for specific bind-
1000 2000 3000 4000
substrate length [bp]
Ob
500 1000 1500 2000
substrate length [bpj
Fig. 4. Dependence of DNA cleavage rates by EcoRI and EcoRV on
the length of the DNA substrate. Measurements with EcoRI were made
using plasmid substrates that had been digested with different other restriction enzymes (Ehbrecht et al., 1985). With EcoRV DNA cleavage
rates of a long PCR product were determined in competition with a 26bp oligodeoxynucleotide (Jeltsch et al., 1996b).
Taken together, linear diffusion apparently is a highly
evolved property of restriction endonucleases that plays a major
role for the biological function of these enzymes.
DNA recognition
RM systems constitute a powerful defense system against
invading foreign DNA. However, restriction endonucleases
would be harmful if cellular DNA was cleaved. Hence, cleavage
of DNA must not and indeed does not occur at sites other than
unmodified recognition sites. Consequently, one of the most fascinating aspects of restriction enzymes is their extraordinary
specificity. Under optimum conditions sites differing in only one
base pair are cleaved by several orders of magnitude more
6
Pingoud and Jeltsch (ELMJ. Biochem. 246)
ing, because binding to the specific site is thermodynamically
strongly favoured as opposed to binding to non-specific sites
even in the absence of divalent metal ions (e.g.: EcoRI: Halford
and Johnson, 1980; Langowski et al., 1980; HindIII: Takasaki,
1994; HinfI: Frankel et al., 1985; MboII: Sektas et al., 1995;
NaeI: Holtz and Topal, 1994; RsrI: Aiken et al., 1991a; SmaI:
Withers and Dunbar, 1995a; XmaI: Withers and Dunbar,
1995b). In gel-shift experiments, specific binding of restriction
enzymes to DNA is detected by formation of only one complex
with a macromolecular DNA which harbours only one site for
this protein. A few restriction enzymes behave differently, for
example EcoRV which in gel-shift experiments in the presence
of EDTA reveals a series of complexes due to the binding of 1,
2, 3, etc., molecules of proteidmolecule of DNA (Taylor et al.,
1991). It was subsequently shown that specific complexes are
formed when a catalytically inactive EcoRV mutant is incubated
with DNA in the presence of Mg” (Thielking et al., 1992) or
when wild-type EcoRV binds to DNA in the presence of Ca2+
(Vipond and Halford, 1995) which does not support cleavage.
This means that with EcoRV the divalent metal ion is intimately
involved not only in catalysis per se but also in specific complex
formation. EcoRV is not unique with respect to its dependence
on divalent metal ions for specific binding: PaeR7 (Ghosh et al.,
1990), TaqI (Zebala et al., 1992), CfrSI (Siksnys and Pleckaityte,
1993) and MunI (Lagunavicius, A., Grazulis, S., Balciunaite, E.,
Vainius, D. and Siksnys, V., personal communication) behave
similarly. Intriguingly, certain EcoRI mutants different from the
wild-type enzyme also need divalent metal ions for specific
binding like EcoRV (Windolph, S. and Alves, J., personal communication). In contrast, some M u d mutants bind specifically
to DNA (Lagunavicius, A. and Siksnys, V., personal communication) as does wild-type MunI at low pH (Lagunavicius, A.,
Grazulis, S., Balciunaite, E., Vainius, D. and Siksnys, V., personal communication). Thus, it appears as if the difference between enzymes capable of specific DNA binding in the absence
of Mgz+ (like EcoRI) and those not capable of specific DNA
binding (like EcoRV or Mud) is not a qualitative but rather only
a quantitative one, because this property can be modulated by
mutation (EcoRI, MunI), by addition of a non-productive divalent metal ion (EcoRV) or by lowering the pH (Mud).
Whether in all instances the same metal ion that is needed
for catalysis is also responsible for specific binding remains to
be seen. The finding that an EcoRV triple mutant, in which all
acidic amino acid residues in the catalytic center of the enzyme
were exchanged to alanine, still binds to DNA in a Mgz+-dependent manner, suggests that additional Mg” binding sites exist
in the EcoRV-DNA complex (Jeltsch et al., 1995a). One Mg”
binding site at the interface of the EcoRV-DNA complex was
located to be in proximity to Tyr219 and the first phosphate
group of the recognition sequence (GpATATC), both being over
1.5 nm away from the catalytic center of the enzyme (Jeltsch et
al., 199Sa). Hence, at least for EcoRV, there are reasons to believe that a second metal ion binding site exists that is distinct
from the metal ion binding site at the catalytic center and is
required for specific binding.
Most of our knowledge of the mechanism of recognition of
DNA by restriction endonucleases has been derived from the
analysis of cocrystal structures (EcoRI: Kim et al., 1990, 1994;
Rosenberg 1991; EcoRV: Winkler, 1992; Winkler et al., 1993;
Kostrewa and Winkler, 199.5; PvuII: Cheng et al., 1994;
BamHI: Newman et al., 1995) which presumably reflect many
features of the respective recognition complex. Structural studies
have been complemented by thermodynamic and kinetic investigations employing chemically modified substrates or enzyme
mutants. While such analyses by themselves provide only a very
crude picture of the recognition process, they are useful for the
refinement of models for the recognition process based on crystallographic data. In the following paragraph we will concentrate
on a discussion of the EcoRI, EcoRV, PvuII and BamHI systems,
with special emphasis on EcoRV, probably the most extensively
studied restriction enzyme to date.
Structures of restriction endonucleases. The sequence
analysis of more than 50 different type I1 restriction enzymes
has produced no evidence for any of the classical DNA-binding
motifs, like the helix-turn-helix, Zn-finger, basic region helixloop-helix, or basic region zipper motifs. Indeed, the four
cocrystal structures solved so far demonstrate that these restriction enzymes use novel and unique strategies to recognize DNA
sequences. Presumably, the fact that specific binding to the DNA
and cleavage of the DNA occurs within a small region is not
compatible with the use of extended regular secondary structure
elements for recognition, which are typical for DNA binding
proteins involved in gene regulation (reviews: Steitz, 1990; Harrison, 1991 ; Freemont et al., 1991). Interestingly, the basic structural motifs used to form the specific contacts of the enzymes
to the DNA, namely an extended peptide chain running through
the major groove (EcoRI), a-helices, adjacent loops and a Cterminal arm (BamHI), loops (EcoRV) and @-sheets(PvuII) are
different in all four enzymes analyzed so far.
The comparison of the crystal structures of EcoRI (Kim et
al., 1990), EcoRV (Winkler et al., 1993), PvuII (Athanasiadis et
al., 1994; Cheng et al., 1994), BamHI (Newman et al., 1994)
and CfrlOI (Bozic et al., 1996) demonstrated that all these enzymes are a@proteins with a central mixed five-stranded @-sheet
flanked by two helices. Whereas the structural similarities between EcoRI and EcoRV are difficult to recognize (Venclovas
et al., 1994), they are quite pronounced between EcoRI and
BamHI, as well as between EcoRV and PvuII (Fig. 5). Intriguingly, the catalytic centers of all four enzymes are very similar,
not only with respect to the amino acid residues involved, but
also with respect to the secondary structure elements on which
these residues are located. Thus, the monomeric subunits of all
restriction enzymes analyzed so far bear a small but nevertheless
striking structural similarity. However, the dimerization modes
of the subunits of EcoRI and BamHI, on the one hand, and
EcoRV and PvuII, on the other hand, are completely dissimilar.
These differences are also reflected in the cocrystal structures
(Fig. 3): while EcoRI (Kim et al., 1990) and BamHI (Newman
et al., 199.5) approach the DNA from the major groove, EcoRV
(Winkler et al., 1993) and PvuII (Cheng et al., 1994) contact the
DNA via the minor groove. It has been argued that this principal
difference might be related to the position of the scissile phosphodiester bonds : the bonds cleaved by enzymes that produce
5‘-overhangs of four nucleotides, like EcoRI and BamHI, are
best approached from the major groove side of the DNA, while
those cleaved by blunt end cutters, like EcoRV and PvuII, are
more accessible from the minor groove side (Anderson, 1993).
The structural similarities of EcoRI and BamHI, and EcoRV and
PvuII, respectively, thus, probably reflect the structural constraints to position two active sites such that they can act in
concert to attack phosphodiester bonds located on two different
strands four base pairs apart or just vis 6 vis, respectively. Based
on this concept, one would expect that enzymes producing 3’overhangs, like PstI, should approach the DNA from the minorgroove side (Anderson, 1993). In Fig. 3 the cocrystal structures
of EcoRI, BamHI, EcoRV and PvuII are shown, and in Fig. 6
schematic representations of the base specific interactions likely
to be involved in the recognition process are depicted.
EcoRI. The structure analyses (Kim et al., 1990, 1994) show
that EcoRI distorts the DNA upon binding to its specific site
(GAATTC): the DNA is kinked and unwound in the AATT se-
Pingoud and Jeltsch ( E m J. Biochem. 246)
7
Fig.5. Comparison of the topologies of the restriction endonucleases EcoRI, BumHI, EcoRV and PvuII (based on Newman et al., 1994 and
Athanasiadis et al., 1994). Catalytically important amino acid residues are marked with a cross (X), those involved in contacts to the bases of the
recognition sequence are marked with circles (0).Regions involved in dimerization contacts are shaded gray. Note the pronounced similarities
between EcoRI and BamHI, on the one hand, and EcoRV and PvuII, on the other hand.
quence, the two central base pairs are unstacked and the major
groove is widened. Overall, the DNA is bent by about 12", less
than estimated from gel-electrophoretic mobility shift assays
(Thompson and Landy, 1988), but similar as seen by atomic
force microscopy imaging (Allison et al., 1996). Several noncontiguous structural elements are involved in DNA contacts :
(a) a bundle of four helices, two from each subunit which penetrate the widened major groove and carry residues involved in
base and backbone interactions at their amino termini; (b) an
extended chain which runs through the major groove of the recognition site; (c) a /I-strand running parallel to the DNA backbone, which contains amino acid residues essential for catalysis
as well as residues engaged in phosphate contacts; (d) two arms
that reach around the DNA and are responsible for backbone
contacts outside of the recognition sequence (Figs 3 and 6). Such
contacts outside of the recognition sequence may explain, in
part, why EcoRI cleaves its sites on DNA with different rates
depending on the sequence context (Forsblom et al., 1976; Halford and Johnson, 1980; Alves et al., 1984; van Cleve and Gumport, 1992). Altogether, there are 18 protein-base hydrogen
bonds, 14 to purines and 4 to pyrimidines, and 10 van-derWaal's contacts to all pyrimidines. It is intriguing to note that
a dodecapeptide comprising the structural element which is of
particular importance for DNA recognition by EcoRI, namely
the extended chain motif together with part of the following ahelix (Met137-Arg145, Fig. 6), binds to the EcoRI recognition
site with high specificity, albeit low affinity (Jeltsch et al.,
1995b). In addition to these base-specific contacts, there are numerous contacts to the backbone of the DNA that could serve to
recognize the specific sequence through a sequence-dependent
backbone conformation. Thus, the recognition process is redun-
dant with more than one direct and/or indirect contact to each
base pair. Many of these contacts have been probed by sitedirected-mutagenesis experiments, the results of which have
confirmed their importance for the recognition process (Wolfes
et al., 1986; Yanofsky et al., 1987; Geiger et al., 1989; Alves et
al., 1989b; Needels et al., 1989; King et al., 1989; Wright et
al., 1989; Osuna et al., 1991; Selent et al., 1992; Jeltsch et al.,
1993a; Flores et al., 1995). Substitution of amino acid residues
involved in recognition contacts in general leads to a dramatic
loss in activity, not, however, to a change in specificity. This
means that any of these contacts could be removed without an
obvious reduction of the level of discrimination, demonstrating
that the recognition process is highly redundant and coupling of
specific binding to catalysis is very tight, as suggested by the
cocrystal structure.
In a complementary approach to site-directed mutagenesis of
the protein, ethylation interference and footprinting experiments
(Lu et al., 1981; Becker et al., 1988; Lesser et al., 1990, 1992),
as well as binding and cleavage experiments with synthetic oligodeoxynucleotides containing modified nucleotides, were used
to find out which structural elements on the DNA are needed
for recognition (Brennan et al., 1986; Fliess et al., 1986;
McLaughlin et al., 1987; Aiken et aI.,1991b; Koziolkewicz and
Stec, 1992; Lesser et al., 1992, 1993; Jeltsch et al., 1993a). The
results of these experiments are in agreement with the recognition model proposed on the basis of the structure analysis. In
addition, it has been shown by ethylation interference experiments that complexes of star sequences and EcoRI have a different conformation than the canonical complex (Lesser et al.,
1990), a result that has been confirmed by crystal structure
analyses (Rosenberg, J. M., personal communication).
8
Pingoud and Jeltsch (Euc J. Biochem. 246)
€wRI
subunit i
DNA
€coRV
subunit2
subunit 1
DNA
subunit 2
Fig. 6. Schematic representation of the interaction of the restriction endonucleases EcoRI, BamHI, EcoRV and PvuII with DNA. Amino acid
residues forming contacts to the bases are coloured green, those which are part of the catalytic center of the restriction enzyme red. With the
exception of the C-terminal arm of BamHL all of these contacts are symmetrically formed by both subunits of the enzyme, but displayed only once.
Pingoud and Jeltsch (Eui: J. Biochern. 246)
A quantitative interpretation of the contribution of individual
interactions to the recognition process can only be done with
great caution, because the modification may perturb the structure, including the water structure in the protein-DNA interface,
or introduce structural constraints incompatible with conformational changes required for the recognition process. Problems of
that sort can be overcome by a combined approach using chemically modified substrates and site-specific mutants. In one instance, a van-der-Waal's contact was probed both by deleting a
group on the DNA and on the protein, with identical effects on
the rate of DNA cleavage (Jeltsch et al., 1993a). Studies on the
EcoRI, as well as the EcoRV system to be described later, have
clearly demonstrated that chemical modification as well as sitedirected-mutagenesis experiments, when interpreted in conjunction with a crystal structure, not only allow one to evaluate the
significance of a protein-DNA contact for specific binding but
in addition can provide very valuable information regarding the
transition state of DNA cleavage, which is not easily accessible
by the structure analysis itself.
Chemical modification studies can also be used for comparative purposes. Thus, it was shown by ethylation interference
studies that the EcoRI endonuclease and the EcoRI methyltransferase recognize their target sequence by different mechanisms (Lu et al., 1981). Using modified oligodeoxynucleotides,
it was demonstrated that EcoRI and RsrI, a closely related
isoschizomer of EcoRI, do not interact with their target sequence
in a completely identical manner (Aiken et al., 1991b).
BamHI. BamHI (Newman et al., 1994, 1995), like EcoRI
with which it shares structural similarity (Figs 3 and S), also
forms most of its interactions to the bases of its recognition sequence (GGATCC) in the major groove but, different from
EcoRI, it does not distort the DNA significantly. Similarly as
observed for EcoRI, major groove contacts are formed from the
amino-termini of four a-helices, two from each subunit, and
adjacent loops. In addition, minor groove contacts are formed,
surprisingly by one subunit only, involving the C-terminal ahelix which in the specific complex is unwound and adopts an
extended conformation (Figs 3 and 6). The C-terminal a-helix
of the other subunit is excluded from entering the minor groove
and folds back to follow the sugar-phosphate backbone. In toto,
there are 12 direct and 6 water-mediated hydrogen bonds to the
bases of the recognition sequence in the major groove and 3
direct hydrogen bonds in the minor groove. Thus, the hydrogenbonding potential in the major groove is completely, and in the
minor groove almost completely, satisfied. In addition, there are
van-der-Waal's contacts to the inner thymine methyl groups and
extensive interactions between the enzyme and the sugar-phosphate backbone, 22 direct hydrogen bonds and approximately
16 water-mediated ones covering a stretch of over 10 base pairs.
It is noteworthy that BamHI makes most of its base and phosphate contacts in a crossover manner, because one subunit forms
the majority of its phosphate contacts to one DNA half-site
(GGA), but almost all of its base contacts to the other DNA
half-site (TCC). The C-terminal arm of only one subunit asymmetrically interacts with both DNA half-sites. While not many
site-directed rnutagenesis or chemical modifications studies have
been performed with the BamHI system, efforts are being undertaken to create BamHI variants with altered specificities. Their
results confirm the importance of some residues for the recognition process (Xu and Schildkraut, 1991a; Schildkraut, I., personal communication). While the structures of BamHI and
EcoRI are very similar, so similar as a matter of fact that a
common evolutionary origin has been proposed (Newman et al.,
1994), they interact with their recognition sequences differently.
In particular, there is no equivalent in BamHI to the extended
9
chain motif and the inner or outer arm of EcoRI. However, both
proteins make use of a four-helix bundle for dimerization as well
as recognition. It is tempting to speculate that this is the basic
recognition module in both enzymes, to which other elements
were added in the course of evolution. Perhaps this module also
occurs in other restriction endonucleases that cut DNA leaving
four nucleotide overhangs.
EcoRV. EcoRV is the only restriction enzyme for which
structure analyses exist of the free enzyme, a non-specific and
two specific complexes, as well as of an enzyme-product complex (Winkler et al., 1993; Kostrewa and Winkler, 1995). The
comparison between the structures of the non-specific and the
specific complexes, respectively, gives a very clear idea of some
of the conformational changes involved in the recognition process (Fig. 2). The most obvious change is the distortion of the
DNA from a regular B-DNA to a highly strained conformation
characterized by a 55" central kink which unwinds the DNA,
unstacks the central two base pairs of the recognition sequence
(GATATC) and bends the DNA into the major groove, making
it narrow and deep, while the minor groove becomes wide and
shallow. The EcoRV-induced bending of its specific DNA substrate has been confirmed by gel electrophoretic mobility shift
assays in the presence of Mg2+ with an inactive EcoRV mutant
(Stover et al., 1993) and in the presence of Ca2+with the wildtype enzyme (Vipond and Halford, 1995), as well as by scanning
force microscopy (Bustamente and Rivetti, 1996).
Not only the conformation of the DNA but also that of the
protein is altered during the transition from the non-specific to
the specific complex. The conformational change is due to the
motion of two flexibly linked subdomains which allows EcoRV
to fully embrace the DNA (Fig. 2). In addition, three loops, more
or less disordered in the free protein and the non-specific complex, become ordered, two of them being intimately involved in
recognition by making specific contacts in the major and minor
groove. The principal recognition element of EcoRV, the socalled recognition loop (R-loop), is responsible for 12 out of the
18 possible direct hydrogen bond contacts to the bases and two
van-der-Waal's contacts to the methyl groups of the outer thymidines as well as for approximately 12 water-mediated hydrogen
bonds to the phosphodiester backbone (Figs 6 and 7). The other
important recognition element, a glutamine-rich loop (Q-loop),
forms four hydrogen bonds to the edges of the bases in the minor
groove and harbours the catalytically important residue Asp74.
It is important to note that in the specific EcoRV-DNA complex
no interactions to the bases of the two central base pairs exist in
the major groove, presumably because the compression of the
major groove through the 55" kink precludes any direct access.
There are many contacts of the protein to the backbone of the
DNA. Not including residues from the R- and Q-loops, approximately 24 amino acid side chains with hydrogen bond donor
functions andor with a positive charge are located sufficiently
close to the phosphate residues to be involved in a favourable
interaction (Fig. 7). Some of these contacts are formed to phosphate residues outside of the recognition sequence. These might
be responsible for the preferences with which EcoRV cleaves
recognition sites located in a certain sequence context (Taylor
and Halford, 1989; Wenz et al., 1996).
The mechanism of DNA recognition by EcoRV as derived
from the crystal structure analysis has been extensively analyzed
by site-directed-mutagenesis experiments (Thielking et al., ;
1990; Selent et al., 1992; Vermote et al., 1992; Wenz et al.,
1994, 1996; Jeltsch et al., 1995a; Lanio et al., 1996). The mutational analysis has shown that substitution of amino acid residues involved in base-specific contacts results in almost inactive
variants (Table 2). Using chemically modified oligodeoxynucleotides (Fliess et al., 1986, 1988; Mazzarelli et al., 1989;
10
Pingoud and Jeltsch ( E m J. Biochem. 246)
Table 2. Compilation of relative catalytic activities of selected EcoRV
mutants. The wild-type activity was taken as 1 ; activities were determined using ],-DNA as substrate.
Mutant
Fig. 7. Schematic representation of the interaction of EcoRV with
specific DNA. Note that all contacts are formed in a symmetrically way
by both subunits, but displayed only once. To allow for a comparison
with biochemical experiments, amino acid residues and functional
groups of the DNA are coloured according to results from mutagenesis
experiments (Table 2) and experiments using chemically modified oligonucleotide substrates (Tables 3 -5). Groups that when replaced lead to
a dramatic loss of activity (at least three orders of magnitude) are coloured red, those causing reduced activities of between one and three
orders of magnitude green and those that can be replaced with minor
effects on activity blue. Note that Thr37, Tyr95, Serll2, Lysll9 and
Argl40 also form contacts to the phosphate groups of the DNA in the
non-specific EcoRV-DNA complex.
Newmann et al., 1990a,b; Waters and Connolly, 1994; Szczelkun and Connolly, 1995) as well as oligodeoxynucleotides with
degenerate recognition sequences (Alves et al., 1995) as substrates for EcoRV, the importance of all exocyclic functional
groups located in the major groove of the recognition sequence
was demonstrated (Tables 3 and 4). The inner A . T base pairs
which are, according to the crystal structures, in the major
groove are not contacted directly by the enzyme, turned out to
be as important for the recognition process as the other base
pairs. The implication of this finding is that sequence recognition does not necessarily require that the edges of the bases are
in a hydrogen bond or van-der-Waal's interaction with the enzyme, but that contacts to the phosphodiester backbone can be
used instead to probe the conformation of the DNA. It can be
assumed that the propensity of the EcoRV recognition sequence
to adopt a particular conformation will exclude other sequences
from a productive interaction with EcoRV (Winkler et al., 1993).
For example, based on the crystal structure, it is hardly conceivable that G . C base pairs substituted for 'the inner A . T base
pairs would allow for an approximately 50" kink which is observed with the canonical sequence.
The role of phosphate contacts for the specific interactions
of EcoRV and its target sequence was systematically analyzed
by site-directed-mutagenesis experiments (Wenz et al., 1996)
Activity
Reference
0.001
0.1 -0.01
0.5
0.01 -0.001
0
0
1
0.02
unpublished result
Thielking et al., 1991
unpublished result
Thielking et al., 1991
Thielking et al., 1991
Thielking et al., 1991
Thielking et al., 1991
Thielking et al., 1991
Base
contacts
K38-A
N70-Q
T106-A
S183-A
N185-A
T186-S
T187-S
N188-A
Catalytic
residues
D74-A
D9C-A
K92-A
0
0
0.0001
Selent et al., 1992
Selent et al., 1992
Selent et al., 1992
Phosphate
contacts
T37-A
S41-A
T93-A
T94-A
Y95-F
T111-A
S112-A
K119-A
R14C-A
R221-A
S223-A
R226-A
0.001
1
0.02
1
1
0.1
0.1
0.2
0.5
2
1
0.1
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
Wenz et al.,
1996
1996
1996
1996
1996
1996
1996
1996
1996
1996
1996
1996
Table 3. Compilation of relative cleavage rates of substrates containing star sites by EcoRV. Table taken from Alves et al. (1995). The
substitutions (indicated in bold letters) were introduced in only one half
of the palindromic recognition site. Rates of cleavage were measured in
the individual strands of the duplexes. Note that at several positions
these rates differ substantially.
Substrate
Sequence
Relative
cleavage
rate
Canonical substrate
TGCAGTAGC GATATC CAGC
ACGTCATCG CTATAGGTCG
1
1
Star substrates
(firsusixth position)
CATATG
GTAATC
AATATC
TTATAG
TATATC
ATATAG
1.5
7.1
1.1
5.3
1.1
1.7
X10-'
Star substrates
(secondlfifth position)
GTTATC
CAATAG
GGTATC
CCATAG
GCTATC
CGATAG
4.3 x
6.7 X lo-'
1.8 x
9.1 XlO-'
6.25 X
3.3 x
Star substrates
(thirdlfourth position)
GAAATC
CTTTAG
GAGATC
CTCTAG
CACATC
CTGTAG
2.2 x
1.1 x 10-4
2.1 xlo-h
5 xlO-h
4.8 X lo-'
6.25 x 10-5
X lo-'
x
x10-6
x10-7
XlO-'
(Table 2). In a complementary approach, DNA cleavage experiments were performed using chemically modified oligodeoxynucleotide substrates in which (&)- and (S,)-phosphorothioate
groups were individually introduced at nine phosphate positions
11
Pingoud and Jeltsch ( E m J. Biochem. 246)
Table 4. Compilation of relative cleavage rates of oligodeoxynucleotide substrates containing modified bases by EcoRV. I, inosine; "G,
6-thioguanine ; "G, 7-deazaguanine ; ""G,3-deazaguanine ; '"P, 2-aminopurine; P, purine; "A, 7-deazaadenine; "A, 3-deazaadenine; 6"A, 6methyladenine; U, uracil; 4'T, 4-thiothymidine; 4HT,5-methyl-2-pyrimidinone; "T, 2-thiothymine; 5mC, 5-methylcytosine; 4HU,2-pyrimidinone.
Relative
cleavage
rate
Substrate
Canonical
substrate
Modified
substrates
-GATATC[ b"Gl ATATC
['"PI ATATC
[ "GI ATATC
[ '=G 1 ATATC
IATATC
Reference
Relative
cleavage
rate
Substrate
Canonical substrate
1
<0.0005
0
0
0.0071
0.033
Table 5. Compilation of relative cleavage rates of oligodeoxynucleotide substrates containing phosphorotbioate-modified phosphate
groups by EcoRV. Table taken from Thorogood et al. (1996). THe numbering scheme regarding the individual phosphate residues is that of
Fig. 7 : 0 refers to the phosphate at the site of cleavage, - 1 and + 1 to
the 5' and 3' adjacent residues, respectively, etc. (R)-P and (S)-P denote
which of the two prochiral non-bridging phosphoryl oxygens had been
replaced: (R)-P refers to the pro-R, (S)-P to the p ro 4 oxygen atom.
Waters and Connolly, 1994
Waters and Connolly, 1994
Waters and Connolly, 1994
Waters and Connolly, 1994
Waters & Connolly, 1994
GPTATC
G [ ' C A I TATC
G L3'A1 TATC
G [ '"A] TATC
0.001
0
0.025
0
Newman et al., 1990
Newman et al., 1990
Newman et al., 1990
Fliess et al., 1988
GAUATC
GA14'T]ATC
GA [',TI ATC
GA [ 2 " T ] ATC
0.033
0.002
0
0.333
Newman et
Newman et
Newman et
Newman et
GATPTC
GAT ["A] T C
GAT ["A] T C
GAT [""A] T C
0.014
0.2
0.033
0
Newman et al., 1990
Newman et al., 1990
Newman et al., 1990
Fliess et al., 1988
GATAUC
GATA [ 4sT I C
GATA [ 4HTIC
GATA 12'T] C
0.014
0
0
0.05
Newman et
Newman et
Newman et
Newman et
GATAT [ ""U]
GATAT [ ""C ]
2
2
Waters and Connolly, 1994
Waters and Connolly, 1994
al.,
al.,
al.,
al.,
al.,
al.,
al.,
al.,
1990
1990
1990
1990
1990
1990
1990
1990
within and flanking the EcoRV recognition site (Thorogood et
al., 1996) (Table 5). The results of these studies demonstrate the
importance of phosphate contacts for recognition and catalysis,
because within the recognition site only two out of 12 phosphoryl oxygens can be replaced by sulfur with minor effects on
the rate of cleavage, and not more than half of the amino acid
residues that form hydrogen bonds or electrostatic interactions
to the phosphates can be exchanged for alanine without a significant loss in activity. This result was reinforced by a similar study
in which methylphosphonate instead of phosphorothioate substitutions were used. This substitution, which does not preserve the
charge of the phosphate group, is tolerated at only one position,
and only as one diastereomer (Schulz, A., Selent, U., Katti, S.
B. and Pingoud, A., unpublished). Given this specificity, it was
possible to engineer an EcoRV variant that cleaves a substrate
modified at that position with the same rate as the wild-type
enzyme cleaves the unmodified oligodeoxynucleotide but that
does not cleave the unmodified DNA (Lanio et al., 1996), one
of the few examples of successful protein engineering with type
I1 restriction enzymes (review: Jeltsch et al., 1996a).
PvuII. PvuII, the smallest restriction endonuclease found so
far, recognizes the sequence CAGCTG and cleaves it in the center to generate products with blunt ends. The enzyme shares
extensive structural similarity with EcoRV (Athanasiadis et al.,
1994; Cheng et al., 1994) (Fig. 5), but, differently from EcoRV,
it does not distort the DNA (Cheng et al., 1994). As in the
EcoRV-DNA complex, the DNA lies in a cleft formed by the
Modified substrates
p G A C GATATC GTC
( R )- PL, :
( S ) -P-,:
( R ) -P->:
( S) -P-,:
GAP& GATATC GTC
GAP& GATATC GTC
GAC PRGATATCGTC
GAC PSGATATC GTC
( R ) -P-,: GAC GPdTATC GTC
( S) -P-,: GAC G P a T A T C GTC
( R ) -P-,: GAC GAPRTATC GTC
( S) -P-,: GAC GAPSTATC GTC
( R ) - P o : GAC GATPRATC GTC
( S )-Po : GAC GATPaTC GTC
( R )-Pb1 : GAC GATAPRTC GTC
( S ) -P+,: GAC GATAPsTC GTC
( R ) - P + 2 : GAC GATATPRCGTC
( S )- P , 2 : GAC GATATPsC GTC
( R ) -P+3 : GAC GATATCPn GTC
(S)
-P+3 : GAC GATATCPs GTC
( R )-P+a : GAC GATATC GPnTC
( S ) -PA4: GAC GATATC GPsTC
1
3
I
3
I
0.04
0.04
0.17
0.5
<O.OOS
0
I
O
0.01
0.1
0.02
0.25
0.5
1
two identical subunits of the PvuII homodimer (Fig. 3). The
DNA-binding regions of each subunit comprise two substructures from different locations in the primary sequence ; binding
and catalytic regions are mutually interspersed. The principal
recognition element of PvuII is a two-stranded antiparallel psheet, somewhat reminiscent of the antiparallel p-ribbon seen in
the E. coli MetT repressor and the bacteriophage P22 Arc repressor (Cheng et al., 1994). Amino acid residues located on this psheet are involved in 12 direct hydrogen bonds and several vander-Waal's contacts to the bases of the recognition sequence in
the major groove, as well as contacts to the sugar-phosphate
backbone. In addition, there are two water-mediated hydrogen
bonds to the inner C residue of the recognition sequence, originating from the dimerization sub-domain (Fig. 6).
PvuII also makes contacts to the DNA outside of the recognition site, both to the bases as well as the phosphates; this may
explain the unexpected finding of Chen et al. (1991) that the
methylation status of the bases outside of the recognition sequence influences the DNA cleavage rate of this enzyme (Cheng
et al., 1994). Several of the amino acid residues suggested to be
involved in DNA recognition on the basis of the cocrystal structure have been subjected to a mutational analysis. The inactivity
of the resulting mutant proteins confirm the recognition model
derived from the structure analysis (Riggs, P. D. and Evans, P.
D., personal communication).
Redesigning the specificity of restriction endonucleases.
Ever since protein engineering became a reality due to the many
advances in recombinant DNA technology, strategies were formulated (Rosenberg et al., 1987) and efforts undertaken to alter
the specificity of restriction enzymes. While initial attempts remained largely unsuccessful, because the cooperative nature of
the recognition process underlying the reaction of restriction enzymes with their DNA substrate was not recognized, results ob-
12
Pingoud and Jeltsch ( E m J. Biochern. 246)
tained recently appear more promising, mainly because the goals
were redefined (review: Jeltsch et al., 1996b). Trying to alter
the specificity of a restriction endonuclease means to re-educating the enzyme to make it forget what it has learnt in the course
of evolution, namely not to cleave sites other than the recognition site, a very difficult task. In contrast, trying to produce a
restriction enzyme that recognizes chemically modified recognition sequences seems to be more realistic, as restriction enzymes
were not confronted with modified DNA during evolution. Indeed, EcoRI and EcoRV mutants were obtained that prefer deoxyuridine over thymidine in their recognition sequence (Jeltsch
et al., 1993a; Wenz et al., 1994), as well as an EcoRV mutant
that preferentially cleaves DNA with a methylphosphonate substitution in one position of the recognition sequence (Lanio et
al., 1996). More demanding, but not unrealistic, will be attempts
to change the selectivity of restriction enzymes, for example redesigning a hexanucleotide cutter to become an octanucleotide
cutter. This means amplifying the natural tendency of restriction
enzymes to prefer recognition sequences in a particular sequence
context. The current status for EcoRV is that enzyme variants
have been obtained that prefer one sequence context by a factor
of 100 over another (Wenz, C. and Pingoud, A., unpublished).
Efforts are presently being undertaken to speed up protein engineering of restriction endonucleases by employing techniques of
evolutionary biotechnology.
In conclusion, DNA recognition by restriction endonucleases
is found to be a highly cooperative and redundant process involving contacts of the protein to the bases and to the DNA
backbone that makes restriction enzymes to be among the most
specific and accurate enzymes known that operate without an
energy-consuming proof-reading mechanism.
Coupling of specific binding to catalysis
As mentioned above, recognition is defined as the process
following binding of the restriction enzyme to its specific site
that leads to the transition state of DNA cleavage separating
the enzyme-substrate complex and the enzyme-product complex.
Coupling of specific binding to catalysis, therefore, is transient
in nature and hardly accessible to experiments. Furthermore, the
cocrystal structures of EcoRI, EcoRV, BamHI and PvuII demonstrate that structural elements responsible for specific binding
are interwoven with structural elements involved in catalysis
(Fig. 6). Thus, it can be anticipated that the transition from nonspecific to specific binding leads to a highly cooperative restructuring of the protein-DNA interface including the release of
many solvent molecules as well as ions, but also to the immobilization of some water molecules at the interface. This became
quite evident in the refinement of the cocrystal structure of
EcoRI (Rosenberg, J. M., personal communication) and the presentation of the well resolved cocrystal structures of BumHI
(Newman et al., 1995) and EcoRV (Kostrewa and Winkler,
1993, which demonstrate that tightly bound water molecules
play an important role in recognition and form an extensive network of hydrogen bonds that interconnects all structural elements involved at the protein-DNA interface, including those
directly responsible for catalysis. A corollary to this observation
is that coupling of specific binding to catalysis cannot be attributed to individual structural elements but rather is the consequence of the mutual induced fit of enzyme and substrate. With
respect to this property, restriction endonucleases resemble many
other proteins specifically interacting with DNA where coupling
of local folding to site-specific DNA binding by a mutual induced fit is also observed (Spolar and Record, 1994). Hence,
even perturbations of the protein-DNA interface which do not
interfere with specific binding and do not affect the catalytic
machinery can be deleterious for the overall reaction, if they
disturb this conformational change. For example, in EcoRI
(Gln115) and EcoRV (Asn188, Thr37) amino acid residues have
been identified by the structure analyses and site-directed-mutagenesis experiments that interconnect various parts of the protein
and whose exchange by alanine strongly disturbs coupling of
specific binding and catalysis (Rosenberg, 1991 ; Winkler et al.,
1993; Thielking et al., 1991 ; Jeltsch et al., 1993a; Wenz et al.,
1996). A consequence of the fact that restriction enzymes do not
make isolated but rather intimately interconnected contacts to
their specific DNA substrate is that AAG values calculated from
binding experiments with chemically modified oligonucleotides
and/or enzyme mutants tend to underestimate the importance of
a contact for the recognition process.
The role of Mg” ions in coupling specific binding and catalysis is important, because Mg’ ’ ions may be needed for specific
binding and are essential for catalysis. EcoRV was the first restriction endonuclease for which it was found that it does not
bind to DNA specifically in the absence of bivalent metal ions
(Taylor et al., 1991). This result suggests that coupling between
specific binding and catalysis is very tight in EcoRV, meaning
that both processes hardly can be separated. This interpretation
is confirmed by the finding that modified oligodeoxynucleotides
which are refractory to cleavage are not bound specifically by
EcoRV (Szczelkun and Connolly, 1995). It must be emphasized,
however, that recognition is a cooperative phenomenon such
that Mg2+ binding cannot be regarded as the sole trigger for
catalysis.
A particularly important aspect of the coupling of specific
binding to catalysis is the intersubunit communication. Even before the first cocrystal structure of EcoRI was solved, it was
recognized that the two identical subunits of EcoRI cooperate in
the binding and cleavage of their substrate (Alves et al., 1982),
presumably because the biological function of restriction enzymes rests on a reliable destruction of phage DNA. This can
only be achieved if double strand breaks are introduced, as
cleavage of only one strand results in nicked products that are
likely to be repaired by the cellular DNA ligase (Taylor et al.,
1990). This means that the recognition process leads to the more
or less simultaneous activation of the two active sites of a homodimeric restriction enzyme which under normal conditions,
cleaves both strands of the DNA in a concerted reaction. Presumably, the proper formation of all specificity-determining contacts between the DNA and the protein is signaled to both catalytic centers. This intersubunit communication structurally could
be mediated by dimerization contacts within the main DNA recognition elements, which are observed in all restriction enzymeDNA complexes known so far (Fig. 5 ) . Evidence for this assumption was provided by the results of experiments performed
with EcoRI and EcoRV using oligodeoxynucleotide substrates
with single mismatched base pairs (Thielking et al., 1990; Alves
et al., 1995). Recently, experiments with artificial EcoRV heterodimers (Wende et al., 1996a) carrying amino acid substitutions
only in one subunit have directly shown that recognition contacts formed by one subunit influence the activity of the catalytic
centers of both subunits (Stahl et al., 1996).
Taken together, specific binding induces a mutual induced
fit of the enzyme-DNA complex, which brings all groups participating i n catalysis into the required proximity and precise orientation. Thereby the catalytic centers are activated and specific
binding is coupled to catalysis in a highly cooperative manner.
Mechanism of DNA cleavage
Active-site architecture. Upon comparison of the first two
restriction endonuclease DNA complexes solved (EcoRI and
13
Pingoud and Jeltsch (Eur: J . Biochern. 246)
EcoRV), it became clear that the active sites of both enzymes
are very similar to each other and are characterized by a PD ...(D/
E)XK motif (Thielking et al., 1991 ; Winkler, 1992). The importance of the conserved amino acids of these motifs (EcoRI:
Pro90, Asp91, Glu111, Lysll3 ; EcoRV: Pro73, Asp74, Asp90,
Lys92) has been investigated by mutagenesis experiments
(Wolfes et al., 1986; King et al., 1989; Wright et al., 1989;
Selent et al., 1992; Grabowski et al., 1995). Both acidic amino
acid residues were found to be essential for cleavage, because
in each case alanine mutants are catalytically completely inactive. The lysine residue is also intimately involved in catalysis,
as alanine mutants display an activity reduced by about three
orders of magnitude. The proline residue has been shown to be
not of importance in EcoR1. A similar stereochemical arrangement of amino acid residues has later been found also in the
structures of BarnHI (Newman et al., 1994, 1995) and PvuII
(Athanasiadis et al., 1994; Cheng et al., 1994), although in both
enzymes the proline is absent (Fig. 10, below). In addition, in
BarnHl the lysine is replaced by glutaniic acid. The importance
of the amino acid residues located in the catalytic center of this
enzyme (Asp94, G l u l l l , Glu113) has been demonstrated by
mutagenesis studies (Xu and Schildkraut, 1991a ; Dorner and
Schildkraut, 1994). In PvuII a catalytic motif (Asp%, Glu68,
Lys70) could be identified in the structure of the enzyme (Athanasiadis et al., 1994; Cheng et al., 1994). Both acidic amino acid
residues were shown to be essential for catalysis by site-directed
mutagenesis (Nastri, H. G., Walker, I. H., Evans, P. D. and
Riggs, P. D., personal communication). In contrast, in the putative catalytic center of the CfrlOI enzyme (Pro133, Asp134,
Ser188, Lysl90), whose apoenzyme structure has been solved
recently, at the position structurally corresponding to Glul11
and Asp90 in EcoRI and EcoRV, respectively, a serine residue
is found (Ser188: Bozic et al., 1996). Presumably, the second
acidic amino acid residue required for catalysis is recruited from
an a-helix nearby (Glu204: Bozic et al., 1996). This suggestion
has been supported by the finding that a double mutant
(S188-E, E204-4) which restores a canonical PD ...(D/E)XK
motif in CfilOI is catalytically active (Siksnys, V., personal communication).
PD ...(D/E)XK motifs can be found in other restriction endonucleases and related enzymes (cf. Anderson, 1993 ; Wittmayer
and Raines, 1996). The occurrence of such motifs might be
taken as a hint for the location of the catalytic center in such
enzymes, but so far, apart from EcoRI and EcoRV, a PD ...(D/
E)XK motif has been shown to be catalytically important by a
mutational analysis only in MunI (Lagunavicius, A. and Siksnys,
V., personal communication), the type IIS restriction endonuclease FokI (Waugh and Sauer, 1993) and the homing endonuclease
I Ppol (Wittmayer and Raines, 1996). On the basis of the structural data available, one has to conclude that the presence of a
PD ...(D/E)XK motif is not sufficient to define active sites of
restriction enzymes because CfrlOI contains a PD ...(D/E)XK
motif that is not part of its the active center and EcoRI contains
two such motifs, one of which is not involved in catalysis.
Furthermore, the mot s not strictly conserved: the proline residue is dispensable in EcoRI and absent in PvuII and BamHI.
The structures of Cfi-101 and BarnHI demonstrate that also the
second part of the motif is variable. In conclusion, it is likely,
that those restriction enzymes not possessing a canonical
PD ...(D/E)XK motif nevertheless might have an active site
structure similar to Pvull, BanzHI or CfrlOI.
Chemistry of catalysis. Cleavage of DNA by restriction endonucleases yields 3’-OH and 5‘-phosphate ends. Hydrolysis of
the phosphodiester bonds by EcoRI (Connolly et al., 1984) and
EcoRV (Grasby and Connolly, 1992) occurs with inversion of
Z:
Z- H
5
X:
H-OR1
H
H
X
X
I
I
Fig. 8. Scheme illustrating the general mechanism of hydrolysis of
phosphodiester bonds (based on Jeltsch et a]., 1992). X, Y and Z-H
are a general base, Lewis acid and general acid, respectively.
configuration at the phosphorous atom, suggesting an attack of a
water molecule in line with the 3’-OH leaving group. In general,
hydrolysis of phosphodiester bonds requires three functional entities: (a) a general base that activates the attacking nucleophile;
(b) a Lewis acid that stabilizes the extra negative charge in the
pentacovalent transition state; and (c) an acid that protonates or
stabilizes the leaving group (Fig. 8). The individual importance
of each function depends on the actual mechanism of the reaction. If the reaction proceeds via an associative pathway, characteristic for the non-enzymatic hydrolysis of phosphate triesters
in solution, all entities are equally important. However, if the
reaction proceeds via a dissociative pathway, characteristic for
the non-enzymatic hydrolysis of phosphate monoesters in solution, activation of the nucleophile is of marginal importance
(Benkovic and Schray, 1973). There is a continuous transition
between both extremes (Herschlag and Jencks, 1989) and, in
solution, transition states for the non-enzymatic hydrolysis of
phosphate diesters are intermediate. Hence, one would expect
that in DNA hydrolysis all three functional entities are required.
Currently, neither the nature of the transition state (more associative or more dissociative) has been identified, nor has the mechanism of DNA cleavage been elucidated unequivocally for any
restriction endonuclease. One general model for the mechanism
of catalysis has been proposed that might be applicable to all
restriction endonuclease whose cocrystal structures have been
solved. This model will be described in the following paragraph.
For EcoRV an alternative model has been suggested that will be
discussed afterwards. These models differ with respect to the
chemical entities responsible for the functions (a, b, c) mentioned above. Finally, we will briefly discuss the molecular
mechanisms leading to a relaxed specificity of restriction endonucleases under certain buffer conditions.
The substrate-assisted catalysis model. Unfortunately, the
crystal structure analyses are only of limited value for deriving
the mechanism of catalysis because, in the complexes crystallized in the absence of Mg”, there is neither an obvious candidate amino acid residue that could activate a water molecule
properly positioned for an in-line attack nor one that could protonate the 3‘-0- leaving group. Mg2+-soakingexperiments have
been performed with all four complexes (EcoRI : Rosenberg,
1991; EcoRV: Kostrewa and Winkler, 1995; PvuII: Cheng, X.,
pel-sonal communication ; BarnHI: Aggarwal, A., personal communication). They have shown that in each case the two conserved acidic amino acid residues serve as ligands for a Mg”
ion that also binds to the phosphate group to be attacked. This
cation is well suited to neutralize the negative charge at the
phosphate group. A similar Lewis acid function may be attrib-
Pingoud and Jeltsch (Eur. J. Biochem. 246)
14
EcoRV
*fd
proton+
H20
4
?+
Asp90
Asp74
Asp91
Fig. 9. Structures of the catalytic centers of EcoRI and EcoRV, after introducing a Mg2+ion and two water molecule into the structures by
modeling, one positioned to allow for an in-line nucleophilic attack on the scissile bond and the other to protonate the leaving group.
Adapted from Jeltsch et al., 1993b. In both models the catalytic amino acid residues (cf. Figs 5 and 6) and parts of the phosphodiester backbone
of the DNA (EcoRI: GJAATTC; EcoRV: GATJATC) are shown. Note the proximity of the attacking water to the phosphate 3' to the scissile
phosphodiester bond as well as the location of the water molecule that serves to protonate the leaving group in the inner hydration sphere of the
Mg2+ ion.
uted to the semi-conserved 1ysine residue. Perhaps the glutamic
acid residue, that replaces the lysine in the active site of BamHI
is protonated and thus able to form a hydrogen bond to the scissile phosphate, similarly as observed for one of the conserved
acidic amino acid residues (Asp90) in the EcoRV-DNA complex
in the absence of Mg2' (Kostrewa and Winkler, 1995).
The geometry of all of these enzyme-DNA-metal-ion
complexes is such that one water molecule from the Mg2+ hydration sphere closely approaches the leaving group oxygen. As
Mgz+-bound water is more acidic (pK, = 11.4, Scott and Klug,
1996) than bulk water, it has been suggested that a water molecule from the inner hydration sphere of the magnesium ion
might protonate the leaving group (Figs 9 and 11B) (Jeltsch et
al., 1992). Apart from the matching stereochemistry there is no
direct evidence supporting this model so far. However, as already pointed out, catalytic activity of all restriction endonucleases is ultimately dependent on the presence of a divalent cation
that has similar properties as Mg*+.Ca2+,for example, does not
support DNA cleavage in any restriction enzyme known so far.
This finding may be taken as evidence for a more intricate role
of the metal ion than simple charge neutralization. Moreover,
star activity in the presence of MnZ+,which is also observed
with many restriction endonucleases, could be explained in a
straightforward manner by this mechanistic model (see below).
As an alternative to protonation, the leaving group might be
stabilized by the formation of a direct ion pair with the metal
ion. This mechanism seems to be operative in the RNA cleavage
reaction catalyzed by the Tetrahymena ribozyme, as shown by
cleavage experiments in which the bridging oxygen atom of the
scissile phosphodiester bond (i.e. the leaving group oxygen) was
replaced by sulfur. These thiolates were hardly attacked in the
presence of Mg". However, they were readily cleaved with
Mn2+as the cofactor. As soft metal ions like Mn2+have a higher
affinity towards soft ligands like sulfur, this result suggests a
direct ion pair formation between the metal ion and the leaving
group (Piccirilli et al., 1993). Similar experiments performed
with EcoRV, however, did not produce any evidence for a direct
interaction of the metal ion and the leaving group oxygen atom,
making such a mechanism unlikely (Szczelkun and Connolly,
1995).
Placing a water molecule into the structures of the EcoRIDNA and EcoRV-DNA complexes at the position needed for an
in-line attack surprisingly revealed that in both cases the water
molecule can form a hydrogen bond to the pro-R, oxygen atom
of the phosphate group 3' to the scissile bond. This observation
has lead to the suggestion that the phosphate group might transiently bind the proton and thereby activate the nucleophile
(Figs 9 and 1IB) (Jeltsch et al., 1992). As in this model a chemical entity of the substrate is essential for catalysis it was called
the substrate-assisted catalysis model. Subsequently, this model
was supported by results obtained with substrates chemically
modified at the position in question. Oligodeoxynucleotide substrates in which these phosphate groups are deleted or modified
are bound but not cleaved by EcoRI and EcoRV (Jeltsch et al.,
1993b). The structures of PvuII and BamHI show a very similar
stereochemical arrangement of the scissile bond and the next
phosphate in 3'-direction (Fig. 10A). Even more suggestive, in
the BamHI complex, an ordered water molecule is observed at
a position allowing for an in line attack (Newman et al., 1995).
This water molecule indeed is hydrogen bonded to the next
phosphate group (Fig. 10B). Furthermore, it has been shown for
BarnHI and PvuII that replacement of the phosphate 3' to the
cleavage site by a methylphosphonate strongly reduces cleavage
efficiency (Jeltsch et al., 1 9 9 5 ~ )Taken
.
together, on the basis of
the biochemical and crystallographic data, it is very likely that
the attacking water molecule is positioned by a hydrogen bond
to the 3'-phosphate. Whether the phosphate also deprotonates
the water during the reaction is unclear, although there is one
result supporting this idea : cleavage experiments using substrates in which this phosphate group was substituted by phosphorothioates revealed that an uncharged phosphoryl oxygen
that could orient well but not deprotonate the water does not
support catalysis. In contrast, a negatively charged sulfur which,
due to its different hydrogen bonding tendency and bond length,
will be less suitable for positioning the water, but which could
abstract a proton, allows for catalysis at a reduced but reasonable
Pingoud and Jeltsch ( E m J. Biochem. 246)
15
Fig. 10. Superposition of the catalytic centers of EcoRI (red), BurnHI (green), EcoRV (yellow) and PVuII (blue). In (A) the Ca positions of
the catalytic amino acid residues and the positions of the attacked phosphorous atoms are superpositioned. In (B) the attacked and 3’ adjacent
phosphorous atoms are overlaid and in addition one water molecule observed in the BamHI-DNA structure is displayed.
rate (EcoRI: Koziolkiewic and Stec, 1992; EcoRV: Thorogood
et al., 1996). These findings suggest that the role of the phosphate group may indeed be deprotonation rather than only positioning of the attacking water molecule.
The main problem with the substrate-assisted catalysis
model is that the pK, of a phosphodiester group (pK, S 2 ) is not
in the range to readily deprotonate a water molecule at neutral
pH. It is, however, known that pKa values are considerably
shifted in proteins. For example, it has been shown that binding
of a nucleic acid substrate to barnase causes a shift of the pK,
of a catalytic glutamic acid residue by about 2.5 (Gordon-Beresford et al., 1996). Furthermore, it can be argued that proton abstraction could be a rare event, as restriction enzymes are slow,
with k,,, values in the order of 0.1- 1 s-’. One important advantage of this mechanism is that it could be valid for all restriction
endonucleases whose structures are known so far. This is an
important aspect, because these restriction enzymes have a similar catalytic center (Fig. lo), suggesting that they employ the
same general mechanism.
Subsequently, substrate-assisted catalysis has been implicated in the DNA cleavage of other restriction enzymes. Results
of experiments with TuqI and oligodeoxynucleotide substrates in
which single phosphate groups were stereospecifically modified
by S-methyl groups (Mayer and Barany, 1994) are in agreement
with a substrate-assisted catalysis mechanism. Methylphosphonate substitutions provided evidence for substrate-assisted catalysis for nine additional restriction enzymes (Bsp1431, CfrSI,
DpnII, Ec0721, MboI, M,I’ NdeII, Sau3AI and XhoII; Jeltsch et
al., 1 9 9 5 ~ )It. should be mentioned that no evidence for substrate-assisted catalysis was found for CfrlOI (Jeltsch et al.,
1995c) whose active site shows the most severe deviations from
the arrangement found in EcoRI, EcoRV, BumHI and PvuII
(Bozic et al., 1996). Perhaps this finding is not a coincidence
but means that the cleavage mechanism of CfrlOI is different
from that of the other enzymes whose structures have been
solved.
The substrate-assisted catalysis model can be summarized as
follows (Fig. 11B). The attacking water molecule is oriented and
deprotonated by the next phosphate group 3‘ to the scissile phosphate. The negative charge of the transition state could be stabilized by the Mg” ion and the semi-conserved lysine. The metal
ion is bound by the two conserved acidic amino acid residues.
The 3‘-0- leaving group is protonated by a Mgz+-bound water.
The model is entirely consistent with all crystallographic data
and supported by biochemical results, but it has not yet been
proven directly. It should be noted that substrate assistance by a
phosphate group is also considered for other enzymes involved
in nucleotidyl or phosphoryl transfer: p2lras and other G-proteins (Schweins et al., 1994), aminoacyl-tRNA synthetases (Perona et al., 1993; Cavarelli et al., 1994) and acylphosphatase
(Thunnisen, M., personal communication).
The two-metal-ion mechanism for EcoRV. EcoRV is the
only restriction enzyme for which results of soaking the crystals
containing restriction-endonuclease-DNA complexes with divalent metal ions have been published in detail so far (Kostrewa
and Winkler, 1995). Soaking experiments with high concentrations of Mg”, Mn2+and/or Ca2+revealed two metal ion interaction sites in the catalytic center of the enzyme-DNA complex.
These sites are formed by Asp901Asp74 and Asp741Glu45.
Soaking experiments with Mg2’ showed significant electron
density at the Asp901Asp74 site. At the Asp741Glu45 site only
a weak density was observed that does not fulfil stringent criteria
for the unequivocal identification of Mg2+ binding sites (Kankare et al., 1996). Moreover, this extra electron density is observed in only one catalytic center of the dimer. Cleavage of
the DNA does not occur after soaking the crystals with Mg”,
presumably because an essential conformational change is prevented by the crystallographic environment of the enzyme
(Kostrewa and Winkler, 1995). In apparent agreement with the
crystallographic results, biochemical experiments provided evidence for the existence of more than one metal ion binding site
in EcoRV, because it was shown that in the presence of Mn2+
addition of Ca2+, which by itself does not support cleavage,
stimulates the DNA cleavage rate of the endonuclease (Vipond
et al., 1995). Based on these findings, a two-metal-ion mechanism was suggested for EcoRV by Kostrewa and Winkler (1995)
and Vipond et al. (1995) (Fig. 1lC) but with different details in
the two versions. In this mechanism a metal ion bound at one
site (Kostrewa and Winkler: Asp741Glu45; Vipond et al.:
Asp901Asp74) is responsible for charge neutralization at the
scissile phosphate. The attacking water is believed to be part of
the hydration sphere of a metal ion bound at the second site
(Kostrewa and Winkler: Asp901Asp74; Vipond et al. : Asp741
Glu45). This proposal, however, has three major drawbacks.
a) This mechanism assigns to Glu45 the role of a central
catalytic residue. However, this role clearly is not in agreement
with results of mutagenesis studies which demonstrate that in the
presence of Mn2+(i.e. under conditions where the Ca2+effect is
observed) an E45+A mutant is nearly as active as wild-type
EcoRV and, in the presence of Mg2+,the activity of this mutant
is reduced by only two or three orders of magnitude (Selent et
Pingoud and Jeltsch ( E m J. Biochem. 246)
16
A
aftackino wafer
(in line)
B
C
Lvs92
0
?
0
I
\
OH2
H- 0.
I
Mg2+ Mg2+
Mg2’
D7
00
\I
00
\I
I
I
c
c
Asp90 Asp74
0 0
\ I
c
00 00
\ I
c
\I
c
I
1
1
Asp90 Asp74 Glu45
Fig. 11. Structure of the catalytic center of EcoRV. (A) The attacking water molecule has been introduced at the position necessary for an in-line
attack (based on Jeltsch et al., 1992). Metal ions are displayed as gray circles. Note that metal ion binding at the Glu45/Asp74 site is observed only
with some combinations of metal ions used (Kostrewa and Winkler, 1995). (B) Schematic illustration of the substrate assistance model for EcoRV.
(C) Schematic representation of the two metal ion catalysis model for EcoRV (adapted from Vipond et al., 1995).
al., 1992; Groll, D., Jeltsch, A,, Selent, U. and Pingoud, A.,
unpublished). This is in sharp contrast to results obtained with
the D74+A and D90-A mutants, which are completely inactive in the presence of Mg” as well as in the presence of
MnZ+(Selent et al., 1992; Groll, D., Jeltsch, A., Selent, U. and
Pingoud, A,, unpublished). A comparison of the structures of
EcoRV and PvuII, which are similar to each other in many details (see above), reveals that in PvuII Leu39 is structurally
equivalent to Glu45 in EcoRV, again suggesting that Glu45
could not be a catalytic residue (Athanasiadis et al., 1994).
b) The stereochemistry of this mechanism is unfavorable, in
particular for the Vipond et al. version of the mechanism, and
cannot be the same as in the two-metal-ion mechanism disscussed by Steitz (1993) for the 5‘-3‘ exonuclease activity of
DNA polymerase I. As shown in Fig. IIA, it is impossible to
place the attacking water in the hydration sphere of the metal
ion in the E45/D74 site, because both entities are 0.89 nm apart
from each other.
c) The two-metal-ion mechanism would not hold for all restriction enzymes. For example, there are no hints of more than
one metal ion bound to the active centers of EcoRI as shown by
MgZ+soaking experiments which, different froin EcoRV, led to
the cleavage of the DNA in the crystal (Rosenberg, 1991). Moreover, in EcoRI and PvulI there are no additional acidic amino
acid residues that could ligand a second metal ion. Possible candidates (EcuRI: Asp59, PvuII: GIu55) were shown not to be
important for catalysis (EcoRI: Grabowski et al., 1996; PvuII:
Nastri, H. G., Walker, I. H., Evans, P. D. and Riggs, P. D., personal communication).
In conclusion, metal ion binding in EcoRV might take place
at a site formed by Asp74 and Glu45, but it is unlikely that a
metal ion bound at this site has an important catalytic role.
Star activity of restriction endonucleases. It has been observed that many restriction enzymes work less accurately under
certain buffer conditions, called star conditions, including the
presence of Mn” ions instead of Mg2+(EcoRI: Hsu and Berg,
1978), buffers of alkaline pH (EcoRI: Polisky et al., 1975) or
the presence of glycerol, other organic solvents (EcoRI: Goodman et al., 1977) or, in general, substances that lead to an
increase in osmotic pressure (Robinson and Sligar, 1993, 1994,
1995). Under star conditions sites are also cleaved that deviate
from the cognate sites by one base pair (EcoRI: Gardner et al.,
1982). Alkaline pH, substitution of Mg” by Mn2+,and presence
of organic solvents are also star conditions for several other restriction endonucleases (BamHI : Xu and Schildkraut, 1991b;
BsuR1: Heininger et al., 1977; CeqI: Izsvak and Duda, 1989;
DdeI: Makula and Meagher, 1980; EcoRV: Halford et al., 1986;
HindIII: Hsu and Berg, 1978; HinfI: Petronzino and Schildkraut, 1990; Kriss et al., 1990; MboII: Sektas et al., 1995; PstI:
Malyguine and Vannier, 1980; PvuII: Nasri and Thomas, 1987;
RsrI: Aiken and Gumport, 1988; SulI: Malyguine and Vannier,
1980; TaqI: Barany, 1988; C a o e t al., 1995; TthlllI: Shinomiya
et a]., 1982).
Based on these observations, star activity appears to be a
general phenomenon of many, if not all, restriction enzymes.
Hence, a general molecular interpretation is needed that is based
on common aspects of the reaction catalyzed by all restriction
enzymes and not on details of the recognition process of each
enzyme. From a molecular point of view, the observation of star
activity means that DNA is cleaved although not all sequencespecific contacts are formed. Thus, under star conditions restriction enzymes seem to require less activation energy to switch
into the catalytically competent conformation. This description
is entirely consistent with the observation that under star conditions some mutants of restriction endonucleases become activated that have a strongly reduced activity under normal cleavage conditions (BamHI: Xu and Schildkraut, 1991b ; EcoRV:
Selent et al., 1992; Vermote et al., 1992; Vipond et al., 1996;
EcoRI: Jeltsch et al., 1993a). Interestingly, in general these mutants are accurate even under star conditions. It appears as if a
situation in which a wild-type restriction enzyme is confronted
with a star substrate is similar to one in which an enzyme mutant
interacts with the canonical substrate; in both cases not all specific contacts between the enzyme and the DNA can be formed
and in both cases cleavage is sometimes possible under star conditions. Hence, under star conditions, some of the specific contacts between the DNA and the protein are dispensable for
catalysis.
As very different conditions are involved, the molecular basis of star activity is likely to be different under each condition.
Mn’+ and alkaline pH might directly influence the catalytic center. One could envisage that Mn’ . . . H,O is better suited than
Mg2’ . . . H,O to protonate the leaving group because Mn’ ‘ bound water has a pK, of 10.6 whereas Mg”-bound water has
a pK., of 11.4 (Scott and Klug, 1996). Moreover, Mn’+ binds
with a higher affinity to oxygen ligands than Mg2+ (Martell and
Smith, 1977). Consequently, it could be demonstrated for EcoRV
+
Pingoud and Jeltsch (Eul: J. Biochem. 246)
that Mn2+ can bind to a catalytic center that is not in a fully
active conformation either because the substrate has a degenerate sequence (star site) (Vermote and Halford, 1992) or the enzyme has an amino acid substitution (Vermote et al., 1992). Thus
a non-ideal conformation of the active site might still lead to
metal ion binding and DNA cleavage in the presence of MnZ+.
Similarly, at alkaline pH a nucleophilic attack could be done
directly by OH-, omitting the need for an activation of water by
the enzyme-substrate complex. In contrast, osmotic pressure is
likely to have a more indirect influence. It tends to dehydrate
the protein-DNA complex, and, thereby, possibly perturbs the
equilibrium between inactive and active conformations. As described above, in the non-specific binding mode, water molecules are present at the enzyme-DNA interface, many of which
are released when the specific complex is formed. Osmotic pressure tends to reduce the water content of the complex. Thereby
it could induce tight pseudo-specific binding, although not all
specific contacts are formed, because under conditions of low
water activity it could be energetically more favorable to have
few unsaturated hydrogen bond partners at the protein-DNA
interface than to recruit many water molecules to form a loose
non-specific complex, in which all hydrogen bond donors and
acceptors can interact with water molecules. Induction of conformational changes are likely to also be the cause of the enhanced star activity observed with some EcoRI mutants containing substitutions at Hisll4, Ala138 or Glu192 (Heitman and
Model, 1991; Flores et al., 1995), because none of these amino
acid residues forms a specific contact to the bases of the EcoRI
recognition sequence.
In conclusion, the phenomenon of star activity of restriction
endonucleases can be explained by direct (OH-, Mn2+)or indirect (osmotic pressure) effects on the catalytic center which
leads to its premature activation.
Conclusions
In the present review we have discussed in detail the enzymology of several type-TI restriction endonucleases and focused
on those enzymes where cocrystal structures are available and
structure/function relationships have been investigated, viz.
EcoRI, EcoRV, BamHI and PvuII. The question, of course, arises
whether principal conclusions derived from the study of these
enzymes hold for other type-I1 restriction endonucleases and related endonucleases. While discussing this point, we would like
to summarize general features of the interaction of these restriction enzymes and DNA regarding target site location, recognition and catalysis.
The ability to interact non-specifically with DNA is common
to these enzymes and all other restriction endonucleases studied
so far in this respect. While this most certainly is the inevitable
side effect of providing a binding site for specific DNA, it could
in addition be the outcome of an optimization process to achieve
fast target site location by linear diffusion, which is dependent
on a certain affinity for non-specific DNA. All restriction endonucleases investigated in this respect are able to scan the DNA
in search for their target site.
The recognition process can be considered to be a mutual
adaptation of the structures of the enzyme and the substrate,
leading quite often to a considerable distortion of the DNA and
restructuring of structural elements of the protein. There is evidence for DNA bending in several other systems than those described here (Aiken et al., 1991 a ; Withers and Dunbar, 1993,
1995a, b), less is known about conformational changes of the
protein in other systems, but it can be anticipated that considerable changes in conformation must take place upon non-specific
and specific DNA binding.
17
Particularly pronounced, of course, are changes in the protein-DNA interface, from which solvent molecules and ions
have to be expelled. As for other DNA-binding proteins, water
and cation release is a major driving force for the recognition
process (Record et al., 1978). Even slight interference with the
restructuring of the interface by increasing the osmotic pressure
leads to star activity with many restriction enzymes, which in
turn can be suppressed by an increase in hydrostatic pressure
(Robinson and Sligar, 1994). However, some water molecules
and some ions, in particular divalent cations, are likely to be
retained in the interface, as they are used for water-mediated
hydrogen bonds between the DNA and the enzyme, as well as
bridging interactions between non-bridging oxygens of the phosphates and carboxylates. Mg*+, the natural metal ion cofactor
for all restriction endonucleases, is probably one of the ions that
is retained in the interface, although it presumably can also diffuse into the enzyme-DNA complex. Mg2+ions can be replaced
by other cations, in particular Mn2+but, as shown in many cases,
with reduction in specificity because Mnz+ forms tighter complexes with its ligands than Mgz+ and, therefore, can be bound
also when the interface is not optimally arranged.
As a result of the restructuring of the protein-DNA interface
the enzyme forms a multitude of new contacts, both to the bases
and the phosphodiester backbone. The interaction with the bases
in the systems analyzed occur mainly in the major groove, in
accordance with a model for DNA recognition proposed by Seeman et al. (1976). In addition, sequence-dependent conformations of the DNA backbone are probed by restriction enzymes,
similarly as originally described for the Trp repressor (Otwinowski et al., 1988). Both direct readout by interactions mainly with
the edges of the bases in the major groove and indirect readout
by interactions with the phosphates are likely to characterize
specific complex formation between DNA and restriction endonucleases in general. Also, it appears as a general principle that
interactions between protein and DNA extend beyond the
boundaries of the recognition sequence. This would in part explain why all restriction enzymes studied in this respect exhibit
preferences for sites in a particular sequence context. Of course,
this phenomenon is also a consequence of the modulation of the
structure of the recognition sequence by the nature of the adjacent sequences. A corollary of the finding that restriction enzymes in general cover a longer stretch of DNA than given by
their recognition sequence is that the extra binding energy can
be used to make DNA cleavage faster. In conjunction with electrostatic effects due to the polyanionic nature of polymeric DNA
(Zhang et al., 1996) this presumably explains why short oligodeoxyiiucleotides are cleaved less efficiently than large oligodeoxynucleotides, again a general observation.
The cocrystal structures solved for restriction enzymes so far
have in common that specific contacts between individual subunits and half-sites (as defined by the C2 symmetry axis) are
cross-over in nature, which means that each subunit makes contacts to both half-sites. This arrangement guarantees that specific
binding leads to activation of both catalytic centers and, therefore, concerted double-strand cleavage. As this is a biological
necessity for restriction enzymes to fulfill their in vivo function,
it can be anticipated that these kinds of cross-over interactions
will be used by restriction enzymes in general.
The number of specificity-determining interactions between
a given restriction enzyme and its recognition sequence, deduced
from the cocrystal structures and confirmed by mutagenesis as
well as chemical modification studies, is close to the maximum
possible. This explains the high specificity of restriction enzymes (as well as frustrations in trying to alter the specificity of
these enzymes). Given the general observation that restriction
endonucleases are very accurate enzymes, it can be assumed that
18
Pingoud and Jeltsch (Eur: J. Biockem. 246)
they all take full advantage of the information content hidden in
their respective recognition sequences, i.e. we can expect that
recognition is highly redundant in all cases. This will be different in related systems, represented by the homing endonucleases which recognize much longer DNA sequences than restriction enzymes. With these enzymes, it has been demonstrated that
in many positions of the recognition sequence base pairs can be
exchanged with negligible effect on cleavage efficiency (review :
Mueller et al., 1993) and that the length of the recognition sequence is not clearly defined (Wende et al., 1996b).
The coupling of specific binding to catalysis in the specific
restriction endonuclease complex is based on conformational
changes and, hence, can only be deduced vaguely from the information provided by the available cocrystal structure analyses,
site-directed mutagenesis and chemical modification studies.
The general scheme that emerges is that the trigger activating
the catalytic centers is the cooperative formation of a highly
ordered and precisely defined interface which includes structural
elements of both enzyme and substrate, as well as solvent molecules and ions. This concept does not allow for individual interactions to have the sole trigger function.
The mechanism of phosphodiester bond cleavage by restriction enzymes has in no case been identified unequivocally, in
spite of the fact that the location of the active site and essential
residues in the active site have been identified in several cases.
This is one reason why it is premature to conclude that restriction enzymes all follow the same mechanism. It is clear, however, that many restriction enzymes make use of the same or a
similar type of active center, represented by the PD ...(D/E)XK
motif. This sequence motif is very loosely defined and, therefore, cannot be used as a signature motif in the primary structure
of restriction enzymes. Presumably, it is no coincidence that all
restriction enzymes whose structures have been solved have basically the same active-site architecture. It is quite likely that
this similarity will extend to related enzymes, homing endonucleases for example, one of which has a bonafide PD ...(D/E)XK
motif (Wittmayer and Raines, 1996).
It is tempting to speculate that type 11restriction (and modification) enzymes which form a very large family of enzymes of
similar function are evolutionarily related. As they occur ubiquitously in almost all prokarytic organisms they must have evolved
early in evolution and/or spread effectively by horizontal gene
transfer (Jeltsch and Pingoud, 1996). Their establishment in a
new host presumably was aided by the fact that the genes for
restriction enzymes can act as selfish genetic elements (Naito et
a]., 1995; Kulakauskas et al., 1995). The amino acid sequence
data available make it very hard to decide whether restriction
enzymes are the outcome of divergent evolution, because their
sequences appear dissimilar, with few exceptions of obviously
related isoschizomers (EcoRYRsrI: Aiken and Gumport, 1988 ;
Stephenson et al., 1989; FnudUNgoPII : Sullivan and Saunders,
1989; XmaIICfi91: Wilson and Murray, 1991 ; BanYHgiCI: Erdmann et al., 1991 ; TaqI/TthHB81: Barany et al., 1992) and one
example of enzymes (BsuFI and MspI), which have a very similar C-terminal amino acid sequence (Kapfer et al., 1991). However, it is intriguing to note that;beyond reasonable doubt, sequences of restriction enzymes with similar recognition sequences are more related to each other than those of restriction
enzymes with dissimilar recognition sequences (Jeltsch et al.,
1995d). This implies that restriction enzymes have common evolutionary roots as exemplified by the Hgi family of restriction
enzymes (Kroger et al., 1984). Recently, structural comparisons
of restriction endonucleases and polynucleotidyltransferase superfamily enzymes, like the HIV integrase, revealed a structural
resemblance (Venclovas and Siksnys, 1995). Even more intriguing is the finding that a NaeI mutant displays DNA topoisom-
erase and recombinase activities (Jo and Topal, 1995). Whether
site-specific endonucleases are evolutionarily related to other
polynucleotidyltransferases, however, remains to be seen.
Thanks are due to Drs R. Roberts and D. Macelis for maintaining
and making available the REBASE-data base (http ://www.neb.com/rebase; Roberts and Macelis, 1996), as well as to Drs A. K. Aggarwal, J.
Alves, A. Bhagwat, T. A. Bickle, J. Brooks, X. Cheng, B. A. Connolly,
R. I. Gumport, W. Guschelbauer, S. Halford, V. Siksnys, Y. Janulaitis,
A. Podhajska, D. N. Rao, P. D. Riggs, J. M. Rosenberg, I. Schildkraut,
W. Szybalski, V. M. Vogt and F. K. Winkler for interesting discussions
and/or communication of results prior to publication. We are grateful to
Drs J. Alves, V. Pingoud, U. Selent, W. Wende and C. Wenz for critically
reading the manuscript. Work in the authors' laboratory has been supported by the Deutsche Forsckungsgemeinsckaft, the Bundesministerium
fur Bildung, Wissenschaft, Forsckung und Tecknologie and the Fonds
der Ckemiscken Industrie.
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