Restriction Endonucleases

Discovery

Restriction enzyme

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or

single stranded DNA at specific recognition nucleotide sequences known as restriction sites.
Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense
mechanism against invading viruses. Inside a bacterial host, the restriction enzymes selectively
cut up foreign DNA in a process called restriction; host DNA is methylated by a modification
enzyme (a methylase) to protect it from the restriction enzyme’s activity. Collectively, these two
processes form the restriction modification system. To cut the DNA, a restriction enzyme makes
two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double
helix.

After isolating the first restriction enzyme, HindII, in 1970, and the subsequent discovery and
characterization of numerous restriction endonucleases, the 1978 Nobel Prize for Physiology or
Medicine was awarded to Daniel Nathans, Werner Arber, and Hamilton O. Smith. Their
discovery led to the development of recombinant DNA technology that allowed, for example, the
large scale production of human insulin for diabetics using E. coli bacteria.Over 3000 restriction
enzymes have been studied in detail, and more than 600 of these are available commercially and
are routinely used for DNA modification and manipulation in laboratories.

Restriction enzymes were discovered 40 years ago during investigations into the phenomenon of
host-specific restriction and modification of bacterial viruses. Restriction enzymes protect
bacteria from infections by viruses, and it is generally accepted that this is their role in nature.
They function as microbial immune systems.. The presence of additional enzymes has a
multiplicative effect; a cell with four or five independent restriction enzymes could be virtually
impregnable.

Among the first restriction enzymes to be purified were EcoRI and EcoRII from Escherichia
coli, and HindII and HindIII from Haemophilus influenzae. These enzymes were found to cleave
DNA at specific sites, generating discrete, gene-size fragments that could be re-joined in the
laboratory. Researchers were quick to recognize that restriction enzymes provided them with a
remarkable new tool for investigating gene organization, function and expression.

As the use of restriction enzymes spread among molecular biologists in the late 1970’s,
companies such as New England Biolabs began to search for more. Except for certain viruses,
restriction enzymes were found only within prokaryotes. Many thousands of bacteria and archaea
have now been screened for their presence. Analysis of sequenced prokaryotic genomes indicates
that they are common - all free-living bacteria and archaea appear to code for them.

Restriction enzymes are exceedingly varied; they range in size from the diminutive PvuII (157
amino acids) to the giant CjeI (1250 amino acids) and beyond. Among over 3,000 activities that
have been purified and characterized, more than 250 different sequence-specificities have been
discovered.

R-M Systems

Restriction enzymes usually occur in combination with one or two modification enzymes (DNA-
methyltrans-ferases) that protect the cell’s own DNA from cleavage by the restriction enzyme.
Modification enzymes recognize the same DNA sequence as the restriction enzyme that they
accompany, but instead of cleaving the sequence, they methylate one of the bases in each of the
DNA strands. The methyl groups protrude into the major groove of DNA at the binding site and
prevent the restriction enzyme from acting upon it. Together, a restriction enzyme and its
"cognate" modification enzyme(s) form a restriction-modification (R-M) system. In some R-M
systems the restriction enzyme and the modification enzyme(s) are separate proteins that act
independently of each other. In other systems, the two activities occur as separate subunits, or as
separate domains, of a larger, combined, restriction-and-modification enzyme.

Types of Restriction Enzymes

Types

Restriction endonucleases are categorized into three general groups (Types I, II and III) based on
their composition and enzyme cofactor requirements, the nature of their target sequence, and the
position of their DNA cleavage site relative to the target sequence.

Restriction enzymes are traditionally classified into four types on the basis of subunit
composition, cleavage position, sequence specificity and cofactor requirements. However, amino
acid sequencing has uncovered extraordinary variety among restriction enzymes and revealed
that at the molecular level there are many more than four different types.

Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes

that cut DNA at random far from their recognition sequences. Originally thought to be rare, we
now know from the analysis of sequenced genomes that they are common. Type I enzymes are
of considerable biochemical interest, but they have little practical value since they do not
produce discrete restriction fragments or distinct gel-banding patterns.

Type II enzymes cut DNA at defined positions close to or within their recognition sequences.
They produce discrete restriction fragments and distinct gel banding patterns, and they are the
only class used in the laboratory for DNA analysis and gene cloning. Rather than forming a
single family of related proteins, Type II enzymes are a collection of unrelated proteins of many
different sorts. Type II enzymes frequently differ so utterly in amino acid sequence from one
another, and indeed from every other known protein, that they exemplify the class of rapidly
evolving proteins that are often indicative of involvement in host-parasite interactions. More than
3000 type II restriction endonucleases have been discovered. They recognize short, usually
palindromic, sequences of 4–8 bp and, in the presence of Mg2+, cleave the DNA within or in
close proximity to the recognition sequence. The orthodox type II enzymes are homodimers
which recognize palindromic sites. Depending on particular features subtypes are classified. All
structures of restriction enzymes show a common structural core comprising four β-strands and
one α-helix. Furthermore, two families of enzymes can be distinguished which are structurally
very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins,
restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for
efficient target site location by facilitated diffusion. Non-specific binding usually does not
involve interactions with the bases but only with the DNA backbone. In contrast, specific
binding is characterized by an intimate interplay between direct (interaction with the bases) and
indirect (interaction with the backbone) readout. Typically ∼15–20 hydrogen bonds are formed
between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to
numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may
also be water mediated. The recognition process triggers large conformational changes of the
enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction
enzymes the catalytic centers, one in each subunit, are represented by the PD . . . D/EXK motif,
in which the two carboxylates are responsible for Mg2+ binding, the essential cofactor for the
great majority of enzymes. The precise mechanism of cleavage has not yet been established for
any enzyme, the main uncertainty concerns the number of Mg2+ ions directly involved in
cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to
inversion of configuration at the phosphorus. The products of the reaction are DNA fragments
with a 3′-OH and a 5′-phosphate.

The most common Type II enzymes are those like HhaI, HindIII and NotI that cleave DNA
within their recognition sequences. Enzymes of this kind are the principle ones available
commercially. Most recognize DNA sequences that are symmetric because they bind to DNA as
homodimers, but a few, (e.g., BbvCI: CCTCAGC) recognize asymmetric DNA sequences
because they bind as heterodimers. Some enzymes recognize continuous sequences (e.g., EcoRI:
GAATTC) in which the two half-sites of the recognition sequence are adjacent, while others
recognize discontinuous sequences (e.g., BglI: GCCNNNNNGGC) in which the half-sites are
separated. Cleavage leaves a 3´-hydroxyl on one side of each cut and a 5´-phosphate on the
other. They require only magnesium for activity and the corresponding modification enzymes
require only S-adenosylmethionine. They tend to be small, with subunits in the 200-350 amino
acid range.

The next most common Type II enzymes, usually referred to as ‘Type IIS" are those like FokI
and AlwI that cleave outside of their recognition sequence to one side. These enzymes are
intermediate in size, 400-650 amino acids in length, and they recognize sequences that are
continuous and asymmetric. They comprise two distinct domains, one for DNA binding, the
other for DNA cleavage. They are thought to bind to DNA as monomers for the most part, but to
cleave DNA cooperatively, through dimerization of the cleavage domains of adjacent enzyme
molecules. For this reason, some Type IIS enzymes are much more active on DNA molecules
that contain multiple recognition sites.

Type IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more than one
subunit. They cleave DNA on both sides of their recognition to cut out the recognition site. They
require both AdoMet and Mg2+ cofactors. Type IIE restriction endonucleases (e.g. NaeI) cleave
DNA following interaction with two copies of their recognition sequence. One recognition site
acts as the target for cleavage, while the other acts as an allosteric effector that speeds up or
improves the efficiency of enzyme cleavage. Similar to type IIE enzymes, type IIF restriction
endonucleases (e.g. NgoMIV) interact with two copies of their recognition sequence but cleave
both sequences at the same time. Type IIG restriction endonucleases (Eco57I) do have a single
subunit, like classical Type II restriction enzymes, but require the cofactor AdoMet to be active.
Type IIM restriction endonucleases, such as DpnI, are able to recognize and cut methylated
DNA. Type IIS restriction endonucleases (e.g. FokI) cleave DNA at a defined distance from their
non-palindromic asymmetric recognition sites. These enzymes may function as dimers.
Similarly, Type IIT restriction enzymes (e.g., Bpu10I and BslI) are composed of two different
subunits. Some recognize palindromic sequences while others have asymmetric recognition sites.

Type IIG restriction enzymes, the third major kind of Type II enzyme, are large, combination
restriction-and-modification enzymes, 850-1250 amino acids in length, in which the two
enzymatic activities reside in the same protein chain. These enzymes cleave outside of their
recognition sequences; those that recognize continuous sequences (e.g., AcuI: CTGAAG) cleave
on just one side; those that recognize discontinuous sequences (e.g., BcgI: CGANNNNNNTGC)
cleave on both sides releasing a small fragment containing the recognition sequence. The amino
acid sequences of these enzymes are varied but their organization are consistent. They comprise
an N-terminal DNA-cleavage domain joined to a DNA-modification domain and one or two
DNA sequence-specificity domains forming the C-terminus, or present as a separate subunit.
When these enzymes bind to their substrates, they switch into either restriction mode to cleave
the DNA, or modification mode to methylate it.

Type III enzymes are also large combination restriction-and-modification enzymes. They cleave
outside of their recognition sequences and require two such sequences in opposite orientations
within the same DNA molecule to accomplish cleavage; they rarely give complete digests.

Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the
McrBC and Mrr systems of E. coli.