Restriction enzymes also called Restriction endonucleases are proteins produced by bacteria that cleave DNA at specific sites along the molecule. In the bacterial cell, restriction enzymes cleave foreign DNA, thus eliminating infecting organisms. Restriction enzymes can be isolated from bacterial cells and used in the laboratory to manipulate fragments of DNA, such as those that contain genes; for this reason they are indispensable tools of recombinant DNA technology.

A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages, or phages. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. The restriction enzyme prevents replication of the phage DNA by cutting it into many pieces. Restriction enzymes were named for their ability to restrict, or limit, the number of strains of bacteriophage that can infect a bacterium.Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule-adenine, cytosine, thymine, and guanine). These regions are called recognition sequences and are randomly distributed throughout the DNA. Different bacterial species make restriction enzymes that recognize different nucleotide sequences.

When a restriction endonuclease recognizes a sequence, it snips through the DNA molecule by catalyzing the hydrolysis (splitting of a chemical bond by addition of a water molecule) of the bond between adjacent nucleotides. Bacteria prevent their own DNA from being degraded in this manner by disguising their recognition sequences. Enzymes called methyltranferases (methylases) add methyl groups (--CH3) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease. The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.
There are three classes of restriction enzymes, designated types I, II, and III. Types I and III enzymes are similar in that both restriction and methylase activities are carried out by one large enzyme complex, in contrast to the type II system, in which the restriction enzyme is independent of its methylase. Type II restriction enzymes also differ from the other two types in that they cleave DNA at specific sites within the recognition site; the others cleave DNA randomly, sometimes hundreds of bases from the recognition sequence.
The 1978 Nobel Prize in Medicine was awarded to Werner Arber, Daniel Nathans and Hamilton Smith for the discovery of restriction endonucleases, leading to the development of recombinant DNA technology. The first practical use of their work was the manipulation of E. coli bacteria to produce human insulin for diabetes.More than 2,500 type II restriction enzymes have been identified from a variety of bacterial species. These enzymes recognize about 200 distinct sequences, which are four to eight bases in length. The names of restriction enzymes are derived from the genus, species, and strain designations of the bacteria that produce them; for example, the enzyme EcoRI is produced by Escherichia coli strain RY13.

Mechanism:
Rather than cutting DNA indiscriminately, a restriction enzyme cuts only double-helical segments that contain a particular nucleotide sequence, and it makes its incisions only within that sequence--known as a "recognition sequence"--always in the same way.
Some enzymes make strand incisions immediately opposite one another, producing "Blunt end" DNA fragments. Most enzymes make slightly staggered incisions, resulting in "sticky ends", out of which one strand protrudes. There are three known evolutionary lineages of restriction enzyme, which each cleave DNA by a different mechanism.
While recognition sequences vary widely, many of them are palindromic; that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. The meaning of "palindromic" in this context is different from what one might expect from its linguistic usage: GTAATG is not a palindromic DNA sequence, but GTATAC is (GTATAC is complementary to CATATG).

The action of restriction enzymes is in many respects as varied as the enzymes themselves. In general, however, the process is one of recognition of the binding site, binding of the enzyme dimer to the DNA, cleavage of the DNA, and enzyme release. To begin, all restriction endonucleases will bind DNA specifically and, with much less strength, non-specifically.
This is a characteristic of many proteins that interact with DNA. It is probable that even non-specific DNA binding will induce a conformational change in the restriction enzyme dimer that will result in the protein adapting to the surface of the DNA strands. These changes are not the same as those that occur when the dimmer binds to the recognition site though. As the dimer slides along the DNA strands, it searches for recognition elements and, when these are encountered, an interaction between the protein and the DNA ensues in which the non-specific complex is converted into a specific complex. This requires significant conformational changes in both the protein and the DNA as well as expulsion of water molecules from the protein/DNA interface so that more intimate contacts can be established. In general, intimate contact is held by 15 - 20 hydrogen bonds that form between the protein and the DNA bases in the recognition site. These bonds are shown to be mediated through specific amino acids, primarily ASP and GLU, held in a proper three-dimensional configuration. There are differences among restriction enzymes with respect to how much water is expelled but, in all cases, it is a substantially greater amount than is expelled during non-specific binding.
Once the enzyme is specifically bound to its cognate DNA sequence there are more differences in the cleavage reaction. To begin, some restriction enzymes will bind one magnesium divalent ion whereas some will bind two. Moreover, other metal ions may or may not be present as well. Interestingly, to date, the actual mechanism by which a restriction enzyme cuts the DNA to which it is bound has not been demonstrated. The conventional wisdom holds that hydrolysis mediated by metal ion binding is the paradigm.

Types of restriction enzymes:
Restriction enzymes are classified biochemically into four types, designated Type I, Type II, Type III, and Type IV.

Type I
The key characteristics of the Type I R-M systems are that these enzymes are multisubunit proteins that function as a single protein complex and usually contain two R subunits, two M subunits and one S subunit. When Type I enzymes act on unmethylated substrates, they function mainly as REases (they may also methylate unmodified sites with a low probability) and have an absolute requirement for ATP during cleavage. They cleave the DNA at variable positions away from their recognition sequence. The location of the cleavage sites is determined by either the collision and stalling of two such complexes during translocation along a DNA chain, or the stalling of a single enzyme on a single-site circular substrate following DNA translocation. The biochemical nature of the termini produced upon cleavage is unknown and the enzymes do not turn over in the cleavage reaction. In contrast, when these complexes encounter a hemimethylated substrate, in which one strand of the recognition sequence is methylated, as would occur immediately after DNA replication of a fully methylated substrate, then the complex functions as a DNA MTase, using S-adenosylmethionine (AdoMet) as the donor of the methyl group. A complex of two M subunits and one S subunit is fully functional as an MTase. Probably the best known Type I enzyme is EcoKI. The REase is referred to as R.EcoKI or EcoKI, but it is important to remember that it is also an MTase. The MTase complex of two HsdM and one HsdS is referred to as M.EcoKI. Four sub-categories of Type I enzymes (A, B, C and D) are in common use. These are based on genetic complementation and their use will be continued.

Type II
The Type II REases recognize specific DNA sequences and cleave at constant positions at or close to that sequence to produce 5'-phosphates and 3'-hydroxyls. Usually they require Mg2+ ions as a cofactor, although some have more exotic requirements. They may act as monomers, dimers or even tetramers and usually act independently of their companion MTase. The MTases usually act as monomers and transfer a methyl group from the donor S-adenosyl-L-methionine directly to double-stranded DNA and form m4C, m5C or m6A. Because of the interest in these Type II REases for recombinant DNA technology, more than 3500 have been characterized. Given the assay that is used to find them, which detects any activity yielding a consistent DNA fragmentation pattern, it is no surprise that they come in a large variety of 'flavors'. Early on it was recognized that while then-normal Type II enzymes recognized palindromic sequences and cleaved symmetrically within them, the Type IIS enzymes cut outside their normally asymmetric sequences and differed in other interesting ways. We now know of additional enzymes that cleave on both sides of their recognition sequence (e.g. BcgI), are activated by AdoMet (e.g. Eco57I), interact with two copies of their recognition sequence (e.g. EcoRII) or have unusual subunit structures (e.g. BbvCI).
These additional kinds of enzymes will be considered subdivisions of Type II. It should be recognized that for the purposes of nomenclature some enzymes would fall into more than one subdivision. Specifically, some of the criteria are based on the sequence cleaved and others on the structure of the enzymes themselves, so not all subdivisions are mutually exclusive, e.g. BcgI is both Type IIB and IIH. Type IIS enzymes, originally designated as enzymes with cleavage sites shifted away from their recognition sequence, will be retained, but a new Type IIA will be defined that includes all Type II REases that recognize asymmetric sequences. A new Type IIP will be used to designate the enzymes that recognize symmetric sequences (palindromes).
The overriding criterion for inclusion as a Type II enzyme would be that it yields a defined fragmentation pattern and cleaves either within or close to its recognition sequence at a fixed site or with known and limited variability. In general, the Type II REases and their associated MTases are separate, independent enzymes, but in several classes (e.g. IIB, IIG and IIH) the R and M genes are fused into a single composite gene. The nomenclature for the subtypes of the Type II enzymes currently known is shown below. It should be noted that these designations are not intended to be exclusive, but rather to permit enzymes with common characteristics to be referred to as a group. Conservation of structural domains with associated enzymatic activities is observed between different classes of Type II enzymes and also between other types of R-M enzymes.

Type IIP
This would be used as a generic description for all enzymes that recognize symmetric sequences, often termed palindromes, and cleave at fixed symmetrical locations either within the sequence or immediately adjacent to it. The recognition sequences and cleavage sites of these enzymes should be represented as in the following example: EcoRI: G AATTC. In full double-stranded form this corresponds to:

5' G A A T T C
3' C T T A A G

Note that enzymes such as SinI (recognition sequence: GGWCC), BglI (recognition sequence: GCCNNNN NGGC) and HindII (recognition sequence: GTYRAC) belong to Type IIP because the recognition mechanism still involves a symmetric homodimer.

Type IIA
This would be used as a generic designation for any Type II enzymes that recognize asymmetric sequences irrespective of whether they cleave away from the sequence or within the sequence. Typically these systems have one REase gene and two MTase genes, one to modify each strand of the asymmetric recognition sequence. However, occasionally two R genes are found as with Bpu10I, or both M genes are fused as with M.FokI. When more than one R or M gene is present the genes and their protein products should be named with either an Arabic 1 or 2 in the prefix of the name. Thus, the two MTases of the SapI system would be named M1.SapI and M2.SapI if the proteins are being referred to, or sapIM1 and sapIM2 for the genes. However, the two subunits of the Bpu10I REase would be designated R.Bpu10IA and R.Bpu10IB and their genes bpu10IAR and bpu10IBR. The recognition sequences and cleavage sites of the Type IIS REases should be represented as in the following example:
HphI: GGTGA(8/7) where the first numeral in the parentheses indicates the position of cleavage on the strand written and the second numeral indicates the cleavage position on the complementary strand. In full double-stranded form this corresponds to:

5' GGTGANNNNNNNN
3' CCACTNNNNNNN

Note that when recognition sequences are assigned, the convention is to write the single-stranded sequence such that cleavage lies downstream of the sequence. If cleavage takes place within the sequence, then the single-strand designation is always written so that the sequence of the strand is first alphabetically.

Type IIB
This would be used for enzymes that cleave on both sides of the recognition sequence. At present there are many well defined members of this class (AloI, BplI, Bst44I, BaeI, BcgI, BsaXI, Bsp24I, CjeI, CjePI, HaeIV, Hin4I and PpiI). In this case the recognition sequence and cleavage sites should be represented as exemplified for BcgI:
BcgI-recognition sequence: (10/12)CGANNNNNNTGC(12/10)
Here the (10/12) preceding the recognition sequence indicates that cleavage occurs 10 bases in front of the sequence on the strand written and 12 bases before the sequence on the complementary strand. The (12/10) following the recognition sequence indicates cleavage 12 bases after the recognition sequence on the strand written and 10 bases after the sequence on the complementary strand. In double-stranded form this would be written:

NNNNNNNNNNCGANNNNNNTGCNNNNNNNNNNNN
NNNNNNNNNNNNGCTNNNNNNACGNNNNNNNNNN

Type IIC
This would be used as a generic term for all enzymes that have a hybrid structure containing both cleavage and modification domains within a single polypeptide. Examples include all of the Type IIB, IIG and some Type IIH enzymes.

Type IIE
This would be used for enzymes that interact with two copies of the recognition sequence, one being the actual target of cleavage, the other being the allosteric effector. The best studied examples are EcoRII and NaeI. FokI, MboII and Sau3AI were found to exhibit similar properties. Other enzymes such as Acc36I, AtuBI, BsgI, BpmI, Cfr9I, Eco57I, HpaII, Ksp632I, NarI, SacII and SauBMKI are likely to be members of this group because they are reported to be stimulated by oligonucleotide duplexes containing the specific recognition site.

Type IIF
This would be used for enzymes that interact with, and cleave coordinately, two copies of their recognition sequence. Examples include BspMI, Cfr10I, NgoMIV, SfiI and SgrAI.

Type IIG
This would be used for enzymes that have both R and M domains fused to form single polypeptides and that may be stimulated or inhibited by AdoMet, but otherwise resemble Type II enzymes. These include Bce83I, BseMII, BseRI, BsgI, BspLU11III, Eco57I, GsuI, MmeI and Tth111II. The recognition sequences may or may not be asymmetric. Thus, both Type IIA and Type IIP enzymes may be of Type IIG.

Type IIH
This would be used for enzymes that contain genetic features resembling Type I enzymes, but biochemically behave as Type II enzymes. At present three examples have been characterized: AhdI and PshAI, both of which comprise a three gene system akin to that of a typical Type I enzyme (G.G.Wilson, unpublished results), and BcgI, which is a two gene system. Several hypothetical systems have gene organizations that resemble that of BcgI.

Type IIM
This would be used for DpnI and similar enzymes that recognize a specific methylated sequence in DNA and cleave at a fixed site. Note that the methyl-dependent enzymes such as McrA, McrBC are not considered members of this subclass, because they do not have well defined recognition sequences and cleavage sites. They are included within the Type IV enzymes.

Type IIS
This would be used for Type IIA enzymes that cleave at least one strand of the DNA duplex outside of the recognition sequence (i.e. cleavage is shifted relative to the recognition sequence). Note that for some enzymes, such as BsmI (recognition sequence: GAATGC), cleavage of the strand written takes place outside of the recognition sequence, whereas cleavage of the complementary strand takes place within the recognition sequence. This is still considered a Type IIS enzyme. However, in most cases both strands are cleaved away from the recognition sequence, which therefore remains intact. These were the earliest sub-classes of the Type II restriction enzymes to be recognized.

Type IIT
This would be used for enzymes that are composed of heterodimeric subunits. This subtype includes enzymes like BbvCI, Bpu10I and BslI

Type III
These systems are composed of two genes (mod and res) encoding protein subunits that function either in DNA recognition and modification (Mod) or restriction (Res). Both subunits are required for restriction, which also has an absolute requirement for ATP hydrolysis. For DNA cleavage, the enzyme must interact with two copies of a non-palindromic recognition sequence and the sites must be in an inverse orientation in the substrate DNA molecule. Cleavage is preceded by ATP-dependent DNA translocation as with the Type I REases. The enzymes cleave at a specific distance away from one of the two copies of their recognition sequence. The Mod subunit can function independently of the Res subunit to methylate DNA: in all known cases the methylated base formed is m6A and full modification is actually hemimethylation. This is not deleterious because of the requirement for two unmodified sites in inverse repeat orientation for cleavage. DNA replication puts all of the unmodified sites in the same orientation. The best-known examples of Type III enzymes are EcoP1I and EcoP15I. Putative Type III R-M systems are easily recognized because of their similarity at the sequence level. When naming the genes for these enzymes the mod gene of EcoP1I would be systematically named ecoP1Imod, but the abbreviation mod is acceptable when it does not result in confusion.

Type IV
These systems are composed of one or two genes encod ing proteins that cleave only modified DNA, including methylated, hydroxymethylated and glucosyl-hydroxymethylated bases. Their recognition sequences have usually not been well defined except for EcoKMcrBC, which recognizes two dinucleotides of the general form RmC (a purine followed by a methylated cytosine-either m4C or m5C) and which are separated by anywhere from 40 to 3000 bases. Cleavage takes place 30 bp away from one of the sites. The best studied example at both the genetic and biochemical level is EcoKMcrBC of E.coli, but on the basis of sequence similarity it is likely that there are many such systems in other bacteria and archaea. As with the genes of the Type I and Type III systems, the abbreviations McrBC for the enzyme and mcrBC for the gene are acceptable.

Factors that Influence Restriction Enzyme Activity

It is not uncommon to have difficulties in digesting DNA with restriction enzymes. At times, the DNA does not appear to cut at all and sometimes it cuts only partially. If the sequence is known, restriction sites can be predicted with accuracy, but in the lab, an enzyme may cut more often than it should or at the wrong sites. In some cases, these unexpected results point to a problem not related to technique - for example, the sequence you have may be incorrect, or a restriction map provided by a colleague could be in error. However, there are a number of commonly-encountered situtions that influence how well restriction enzymes cut, and it is important to be aware of these for troubleshooting.

Buffer Composition
Different restriction enzymes have differing preferences for ionic strength (salt concentration) and major cation (sodium or potassium). A battery of 3 to 4 different buffers will handle a large number of available enzymes, although there are a few that require a unique buffer environment. In all cases, a major function of the buffer is to maintain pH of the reaction (usually at 8.0). Additionally, some enzymes are more fussy about having their optimal buffer than other enzymes. Clearly, use of the wrong buffer can lead to poor cleavage rates.

Incubation Temperature
Most restriction enzymes cut best at 37C, but there are many exceptions. Enzymes isolated from thermophilic bacteria cut best at temperatures ranging from 50 to 65C. Some other enzymes have a very short half life at 37C and its recommended that they be incubated at 25C.

Influence of DNA Methylation
Almost all strains of E. coli bacteria used for propagating cloned DNA contain two site-specific DNA methylases:
Dam methylase adds a methyl group to the adenine in the sequence GATC, yielding a sequence symbolized as GmATC.
Dcm methylase methylates the internal cytosine in CC(A/T)GG, generating the sequence CmC(A/T)GG.
The practical importance of this phenomenon is that a number of restriction endonucleases will not cleave methylated DNA.

Star Activity
When DNA is digested with certain restriction enzymes under non-standard conditions, cleavage can occur at sites different from the normal recognition sequence - such aberrant cutting is called "star activity". An example of an enzyme that can exhibit star activity is EcoRI; in this case, cleavage can occur within a number of sequences that differ from the canonical GAATTC by single base substitutions.
So what constitutes non-standard conditions? Examples that may induce star activity include:
" High pH (>8.0) or low ionic strength (e.g. if you forget to add the buffer)
" Glycerol concentrations > 5% (enzymes are usually sold as concentrates in 50% glycerol)
" Extremely high concentration of enzyme (>100 U/ug of DNA)
" Presence of organic solvents in the reaction (e.g. ethanol, DMSO)

Digestion with Multiple Enzymes
Digesting DNA with two enzymes is a commonplace task, and oftentimes the two enzymes have different buffer requirements. There are at least three ways to handle this situation:
" Digest with both enzymes in the same buffer. In many cases, even those a given buffer is not optimal for an enzyme, you can still get quite good cleavage rates. Enzyme manufacturer catalogs usually contain a reference table recommended the best single buffer for conducting specific double digests.
" Cut with one enzyme, then alter the buffer composition and cut with the second enzyme. This usually applies to situations where one enzyme like a low salt buffer and the other a high salt buffer, in which case you can digest with the first enzyme for a time, add a calculated amount of concentrated NaCl and cut with the second enzyme.
" Change buffers between digestion with two enzymes. In some cases, two enzymes will have totally incompatible buffers. In that case, perform one digestion, recover the DNA (usually by precipitation) and suspend in the buffer appropriate for the second enzyme.

Variability In Digestion of Different DNA Substrates
The efficiency with which a restriction enzyme cuts its recognition sequence at different locations in a piece of DNA can vary 10 to 50-fold. This is apparently due to influences of sequences bordering the recognition site, which perhaps can either enhance or inhibit enzyme binding or activity.
A related situation is seen when restriction recognition sites are located at or very close to the ends of linear fragments of DNA. Most enzymes require a few bases on either side of their recognition site in order to bind and cleave. Many of the companies that sell enzymes provide a table in their catalog that presents "end requirements" for a variety of enzymes.

Uses of Restriction Enzymes

Physical Mapping of DNA.
 RE digest permits the purification of specific DNA sequences and the mapping of sequences relative to one another. RE mapping can be achieved by digesting the DNA with single RE followed by double or triple digests.

Cloning of DNA.
RE permit the isolation of a particular DNA sequence creating a fragment that can now be inserted into a region of a bacterial plasmid or viral DNA that will serve as vectors.

Hybridization.
A particular gene may be located along a DNA molecule by hybridizing a particular DNA or mRNA probe to DNA fragments generated by RE digestion. E. Southern developed a technique in which the DNA restriction fragments separated by agarose gel electrophoresis were denatured (strands separated) in place by placing the entire gel into an alkali solution. The denatured DNA fragments can then be transferred and bound to a nitrocellulose membrane in precisely their arrangement in the gel by causing the fragments to diffuse out of the gel onto the membrane. After transfer, the membrane is placed in a solution containing a labeled DNA or RNA probe. The probe will base-pair (hybridize) only with the complementary sequence of DNA.

Sequence Analysis of DNA.
The DNA molecule to be sequenced is cleaved by a RE into a number of fragments. These fragments are then separated by electrophoresis, isolated and may be radioactively labeled at the 5' end using T4 polynucleotide kinase (an enzyme that can transfer 32P from 32P ATP to 5' end of DNA chain. After labeling the DNA to be sequenced, the fragment is cleaved by a second restriction enzyme to separate the two 5' ends. DNA sequencing can then be carried out by the Maxam and Gilbert technique or the Sanger dideoxy method.