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.