Restriction enzymes were discovered about 30 years ago during investigations into thephenomenon of host-specific restriction and modification of bacterial viruses.
Bacteria initially resist infections by new viruses, and this "restriction" of viral growthstemmed from endonucleases within the cells that destroy foreign DNA molecules.Among the first of these "restriction enzymes" to be purified were EcoRI and EcoR IIfromEscherichia coli, and Hind II and Hind III fromHaemophilus influenzae. Theseenzymes were found to cleave DNA at specific sites, generating discrete, gene-sizefragments that could be re-joined in the laboratory.
Researchers were quick to recognize that restriction enzymes provided them with aremarkable new tool for investigating gene organization, function and expression. As theuse 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 certainviruses, restriction enzymes were found only within prokaryotes.
Many thousands of bacteria and archae 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 areexceedingly varied; they range in size from the diminutive Pvu II (157 amino acids) to thegiant Cje I (1250 amino acids) and beyond. Among over 3,000 activities that have beenpurified and characterized, more than 250 different sequence-specificities have beendiscovered. Of these, over 30% were discovered and characterized at New EnglandBiolabs.
第3页：Thesearchfornewspeci
The search for new specificities continues, both biochemically, by theanalysis of cell-extracts, and computationally, by the analysis ofsequenced genomes. Although most activities encountered today turnout to be duplicates--isoschizomers--of existing specificities, restrictionenzymes with new specificities are found with regularity. Beginning inthe early 1980’s, New England Biolabs embarked on a program to cloneand overexpress the genes for restriction enzymes. Cloning improvesenzyme purity by separating enzymes from contaminating activitiespresent in the same cells. It also improves enzyme yields and greatlysimplifies purification, and it provides the genes for sequencing andanalysis, and the proteins for x-ray crystallography.
Restriction enzymes protect bacteria from infections by viruses, and it isgenerally accepted that this is their role in nature. They function as microbialimmune systems. When a strain ofE.colilacking a restriction enzyme isinfected with a virus, most virus particles can initiate a successful infection.When the same strain contains a restriction enzyme, however, the probability ofsuccessful infection plummets. The presence of additional enzymes has amultiplicative effect; a cell with four or five independent restriction enzymescould be virtually impregnable.
第4页：Restrictionenzymesus
Restriction enzymes usually occur in combination with oneor two modification enzymes (DNA-methyltransferases)that protect the cell’s own DNA from cleavage by therestriction enzyme.
Modification enzymes recognize the same DNA sequenceas the restriction enzyme that they accompany, but insteadof cleaving the sequence, they methylate one of the basesin each of the DNA strands. The methyl groups protrudeinto the major groove of DNA at the binding site andprevent 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 themodification enzyme(s) are separate proteins that actindependently of each other.
In other systems, the two activities occur as separatesubunits, or as separate domains, of a larger, combined,restriction-and-modification enzyme.
第5页：Restrictionenzymesar
Restriction enzymes are traditionally classified into three types on thebasis of subunit composition, cleavage position, sequence-specificityand cofactor-requirements. However, amino acid sequencing hasuncovered extraordinary variety among restriction enzymes andrevealed that at the molecular level there are many more than threedifferent kinds.
Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognitionsequences. Originally thought to be rare, we now know from theanalysis of sequenced genomes that they are common. Type Ienzymes are of considerable biochemical interest but they have littlepractical value since they do not produce discrete restriction fragmentsor distinct gel-banding patterns.
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Type II enzymes cut DNA at defined positions close to or within their recognitionsequences. They produce discrete restriction fragments and distinct gel bandingpatterns, and they are the only class used in the laboratory for DNA analysisand gene cloning. Rather then forming a single family of related proteins, typeIIenzymes are a collection of unrelated proteins of many different sorts. Type IIenzymes frequently differ so utterly in amino acid sequence from one another,and indeed from every other known protein, that they likely arose independentlyin the course of evolution rather than diverging from common ancestors.
The most common type II enzymes are those like HhaI, Hind III and Not I thatcleave DNA within their recognition sequences.
Enzymes of this kind are the principle ones available commercially. Mostrecognize DNA sequences that are symmetric because they bind to DNA ashomodimers, but a few, (e.g., BbvC I: CCTCAGC) recognize asymmetric DNAsequences because they bind as heterodimers. Some enzymes recognizecontinuous sequences (e.g.,EcoRI: GAATTC) in which the two half-sites of therecognition sequence are adjacent, while others recognize discontinuoussequences (e.g., Bgl I: GCCNNNNNGGC) in which the half-sites are separated.Cleavage leaves a 3´-hydroxyl on one side of each cut and a 5´-phosphate onthe other.
They require only magnesium for activity and the corresponding modificationenzymes require only S-adenosylmethionine. They tend to be small, withsubunits in the 200–350 amino acid range.
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The next most common type II enzymes, usually referred to as ‘type IIs" are those likeFok I and Alw I that cleave outside of their recognition sequence to one side.These enzymes are intermediate in size, 400–650 amino acids in length, and theyrecognize sequences that are continuous and asymmetric. They comprise two distinctdomains, one for DNA binding, the other for DNA cleavage. They are thought to bind toDNA as monomers for the most part, but to cleave DNA cooperatively, throughdimerization of the cleavage domains of adjacent enzyme molecules. For this reason,some type IIs enzymes are much more active on DNA molecules that contain multiplerecognition sites.
The third major kind of type II enzyme, more properly referred to as "type IV" are large,combination restriction-and-modification enzymes, 850–1250 amino acids in length, inwhich the two enzymatic activities reside in the same protein chain. These enzymescleave outside of their recognition sequences; those that recognize continuous sequences(e.g., Eco57 I: CTGAAG) cleave on just one side; those that recognize discontinuoussequences (e.g., Bcg I: CGANNNNNNTGC) cleave on both sides releasing a smallfragment containing the recognition sequence. The amino acid sequences of theseenzymes are varied but their organization are consistent. They comprise an N-terminalDNA-cleavage domain joined to a DNA-modification domain and one or two DNAsequence-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 tocleave the DNA, or modification mode to methylate it.
第8页：TypeIIIenzymes
Type III enzymes
Type III enzymes are also large combinationrestriction-and-modification enzymes. Theycleave outside of their recognitionsequences and require two such sequencesin opposite orientations within the sameDNA molecule to accomplish cleavage; theyrarely give complete digests. No laboratoryuses have been devised for them, and noneare available commercially.
