n Enzymes

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n Enzymes

Genetics | 2003 | Guilfoile, Patrick G.

Copyright Genetics Society of America.

Restriction Enzymes

Restriction enzymes are bacterial proteins that recognize specific DNA sequences and cut DNA

at or near the recognition site. These enzymes are widely used in molecular genetics for

analyzing DNA and creatingrecombinant DNA molecules.

Biological unction and !istorical Bac"ground

Restriction enzymes apparently e#ol#ed as a primiti#e immune system in bacteria. $f #iruses

enter a bacterial cell containing restriction enzymes% the #iral DNA is fragmented. Destruction of

the #iral DNA pre#ents destruction of the bacterial cell by the #irus. The term &restriction&

deri#es from the phenomenon in which bacterial #iruses are restricted from replicating in certain

strains of bacteria by enzymes that clea#e the #iral DNA% but lea#e the bacterial DNA untouched.

$n bacteria% restriction enzymes form a system with modification enzymes thatmethylate the

bacterial DNA. 'ethylation of DNA at the recognition sequence typically protects the microbe

from clea#ing its own DNA.

(ince the )*+,s% restriction enzymes ha#e had a #ery important role in recombinant DNA

techniques% in both the creation and analysis of recombinant DNA molecules. The first restriction

enzyme was isolated and characterized in )*-% and o#er /%0,, restriction enzymes ha#e been

disco#ered since. 1f these enzymes% o#er 20, are currently commercially a#ailable.

Nomenclature and 3lassification

Restriction enzymes are named based on the organism in which they were disco#ered. ore4ample% the enzyme Hin d $$$ was isolated from Haemophilus influenzae% strain Rd. The first

three letters of the name are italicized because they abbre#iate the genus and species names of

the organism. The fourth letter typically comes from the bacterial strain designation. The Roman

numerals are used to identify specific enzymes from bacteria that contain multiple restriction

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enzymes. Typically% the Roman numeral indicates the order in which restriction enzymes were

disco#ered in a particular strain.

There are three classes of restriction enzymes% labeled types $% $$% and $$$. Type $ restriction

systems consist of a single enzyme that performs both modification 5methylation6 and restriction

acti#ities. These enzymes recognize specific DNA sequences% but clea#e the DNA strand

randomly% at least )%,,, base pairs 5bp6 away from the recognition site. Type $$$ restriction

systems ha#e separate enzymes for restriction and methylation% but these enzymes share a

common subunit. These enzymes recognize specific DNA sequences% but clea#e DNA at random

sequences appro4imately twenty7fi#e bp from the recognition sequence. Neither type $ nor type

$$$ restriction systems ha#e found much application in recombinant DNA techniques.

Type $$ restriction enzymes% in contrast% are hea#ily used in recombinant DNA techniques. Type$$ enzymes consist of single% separate proteins for restriction and modification. 1ne enzyme

recognizes and cuts DNA% the other enzyme recognizes and methylates the DNA. Type $$

restriction enzymes clea#e the DNA sequence at the same site at which they recognize it. The

only e4ception are type $$s 5shifted6 restriction enzymes% which clea#e DNA on one side of the

recognition sequence% within twenty nucleotides of the recognition site. Type $$ restriction

enzymes disco#ered to date collecti#ely recognize o#er 8,, different DNA sequences.

Type $$ restriction enzymes can clea#e DNA in one of three possible ways. $n one case% these

enzymes clea#e both DNA strands in the middle of a recognition sequence% generating blunt

ends. or e4ample9 5The notations 2′ and /′ are used to indicate the orientation of a DNA

molecule. The numbers 2 and / refer to specific carbon atoms in the deo4yribose sugar in DNA.6

These blunt ended fragments can be :oined to any other DNA fragment with blunt ends% ma"ing

these enzymes useful for certain types of DNA cloning e4periments.

Type $$ restriction enzymes can also clea#e DNA to lea#e a /′ 5&three prime&6 o#erhang. 5An

o#erhang means that the restriction enzyme lea#es a short single7stranded &tail& of DNA at thesite where the DNA was cut.6 These /′o#erhanging ends can only :oin to another compatible

/′ o#erhanging end 5that is% an end with the same sequence in the o#erhang6. inally% some type

$$ enzymes can generate 2′ o#erhanging DNA ends% which can only be :oined to a compatible

2′ end.

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$n the type $$ restriction enzymes disco#ered to date% the recognition sequences range from 0 bp

to * bp long. 3lea#age will not occur unless the full length of the recognition sequence is

encountered. Enzymes with a short recognition sequence cut DNA frequently; restriction

enzymes with or * bp sequences typically cut DNA #ery infrequently% because these longer

sequences are less common in the target DNA.

<se of Restriction Enzymes in Biotechnology

The ability of restriction enzymes to reproducibly cut DNA at specific sequences has led to the

widespread use of these tools in many molecular genetics techniques. Restriction enzymes can be

used to map DNA fragments or genomes. 'apping means determining the order of the

restriction enzyme sites in the genome. These maps form a foundation for much other genetic

analysis. Restriction enzymes are also frequently used to #erify the identity of a specific DNA

fragment% based on the "nown restriction enzyme sites that it contains.

=erhaps the most important use of restriction enzymes has been in the generation of recombinant

DNA molecules% which are DNAs that consist of genes or DNA fragments from two different

organisms. Typically% bacterial DNA in the form of a plasmid 5a small% circular DNA molecule6 is

:oined to another piece of DNA 5a gene6 from another organism of interest. Restriction enzymes

are used at se#eral points in this process. They are used to digest the DNA from the e4perimental

organism% in order to prepare the DNA for cloning. Then a bacterial plasmid or bacterial #irus isdigested with an enzyme that yields compatible ends. These compatible ends could be blunt 5no

o#erhang6% or ha#e complementary o#erhanging sequences. DNA from the e4perimental

organism is mi4ed with DNA from the plasmid or #irus% and the DNAs are :oined with an

enzyme called DNAligase . As noted abo#e% the identity of the recombinant DNA molecule is

often #erified by restriction enzyme digestion.

Restriction enzymes also ha#e applications in se#eral methods for identifying indi#iduals or

strains of a particular species. =ulsed field gel electrophoresis is a technique for separating large

DNA fragments% typically fragments resulting from digesting a bacterial genome with a rare7

cutting restriction enzyme. The reproducible pattern of DNA bands that is produced can be used

to distinguish different strains of bacteria% and help pinpoint if a particular strain was the cause of

a widespread disease outbrea"% for e4ample.

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Restriction fragment length polymorphism 5R>=6 analysis has been widely used for

identification of indi#iduals 5humans and other species6. $n this technique% genomic DNA is

isolated% digested with a restriction enzyme% separated by size in an agarose gel% then transferred

to a membrane. The digested DNA on the membrane is allowed to bind to a radioacti#ely or

fluorescently labeled probe that targets specific sequences that are brac"eted by restriction

enzyme sites. The size of these fragments #aries in different indi#iduals% generating a &biological

bar code& of restriction enzyme7digested DNA fragments% a pattern that is unique to each

indi#idual.

Restriction enzymes are li"ely to remain an important tool in modern genetics. The

reproducibility of restriction enzyme digestion has made these enzymes critical components of

many important recombinant DNA techniques.

see also Biotechnology; 3loning ?enes; ?el Electrophoresis; 'apping; 'ethylation; Nucleases;

=olymorphisms; Recombinant DNA.

Patrick G. Guilfoile

Bibliography

Bloom% 'ar" @.% ?reg A. reyer% and Da#id A. 'ic"los. Laboratory DNA Science: An

Introuction to !ecombinant DNA "echni#ues an $ethos of Genome Analysis. 'enlo =ar"%3A9 Addison7esley% )**-.

3ooper% ?eoffrey. "he %ell: A $olecular Approach. ashington% D39 A(' =ress% )**+.

reuzer% !elen% and Adrianne 'assey. !ecombinant DNA an &iotechnolo'y( 8nd ed.

ashington% D39 A(' =ress% 8,,,.

>odish% !ar#ey% et al. $olecular %ell &iolo'y( 0th ed. New Cor"9 . !. reeman% 8,,,.

1ld% R. .% and (. B. =rimrose. Principles of Gene $anipulation( 2th ed. >ondon9 Blac"well

(cientific =ublications%

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