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Restriction enzyme


A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the phosphate backbones of the double helix without damaging the bases. The chemical bonds that the enzymes cleave can be reformed by other enzymes known as ligases, so that restriction fragments carved from different chromosomes or genes can be spliced together, provided their ends are complementary (more below). Many of the procedures of molecular biology and genetic engineering rely on restriction enzymes. The term restriction comes from the fact that these enzymes were discovered in E. coli strains that appeared to be restricting the infection by certain bacteriophages. Restriction enzymes therefore are believed to be a mechanism evolved by bacteria to resist viral attack and to help in the removal of viral sequences.

Contents

Sites of cleavage

Rather than cutting DNA indiscriminately, a restiction 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.

Fragment complementarity and splicing

Because recognition sequences differ between restriction enzymes, the length and the exact sequence of a sticky-end "overhang", as well as whether it is the 5' or the 3' strand that overhangs, depends on which enzyme produced it. Base-pairing between overhangs with complementary sequences enables two fragments to join or "to be spliced," which they tend to do spontaneously in a test tube. Thus, a sticky-end fragment will readily reunite with the fragment from which it was originally cleaved, but it will also attach to any other fragment generated by the same restriction enzyme; because cuts made by a given type of enzyme always produce identical ends with identical sequences. These rules enable molecular biologists to anticipate which fragments will join and how they will join--and to choose enzymes to produce fragments they can splice. This knowledge represents more or less the essence of genetic engineering.

Restriction enzymes as tools

See the main article on restriction digests.

Recognition sequences typically are only four to twelve nucleotides long. Because there are only so many ways to arrange the four nucleotides--A,C,G and T--into a four or eight or twelve nucleotide sequence, recognition sequences tend to "crop up" by chance in any long sequence. Furthermore, restriction enzymes specific to hundreds of distinct sequences have been identified and synthesized for sale to laboratories. As a result, potential "restriction sites" appear in almost any gene or chromosome. Meanwhile, the sequences of some artificial plasmids include a "linker" that contains dozens of restriction enzyme recognition sequences within a very short segment of DNA. So no matter the context in which a gene naturally appears, there is probably a pair of restriction enzymes that can snip it out, and which will produce ends that enable the gene to be spliced into a plasmid (i.e. which will enable what molecular biologists call "cloning" of the gene).

Many Recognition sequences are palindromic

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.

Types of restriction enzymes

Restriction enzymes are classified biochemically into three types, designated Type I, Type II and Type III. In type I and III systems, both the methylase and restriction activities are carried out by a single large enzyme complex. Although these enzymes recognize specific DNA sequences, the sites of actual cleavage are at variable distances from these recognition sites, and can be hundreds of bases away. In type II systems, the restriction enzyme is independent of its methylase, and cleavage occurs at very specific sites that are within or close to the recognition sequence. The vast majority of known restriction enzymes are of type II, and it is these that find the most use as laboratory tools.

Naming

Restriction enzymes are named based on the bacteria in which they are isolated in the following manner:

E Escherichia (genus)
co coli (species)
R RY13 (strain)
I First identified Order ID'd in bacterium


Examples

Enzyme	 Source	                 Recognition Sequence  Cut
EcoRI   Escherichia coli	        5'GAATTC              5'---G     AATTC---3'          
                                   3'CTTAAG	          3'---CTTAA     G---5'        
BamHI   Bacillus amyloliquefaciens 5'GGATCC              5'---G     GATCC---3'
                                   3'CCTAGG              3'---CCTAG     G---5'  
HindIII Haemophilus influenzae     5'AAGCTT              5'---A     AGCTT---3'
                                   3'TTCGAA	         3'---TTCGA     A---5'  
MstII   Microcoleus species        5'CCTNAGG
                                   3'GGANTCC	
TaqI    Thermus aquaticus          5'TCGA                5'---T   CGA---3'
                                   3'AGCT	         3'---AGC   T---5'
NotI    Nocardia otitidis          5'GCGGCCGC
                                   3'CGCCGGCG	
AluI*   Arthrobacter luteus        5'AGCT                5'---AG  CT---3'	
                                   3'TCGA                3'---TC  GA---5'	
* = blunt ends

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