Restriction enzyme

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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. They are part of what is called the restriction modification system.

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 diabetics.

[edit] Sites of cleavage

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.

[edit] Fragment complementarity and splicing

EcoRI restriction enzyme cleavage produces "sticky" ends.

SmaI restriction enzyme cleavage produces "blunt" ends.

Because recognition sequences and cleavage sites differ between restriction enzymes, the length and the exact sequence of a sticky-end "overhang", as well as whether it is the 5' end or the 3' end strand that overhangs, depends on which enzyme produced it. Base-pairing between overhangs with complementary sequences enables two fragments to be joined or "spliced" by a DNA ligase. A sticky-end fragment can be ligated not only to the fragment from which it was originally cleaved, but also to any other fragment with a compatible sticky end.The sticky end is also called a cohesive end or complementry end in some reference. If a restriction enzyme has a non-degenerate palindromic cleavage site, all ends that it produces are compatible. Ends produced by different enzymes may also be compatible. Knowledge of cleavage sites allows molecular biologists to anticipate which fragments can be joined in which ways, and to choose enzymes appropriately.

[edit] 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).

Another use of restriction enzymes can be to find specific SNPs. If a restriction enzyme can be found such that it cuts only one possible allele of a section of DNA (that is, the alternate nucleotide of the SNP causes the restriction site to no longer exist within the section of DNA), this restriction enzyme can be used to genotype the sample without completely sequencing it. The sample is first run in a restriction digest to cut the DNA, then gel electrophoresis is performed on this digest. If the sample is homozygous for the common allele, the result will be two bands of DNA, because the cut will have occurred at the restriction site. If the sample is homozygous for the rarer allele, the sample show only one band, because it will not have been cut. If the sample is heterozygous at that SNP, there will be three bands of DNA.

[edit] 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 (GTATAC is complementary to CATATG).

[edit] Types of restriction enzymes

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

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. Both require ATP for their proper function. Type I restriction enzymes produce DNA cleavage following translocation of the DNA, which makes them important molecular motors. The cleavage of DNA appears to occur after blockage of the translocation activity (often following collision with another translocating Type R-M enzyme, but also due to other factors). These enzymes read the methylation status of their recognition sequnce, compare the methylation status of two adenines within the recognition sequence, and if both adenines are unmethylated (a signal that the DNA is none host DNA, the enzyme undergoes a conformational switch that turns the enzyme into a molecular motor and endonuclease. However, if either one of the adenines is methylated (a signal that the DNA is host DNA) then the enzyme acts as a maintenance methylase and methylates the other adenine.

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. The most known one is EcoRI which is staggered and its recognition sequence is 5'-GAATTC-3'. Type II enzymes are further classified according to their recognition site. Most type II enzymes cut palindromic DNA sequences, while type IIa enzymes recognise non-palindromic sequences and cleaveage outside of the recognition site, and type IIb ones cut sequences twice at both sites outsite the recognition sequence.

In type IV systems, the restriction enzymes target only methylated DNA.

[edit] Naming

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

[edit] Examples

5'GAATTC
3'CTTAAG

5'---G     AATTC---3'
3'---CTTAA     G---5'

5'GGATCC
3'CCTAGG

5'---G     GATCC---3'
3'---CCTAG     G---5'

5'AAGCTT
3'TTCGAA

5'---A     AGCTT---3'
3'---TTCGA     A---5'

5'CCTNAGG
3'GGANTCC

5'TCGA
3'AGCT

5'---T   CGA---3'
3'---AGC   T---5'

5'GCGGCCGC
3'CGCCGGCG

5'GANTC
3'CTNAG

5'AGCT
3'TCGA

5'---AG  CT---3'
3'---TC  GA---5'

[edit] External links

• Restriction Enzyme Digestion of DNA Protocol
• Restriction enzyme tools
• Restriction enzymes: protein data bank molecule of the month
• REBASE - The Restriction Enzyme Database
• Restriction enzyme finder
• WatCut - An online tool for restriction analysis
• Restriction digest of DNA - Online tool, free source code (PHP)
• Restriction Homepage - Six different tools
• Restriction Endonucleases: Molecular Scissors for Specifically Cutting DNA - a review from the Science Creative Quarterly