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A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA following its specific recognition of short nucleotide sequences, known as restriction sites, in the DNA. 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.
The Nobel Prize in Medicine was awarded, in 1978, to Daniel Nathans, Werner Arber, and Hamilton Smith for the discovery of restriction endonucleases. Their discovery lead 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 100 restriction enzymes have since been purified and characterized from different types and strains of bacteria, and are routinely used for DNA modification and manipulation in laboratories.
- 1 Restriction enzymes as tools
- 2 Recognition sites
- 3 Nomenclature
- 4 Enzyme Mechanisms
- 5 Splicing together cleaved DNA fragments
- 6 Examples
- 7 See also
- 8 References
- 9 External links
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 will 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. This is an example of restriction mapping, see the article on restriction maps
Restriction enzymes recognize a specific sequence of nucleotides and produce a double stranded cut in the DNA that prevents the phage from replicating. While recognition sequences vary widely, with lengths between 4 and 8 nucleotides, many of them are palindromic; that is, the sequence on one strand reads the same in the reverse 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):5'-GTATAC-3' |||||| 3'-CATATG-5'
NomenclatureE Escherichia (genus) co coli (species) R RY13 (strain) I First identified (order of identification in the bacterium)
Since their discovery in the 1970s, more than 100 different restriction enzymes have been identified in different bacteria. Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain. For example, the name of the EcoRI restriction enzyme was derived as shown in the box to the right.
- Type I restriction enzymes cut DNA about 100 nucleotides after the recognition site and requires ATP.
- Type II restriction enzymes cut DNA at the recognition site or near the recognition site (Type IIS) and for this reason are most often used in scientific experimentation.
- Type III restriction enzymes cut DNA about 20-30 base pairs after the recognition site and requires ATP.
Splicing together cleaved DNA fragments
The chemical bonds cleaved by restriction enzymes can be reformed by other enzymes known as DNA ligases, allowing restriction fragments carved from different chromosomes or genes to be spliced together, provided their ends are complementary (more below). Many of the procedures of molecular biology and genetic engineering rely on restriction enzymes.
Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation (5' end or the 3' end) of a sticky-end "overhang" of an enzyme restriction.
Examples of restriction enzymes include:Enzyme Source Recognition Sequence Cut EcoRIEscherichia coli5'GAATTC 3'CTTAAG 5'---G AATTC---3' 3'---CTTAA G---5' EcoRIIEscherichia coli5'CCWGG 3'GGWCC 5'--- CCWGG---3' 3'---GGWCC ---5' BamHIBacillus amyloliquefaciens5'GGATCC 3'CCTAGG 5'---G GATCC---3' 3'---CCTAG G---5' HindIIIHaemophilus influenzae5'AAGCTT 3'TTCGAA 5'---A AGCTT---3' 3'---TTCGA A---5' TaqIThermus aquaticus5'TCGA 3'AGCT 5'---T CGA---3' 3'---AGC T---5' NotI Nocardia otitidis 5'GCGGCCGC 3'CGCCGGCG 5'---GC GGCCGC---3' 3'---CGCCGG CG---5' HinfI Haemophilus influenzae5'GANTC 3'CTNAG 5'---G ANTC---3' 3'---CTNA G---5' Sau3A Staphylococcus aureus5'GATC 3'CTAG 5'--- GATC---3' 3'---CTAG ---3' PovII* Proteus vulgaris5'CAGCTG 3'GTCGAC 5'---CAG CTG---3' 3'---GTC GAC---5' SmaI* Serratia marcescens5'CCCGGG 3'GGGCCC 5'---CCC GGG---3' 3'---GGG CCC---5' HaeIII*Haemophilus aegyptius 5'GGCC 3'CCGG 5'---GG CC---3' 3'---CC GG---5' AluI* Arthrobacter luteus 5'AGCT 3'TCGA 5'---AG CT---3' 3'---TC GA---5' EcoRV*Escherichia coli5'GATATC 3'CTATAG 5'---GAT ATC---3' 3'---CTA TAG---5' KpnIKlebsiella pneumoniae5'GGTACC 3'CCATGG 5'---GGTAC C---3' 3'---C CATGG---5' PstIProvidencia stuartii 5'CTGCAG 3'GACGTC 5'---CTGCA G---3' 3'---G ACGTC---5' SacIStreptomyces achromogenes5'GAGCTC 3'CTCGAG 5'---GAGCT C---3' 3'---C TCGAG---5' SalIStreptomyces albus 5'GTCGAC 3'CAGCTG 5'---G TCGAC---3' 3'---CAGCT G---5' ScaIStreptomyces caespitosus 5'AGTACT 3'TCATGA 5'---AGT ACT---3' 3'---TCA TGA---5' SphIStreptomyces phaeochromogenes 5'GCATGC 3'CGTACG 5'---G CATGC---3' 3'---CGTAC G---5' StuI Streptomyces tubercidicus 5'AGGCCT 3'TCCGGA 5'---AGG CCT---3' 3'---TCC GGA---5' XbaIXanthomonas badrii 5'TCTAGA 3'AGATCT 5'---T CTAGA---3' 3'---AGATC T---5' * = blunt ends N = C or G or T or A W = A or T
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- MeSH DNA Restriction Enzymes
- Firman K (2007-11-24). Type I Restriction-Modification. University of Portsmouth. Retrieved on 2008-06-06.
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- Roberts RJ, Vincze T, Posfai, J, Macelis D. REBASE. Retrieved on 2008-06-06. “Restriction Enzyme Database”
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- Palmer M. WatCut. University of Waterloo, Ontario, Canada. Retrieved on 2008-06-06. “An on-line tool for restriction analysis, silent mutation scanning, SNP-RFLP analysis”
- Vincze,T, Posfai J, Roberts RJ. NEBcutter V2.0. New England Biolabs Inc.. Retrieved on 2008-06-06. “Restriction enzyme finder”
- Restriction enzyme digest of DNA software. BioPHP: PHP for Bioinformatics. Retrieved on 2008-06-06. “Online tool, free source code”
- pDRAW32. AcaClone software. Retrieved on 2008-06-06. “Freeware DNA cloning, sequence analysis and plasmid/DNA plotting software”
Exoribonuclease(Oligonucleotidase) 3.1.21-31: EndonucleaseEndodeoxyribonuclease(Deoxyribonuclease I, Deoxyribonuclease II, Deoxyribonuclease IV, Restriction enzyme, UvrABC endonuclease)
Endoribonuclease(RNase III, RNase H, RNase P, RNase A, RNase T1, RNA-induced silencing complex)
either deoxy- or ribo- (Aspergillus nuclease S1, Micrococcal nuclease)
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