Restriction enzyme

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Restriction enzyme bound to DNA
Restriction enzyme bound to DNA


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.[1][2] 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.[3] 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.[4][5][6]

The Nobel Prize in Medicine was awarded, in 1978, to Daniel Nathans, Werner Arber, and Hamilton Smith for the discovery of restriction endonucleases.[7] 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.[8] 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.

Contents

[edit] Restriction enzymes as tools

See the main article on restriction digests.
EcoRI digestion produces "sticky" ends.
EcoRI digestion produces "sticky" ends.
SmaI restriction enzyme cleavage produces "blunt" ends.
SmaI restriction enzyme cleavage produces "blunt" ends.

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).[9][10]

Another use of restriction enzymes can be to find specific SNPs.[11][12] 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

[edit] Recognition sites

Restriction enzymes recognize a specific sequence of nucleotides[5] 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.[13] 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'

Bacteria prevent their own DNA from being cut by modifying their nucleotides via methylation.[1]

[edit] Nomenclature

E 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.[14][15] For example, the name of the EcoRI restriction enzyme was derived as shown in the box to the right.

[edit] Enzyme Mechanisms

There are three groups of restriction enzyme that vary in the way recognise their restriction sites and where they cut the DNA:[16][17]

  • Type I restriction enzymes cut DNA about 100 nucleotides after the recognition site and requires ATP.[18]
  • 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.[13]
  • Type III restriction enzymes cut DNA about 20-30 base pairs after the recognition site and requires ATP.[19]

[edit] 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.[20][21][22]

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.

[edit] Examples

Examples of restriction enzymes include:[23]

Enzyme Source Recognition Sequence Cut
EcoRI Escherichia coli 
5'GAATTC
3'CTTAAG

5'---G     AATTC---3'
3'---CTTAA     G---5'
EcoRII Escherichia coli 
5'CCWGG
3'GGWCC

5'---     CCWGG---3'
3'---GGWCC     ---5'
BamHI Bacillus amyloliquefaciens 
5'GGATCC
3'CCTAGG

5'---G     GATCC---3'
3'---CCTAG     G---5'
HindIII Haemophilus influenzae 
5'AAGCTT
3'TTCGAA

5'---A     AGCTT---3'
3'---TTCGA     A---5'
TaqI Thermus aquaticus 
5'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 influenzae 
5'GANTC
3'CTNAG

5'---G   ANTC---3'
3'---CTNA   G---5'
Sau3A Staphylococcus aureus 
5'GATC
3'CTAG

5'---     GATC---3'
3'---CTAG     ---3'
PovII* Proteus vulgaris 
5'CAGCTG
3'GTCGAC

5'---CAG  CTG---3'
3'---GTC  GAC---5'
SmaI* Serratia marcescens 
5'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 coli 
5'GATATC
3'CTATAG

5'---GAT  ATC---3'
3'---CTA  TAG---5'
KpnI[24] Klebsiella pneumoniae 
5'GGTACC
3'CCATGG

5'---GGTAC  C---3'
3'---C  CATGG---5'
PstI[24] Providencia stuartii 
5'CTGCAG
3'GACGTC

5'---CTGCA  G---3'
3'---G  ACGTC---5'
SacI[24] Streptomyces achromogenes 
5'GAGCTC
3'CTCGAG

5'---GAGCT  C---3'
3'---C  TCGAG---5'
SalI[24] Streptomyces albus 
5'GTCGAC
3'CAGCTG

5'---G  TCGAC---3'
3'---CAGCT  G---5'
ScaI[24] Streptomyces caespitosus 
5'AGTACT
3'TCATGA

5'---AGT  ACT---3'
3'---TCA  TGA---5'
SphI[24] Streptomyces phaeochromogenes 
5'GCATGC
3'CGTACG

5'---G  CATGC---3'
3'---CGTAC  G---5'
StuI [25][26] Streptomyces tubercidicus 
5'AGGCCT
3'TCCGGA

5'---AGG  CCT---3'
3'---TCC  GGA---5'
XbaI[24] Xanthomonas 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

[edit] See also

[edit] References

  1. ^ a b Arber W, Linn S (1969). "DNA modification and restriction". Annu. Rev. Biochem. 38: 467–500. doi:10.1146/annurev.bi.38.070169.002343. PMID 4897066. 
  2. ^ Krüger DH, Bickle TA (September 1983). "Bacteriophage survival: multiple mechanisms for avoiding the deoxyribonucleic acid restriction systems of their hosts". Microbiol. Rev. 47 (3): 345–60. PMID 6314109. 
  3. ^ Kobayashi I (September 2001). "Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution". Nucleic Acids Res. 29 (18): 3742–56. doi:10.1093/nar/29.18.3742. PMID 11557807. 
  4. ^ Roberts RJ (November 1976). "Restriction endonucleases". CRC Crit. Rev. Biochem. 4 (2): 123–64. PMID 795607. 
  5. ^ a b Kessler C, Manta V (August 1990). "Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3)". Gene 92 (1-2): 1–248. doi:10.1016/0378-1119(90)90486-B. PMID 2172084. 
  6. ^ Pingoud A, Alves J, Geiger R (1993). "Chapter 8: Restriction Enzymes", in Burrell, Michael: Enzymes of Molecular Biology, Methods of Molecular Biology 16. Totowa, NJ: Humana Press, pages 107-200. ISBN 0-89603-234-5. 
  7. ^ The Nobel Prize in Physiology or Medicine. The Nobel Foundation (1978). Retrieved on 2008-06-07. “for the discovery of restriction enzymes and their application to problems of molecular genetics”
  8. ^ Villa-Komaroff L, Efstratiadis A, Broome S, Lomedico P, Tizard R, Naber SP, Chick WL, Gilbert W. (August 1978). "A bacterial clone synthesizing proinsulin". Proc. Natl. Acad. Sci. U.S.A. 75 (8): 3727–31. PMID 358198. 
  9. ^ Geerlof A. Cloning using restriction enzymes. European Molecular Biology Laboratory - Hamburg. Retrieved on 2008-06-07.
  10. ^ Russell, David W.; Sambrook, Joseph (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory. ISBN 0-87969-576-5. 
  11. ^ Wolff JN, Gemmell NJ (February 2008). "Combining allele-specific fluorescent probes and restriction assay in real-time PCR to achieve SNP scoring beyond allele ratios of 1:1000". BioTechniques 44 (2): 193–4, 196, 199. PMID 18330346. 
  12. ^ Zhang R, Zhu Z, Zhu H, Nguyen T, Yao F, Xia K, Liang D, Liu C (July 2005). "SNP Cutter: a comprehensive tool for SNP PCR-RFLP assay design". Nucleic Acids Res. 33 (Web Server issue): W489–92. doi:10.1093/nar/gki358. PMID 15980518. 
  13. ^ a b Pingoud A, Jeltsch A (September 2001). "Structure and function of type II restriction endonucleases". Nucleic Acids Res. 29 (18): 3705–27. doi:10.1093/nar/29.18.3705. PMID 11557805. 
  14. ^ Smith HO, Nathans D (December 1973). "Letter: A suggested nomenclature for bacterial host modification and restriction systems and their enzymes". J. Mol. Biol. 81 (3): 419–23. PMID 4588280. 
  15. ^ Roberts RJ, Belfort M, Bestor T, Bhagwat AS, Bickle TA, Bitinaite J, Blumenthal RM, Degtyarev SKh, Dryden DT, Dybvig K, Firman K, Gromova ES, Gumport RI, Halford SE, Hattman S, Heitman J, Hornby DP, Janulaitis A, Jeltsch A, Josephsen J, Kiss A, Klaenhammer TR, Kobayashi I, Kong H, Krüger DH, Lacks S, Marinus MG, Miyahara M, Morgan RD, Murray NE, Nagaraja V, Piekarowicz A, Pingoud A, Raleigh E, Rao DN, Reich N, Repin VE, Selker EU, Shaw PC, Stein DC, Stoddard BL, Szybalski W, Trautner TA, Van Etten JL, Vitor JM, Wilson GG, Xu SY (April 2003). "A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes". Nucleic Acids Res. 31 (7): 1805–12. doi:10.1093/nar/gkg274. PMID 12654995. 
  16. ^ Boyer HW (1971). "DNA restriction and modification mechanisms in bacteria". Annu. Rev. Microbiol. 25: 153–76. doi:10.1146/annurev.mi.25.100171.001101. PMID 4949033. 
  17. ^ Yuan R (1981). "Structure and mechanism of multifunctional restriction endonucleases". Annu. Rev. Biochem. 50: 285–319. doi:10.1146/annurev.bi.50.070181.001441. PMID 6267988. 
  18. ^ Murray NE (June 2000). "Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle)". Microbiol. Mol. Biol. Rev. 64 (2): 412–34. doi:10.1128/MMBR.64.2.412-434.2000. PMID 10839821. 
  19. ^ Dryden DT, Murray NE, Rao DN (September 2001). "Nucleoside triphosphate-dependent restriction enzymes". Nucleic Acids Res. 29 (18): 3728–41. doi:10.1093/nar/29.18.3728. PMID 11557806. PMC:55918. 
  20. ^ Primrose, Sandy B.; Old, R. W. (1994). Principles of gene manipulation: an introduction to genetic engineering. Oxford: Blackwell Scientific. ISBN 0-632-03712-1. 
  21. ^ Micklos, David A.; Bloom, Mark V.; Freyer, Greg A. (1996). Laboratory DNA science: an introduction to recombinant DNA techniques and methods of genome analysis. Menlo Park, Calif: Benjamin/Cummings Pub. Co. ISBN 0-8053-3040-2. 
  22. ^ Adrianne Massey; Helen Kreuzer (2001). Recombinant DNA and Biotechnology: A Guide for Students. Washington, D.C: ASM Press. ISBN 1-55581-176-0. 
  23. ^ Roberts RJ (January 1980). "Restriction and modification enzymes and their recognition sequences". Nucleic Acids Res. 8 (1): r63–r80. doi:10.1093/nar/8.1.197-d. PMID 6243774. 
  24. ^ a b c d e f g Monty Krieger; Matthew P Scott; Matsudaira, Paul T.; Lodish, Harvey F.; Darnell, James E.; Lawrence Zipursky; Kaiser, Chris; Arnold Berk (2004). Molecular Cell Biology, 5th ed, New York: W.H. Freeman and Company. ISBN 0-7167-4366-3. 
  25. ^ Stu I from Streptomyces tubercidicus. Sigma-Aldrich. Retrieved on 2008-06-07.
  26. ^ Shimotsu H, Takahashi H, Saito H (November 1980). "A new site-specific endonuclease StuI from Streptomyces tubercidicus". Gene 11 (3-4): 219–25. doi:10.1016/0378-1119(80)90062-1. PMID 6260571. 

[edit] External links

General:

Databases:

  • Roberts RJ, Vincze T, Posfai, J, Macelis D. REBASE. Retrieved on 2008-06-06. “Restriction Enzyme Database”

Software:

v • d • e
Hydrolase, esterase: nucleases (EC 3.1.11 - 3.1.31) - includes deoxyribonuclease and ribonuclease
3.1.11-16: Exonuclease
3.1.21-31: Endonuclease