Restriction enzyme
| Restriction enzyme | ||
|---|---|---|
|
||
| The restriction endonuclease EcoRI (homodimer) bound to DNA | ||
| Identifier | ||
| Gene name (s) | T2; R. | |
| Enzyme classification | ||
| EC, category | 3.1.21.4 , endonuclease | |
| Response type | hydrolysis | |
| Substrate | DNA | |
| Products | two pieces of DNA | |
| Occurrence | ||
| Parent taxon | bacteria | |
Restriction enzymes , more precisely also restriction endonucleases (REN), are enzymes that recognize and cut DNA at certain positions. Restriction endonucleases occur, among other things, in bacteria and archaea , where they serve the defense against bacteriophages . The restriction enzymes recognize foreign DNA by the missing methylation pattern or by an otherwise non-existent DNA sequence and then hydrolyze the foreign DNA. They therefore always appear in the bacterium together with typical DNA methyltransferases , which imprint characteristic patterns on the bacterial DNA.
properties
In order for a bacterium to have restriction enzymes as a defense system, at least three functionally distinguishable protein areas are necessary: restriction, methylation and sequence recognition domains. These three domains can either be located on just one protein or distributed over several.
Each restriction endonuclease recognizes a specific DNA base sequence. The specificity of a restriction enzyme in a restriction digest depends , among other things, on the buffer used and the cofactors .
Restriction enzymes are used in biochemistry to cut DNA at defined locations, e.g. B. in a restriction analysis (a restriction digest with subsequent agarose gel electrophoresis ) or a cloning . Hence these enzymes are also known as molecular scissors . A ligase is used to rejoin the cut ends covalently (with sticky ends only after hybridization ) . In the case of wrong environmental conditions, non-specific restrictions can also be carried out, which is known as star activity . The specificity of restriction endonucleases can be specifically adapted to a desired DNA sequence by means of a protein design, e.g. B. in zinc finger nucleases .
The positions of the interfaces of individual restriction enzymes can be shown in restriction maps of a DNA. Such maps are available for genomes and plasmids , for example . Using the length of the DNA fragments that arise when the DNA is cut by restriction enzymes, DNA sections can be identified in comparison with a restriction map.
Classification
According to their properties, a distinction is made between four main types, which are further divided into several sub-types:
- Type I cuts the DNA at a random location far from the recognition sequence. Requires ATP and transfers a methyl group from S-adenosyl methionine.
- Type II cuts the DNA within or in the immediate vicinity of the recognition sequence. Does not require ATP and has no methyltransferase activity.
- Type III cuts the DNA about 20 to 25 base pairs from the recognition sequence. Requires ATP and transfers a methyl group from S-adenosyl methionine.
- Type IV only cuts methylated / hydroxymethylated / glucosyl-hydroxymethylated DNA - in contrast to types I-III, which are inhibited by methylation patterns.
Type II has type IIS and type IIG as known subtypes, which cut outside of the recognition sequence.
In common parlance, the term restriction enzyme is usually equated with restriction endonucleases of type II, since the enzymes of types I and III are of little importance in molecular biology. The names of the restriction enzymes indicate their origin. The first letter stands for the genus , the second and third for the species , it is extended by name additions and the chronological order of the discovery. The enzyme EcoRI is, for example, the first enzyme that was found in the strain Escherichia coli R (rough) and SmaI the first enzyme from Serratia marcescens . Restriction enzymes of different origins with identical recognition sequences and the same cutting pattern are called isoschizomers . If they cut within the same sequence but leave different cut ends , they are called neoschizomers .
The recognition sequences of type II restriction endonucleases usually consist of palindromic sequences of four, six or eight base pairs. The cut can be straight (English blunt ends, German blunt ends or smooth ends, e.g. SmaI). Such blunt ends result in a lower ligation yield in the course of cloning . The recognition sequence of SmaI is: 5'-CCCGGG-3 ' . The cut is made between the C and the G :
The cut can also be offset ( English sticky ends , German sticky ends , e.g. EcoRI ). The recognition sequence of EcoRI is: 5'-GAATTC-3 ' . The cut is made between the G and the A :
Sticky ends are easier to ligate because they can hybridize with each other and therefore come together more often.
Examples
| enzyme | source | Recognition sequence | cut | end up |
|---|---|---|---|---|
| EcoRI | Escherichia coli |
5'-GAATTC-3' 3'-CTTAAG-5' |
5'-G AATTC-3' 3'-CTTAA G-5' |
5 'overhang with four bases (sticky ends) |
| EcoRV | Escherichia coli |
5'-GATATC-3' 3'-CTATAG-5' |
5'-GAT ATC-3' 3'-CTA TAG-5' |
no overhang (smooth ends) |
| BamHI | Bacillus amyloliquefaciens |
5'GGATCC 3'CCTAGG |
5'---G GATCC---3' 3'---CCTAG G---5' |
5 'overhang with four bases (sticky ends) |
| HindIII | Haemophilus influenzae |
5'AAGCTT 3'TTCGAA |
5'---A AGCTT---3' 3'---TTCGA A---5' |
5 'overhang with four bases (sticky ends) |
| HaeIII | Haemophilus aegyptius |
5'GGCC 3'CCGG |
5'---GG CC---3' 3'---CC GG---5' |
no overhang (smooth ends) |
| NdeI | Neisseria denitrificans |
5'-CATATG-3' 3'-GTATAC-5' |
5'-CA TATG-3' 3'-GTAT AC-5' |
5 'overhang with two bases (sticky ends) |
| SacI | Streptomyces achromogenes |
5'-GAGCTC-3' 3'-CTCGAG-5' |
5'-GAGCT C-3' 3'-C TCGAG-5' |
3 'overhang with four bases (sticky ends) |
| SmaI | Serratia marcescens |
5'-CCCGGG-3' 3'-GGGCCC-5' |
5'-CCC GGG-3' 3'-GGG CCC-5' |
no overhang (smooth ends) |
| PvuI | Proteus vulgaris |
5'-CGATCG-3' 3'-GCTAGC-5' |
5'-CGAT CG-3' 3'-GC TAGC-5' |
3 'overhang with two bases (sticky ends) |
| SphI | Streptomyces phaeochromogenes |
5'-GCATGC-3' 3'-CGTACG-5' |
5'-GCATG C-3' 3'-C GTACG-5' |
3 'overhang with four bases (sticky ends) |
| XbaI | Xanthomonas badrii |
5'-TCTAGA-3' 3'-AGATCT-5' |
5'-T CTAGA-3' 3'-AGATC T-5' |
5 'overhang with four bases (sticky ends) |
history
The development of molecular biology began with the discovery of restriction enzymes in 1967/68 through isolation from bacteria . They enable the targeted production of DNA fragments (restriction fragments), which can then be isolated and, since 1972, put together to new constructions with the help of ligases. Enzymes that create sticky ends are especially helpful because the overlapping ends are easy to join together. The first article by a research group at Stanford University School of Medicine appeared in 1973 in the Proceedings of the National Academy of Sciences . For their pioneering work on "discovery of restriction enzymes and their application in molecular genetics" got Werner Arber , Daniel Nathans and Hamilton O. Smith in 1978 the Nobel Prize in Physiology or Medicine .
The name restriction enzyme comes from the bacterial restriction modification system that is used to defend against foreign (viral) DNA. Many bacteria have strain-specific restriction endonucleases. The corresponding recognition sequences in one's own DNA are modified ( methylated ) and are therefore not cut. When viruses that multiply in bacteria ( bacteriophages ) inject their DNA into the cells, the DNA is not methylated and is broken down. Only viruses that come from bacteria of the same strain have the correct methylation pattern and can continue to multiply. The replication of the viruses is limited or restricted to this strain. (Restriction = restriction).
Individual evidence
- ↑ Applied Microbial Systematics, FG Priest, Michael Goodfellow, ISBN 0-7923-6518-6 , limited preview in the Google book search
- ↑ Cornel Mülhardt: The Experimentator: Molecular Biology / Genomics , Springer 2008, ISBN 3-8274-2036-9 , page 48 ( preview on Google Books ).
- ↑ Roberts, RJ et al. (2003): A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. (PDF; 93 kB) In: Nucleic Acids Res. Vol. 31, pp. 1805-1812. PMID 12654995
- ↑ Cornel Mülhardt: General Microbiology , Georg Fuchs, Hans G. Schlegel, Georg Thieme Verlag, 2006, ISBN 3-13-444608-1 , page 468, limited preview in the Google book search.
- ↑ Cornel Mülhardt: The Experimentator: Molecular Biology / Genomics , Springer 2008, ISBN 3-8274-2036-9 , page 48 ( preview on Google Books ).
- ↑ Restriction Enzymes - Tools of Molecular Biology. In: New England Biolabs GmbH. Retrieved on February 27, 2020 (German).
- ↑ Hans-Peter Kröner: Human Genetics. In: Werner E. Gerabek , Bernhard D. Haage, Gundolf Keil , Wolfgang Wegner (eds.): Enzyklopädie Medizingeschichte . De Gruyter, Berlin 2005, ISBN 3-11-015714-4 , pp. 635–641, here: p. 640 ( human genetics in the molecular biological epoch ).
- ↑ Information from the Nobel Foundation on the 1978 award ceremony to Werner Arber, Daniel Nathans and Hamilton O. Smith (English)
Web links
- REBASE - the most comprehensive database of all known restriction enzymes, including availability from all commercial manufacturers
- NEBCutter - web-based program for cutting DNA with all available restriction enzymes; observes methylation sensitivities; Simulation of gels
- WatCut - Web-based program for cutting DNA with restriction enzymes