Restriction enzyme

Restriction enzymes, restriction endonucleases also more accurate (REN ) are enzymes that can recognize and cleave DNA at specific positions. Restriction endonucleases occur among others in bacteria and archaea, where they serve the phage defense. Restriction enzymes recognize foreign DNA methylation patterns in the absence of or at an otherwise non- occurring DNA sequence, and then hydrolyze the foreign DNA. Always therefore occur in the bacterium along with typical DNA methyltransferases, which impart the bacteria 's own DNA characteristic pattern.

Properties

In order for a bacterium by restriction enzymes can have as a defense system, at least three functionally distinct protein domains are required: restriction, methylation and sequence recognition domain. These three domains can be hosted either on a single protein or be distributed over several.

Each restriction endonuclease recognizes a specific DNA base sequence. The specificity depends among others on the buffer used and the cofactors. Incorrect ambient conditions can also be restricted non-specific, which is referred to as the star activity. The specificity of restriction endonucleases can be adapted by a design of protein targeted to a desired DNA sequence, such as Zinkfingernukleasen.

The positions of the interfaces of individual restriction enzymes can be represented in a DNA restriction maps. Such cards are available, for example, genomes and plasmids. The length of DNA fragments that arise during the cutting of the DNA by restriction enzymes, DNA fragments can be identified as compared with a restriction map.

Classification

In biotechnological processes in molecular biology, the restriction enzymes are used to break DNA molecules at defined locations. Therefore, these enzymes are also referred to as molecular scissors. To rejoin the cut ends back covalently used a ligase (with sticky ends only after hybridization ).

According to their properties there are four main types, which further split into several subtypes:

• Type I cuts the DNA far from the recognition sequence at a random position. Requires ATP and transfers a methyl group from S- adenosyl -methionine.
• Type II cuts the DNA within or close to the recognition sequence. Does not require ATP and has no methyltransferase activity.
• Type III cuts the DNA is about 20 to 25 base pairs away from the recognition sequence. Requires ATP and transfers a methyl group from S- adenosyl -methionine.
• Type IV cuts only methylated / hydroxymethylated / glucosyl - hydroxymethylated DNA - as opposed to the types I- III, which are inhibited by methylation pattern.

In common parlance, the term restriction enzyme is usually equated with the restriction endonucleases of type II, since the enzymes of types I and III have only minor importance in molecular biology. The names of the restriction enzymes indicate their origins. The first letter stands for the genus, the second and third of the way it is extended by additions to names and the chronological order of discovery. The enzyme EcoRI, 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 from different backgrounds with the same recognition sequence and the same patterns are called isoschizomers. Cut them in the same sequence, but leave different cut ends, they are called Neoschizomere.

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, Ger. Blunt ends or blunt ends, such as SmaI ). The recognition sequence from Sma I is: 5'- CCCGGG -3 '. The cut is made between the C and G:

The section may also be offset (english sticky ends, German sticky ends, such as EcoRI). The recognition sequence of EcoRI is: 5'- GAATTC -3 '. The cut is made between the G and A:

Sticky ends are easier ligatable because they can hybridize with each other and therefore get together more often.

Examples

5'- GAATTC -3 ' 3'- CTTAAG -5 ' 5' -G AATTC -3 ' 3'- CTTAA G -5 ' 5 'overhang of four bases ( sticky ends ) BamHI Bacillus amyloliquefaciens 5'GGATCC 3'CCTAGG 5' --- G --- GATCC 3 ' 3' --- CCTAG G --- 5 ' 5 'overhang of four bases ( sticky ends ) HindIII Haemophilus influenzae 5'AAGCTT 3'TTCGAA 5' --- A --- 3 AGCTT ' 3 --- A --- TTCGA 5 ' 5 'overhang of four bases ( sticky ends ) HaeIII Haemophilus aegyptius 5'GGCC 3'CCGG 5' --- CC --- GG 3 ' 3 --- CC GG --- 5 ' no overhang ( blunt ends ) NdeI Neisseria denitrificans 5'- CATATG -3 ' 3'- GTATAC -5 ' 5' -CA -3 TATG ' 3'- GTAT AC-5 ' 5 'overhang with two bases ( sticky ends ) SmaI Serratia marcescens 5'- CCCGGG -3 ' 3'- GGGCCC -5 ' 5'- CCC GGG -3 ' 3'- GGG CCC -5 ' no overhang ( blunt 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 of four bases ( sticky ends ) history

With the discovery of restriction enzymes, the development of molecular biology began. They allow the selective production of DNA fragments, which can then be isolated and assembled into new designs. Enzymes that produce sticky ends, are particularly helpful, as is easy to let the overlapping ends together. For her seminal work on the " discovery of restriction enzymes and their application in molecular genetics " got Werner Arber, Daniel Nathans and Hamilton Smith Othanel the 1978 Nobel Prize in Physiology or Medicine.

The name restriction enzyme comes from the bacterial restriction-modification system that is used to prevent foreign ( viral ) DNA. Many bacteria possess strain-specific restriction endonucleases. In their own DNA, the corresponding recognition sequences are modified ( methylated ) and are therefore not cut. If viruses that replicate in the bacteria ( bacteriophage ), to inject their DNA into the cells, this is not methylated and is degraded. Only viruses that come from bacteria of the same strain, have the correct methylation patterns and can continue to multiply. The proliferation of the virus is so limited or restricted to this strain. ( = Restriction restriction ).