Restriction Enzymes: Definition, Ligasas, Identity and Properties “Adhesive Ends”

It is a protein produced by bacteria that cleaves DNA at specific sites.

Bacteria use restriction enzymes to defend against bacterial viruses called bacteriophages (or phages).

A restriction enzyme acts as a biochemical scissor. It is also called restriction endonuclease. It can recognize specific base sequences in the DNA and cut the DNA at that site (the restriction site).

When a phage infects a bacterium, it inserts its DNA into the bacteria so that it can be replicated. The restriction enzyme prevents replication of phage DNA by cutting it in many places.

Restriction enzymes were named for their ability to restrict or limit the number of bacteriophage strains that can infect bacteria.

Restriction enzymes can be isolated from bacteria and used in the laboratory to cut DNA. They are indispensable tools in recombinant DNA technology and genetic engineering.

Each restriction enzyme recognizes a short, specific sequence of nucleotide bases (the four basic chemical subunits of the linear double-stranded DNA molecule: adenine, cytosine, thymine, and guanine).

These stretches in DNA are called recognition sequences and are distributed randomly throughout the DNA. Different bacterial species produce restriction enzymes that recognize different nucleotide sequences.

After a restriction endonuclease recognizes a sequence, it cuts through the DNA molecule catalysing hydrolysis (splitting of a chemical bond by the addition of a water molecule) of the bond between adjacent nucleotides.

Bacteria prevent their own DNA from degrading in this way by disguising their recognition sequences.

Enzymes called methylases add methyl groups (-CH3) to adenine or cytosine bases within the recognition sequence, which in this way is modified and protected from the endonuclease.

The restriction enzyme and its corresponding methylase constitute the restriction-modification system of a bacterial species.

All restriction enzymes are different. There are three classes of restriction enzymes, designated types I, II and III.

The enzymes of Types I and III are similar in that both restriction and methylase activities are carried out by a large enzyme complex, in contrast to the type II system, in which the restriction enzyme is independent of its methylase.

Type II restriction enzymes also differ from the other two types in that they cleave DNA at specific sites within the recognition site; the others splint the DNA randomly, sometimes hundreds of bases of the recognition sequence.

Restriction enzymes were discovered and originally characterized by molecular biologists Werner Arber, Hamilton O. Smith and Daniel Nathans, who shared the 1978 Nobel Prize for Medicine.

The ability of restriction enzymes to cut DNA at precise locations has allowed researchers to isolate fragments that contain genes and recombine them with other DNA molecules.

More than 2,500 type II restriction enzymes have been identified from a variety of bacterial species. These enzymes recognize approximately 200 different sequences, which have a length of four to eight bases.

Identity of restriction enzymes

The restriction enzymes are named for the organism from which they were isolated for the first time.

For example:

  • Eco RI is isolated from the RY13 strain of E. coli.
  • Eco refers to the genus and the species (1st letter of the genus, 1st two letters of the specific epithet).
  • R is the E. coli strain.
  • I (Roman numeral) indicates that it was the first enzyme of that type isolated from E. coli RY13.
  • Bam HI is isolated from the H strain of Bacillus amyloliquefaciens.
  • Sau3A is isolated from Staphylococcus aureas strain 3A.

Some restriction enzymes also cut the DNA to form “blunt” ends (without single-stranded tails), which can also be inserted into the target DNA by the action of DNA ligase.

DNA ligase is not fussy, it can not differentiate between host and host DNA (who would imagine that it would ever have to?), And this allows the creation of chimeric DNA – DNA from two different sources.

Each enzyme recognizes and cuts specific DNA sequences. For example, Bam HI recognizes the double-stranded sequence:

  • 5 ‘- GGATCC – 3’
  • 3 ‘- CCTAGG – 5’

Most restriction enzymes are specific to a single restriction site. Restriction sites are recognized no matter where the DNA came from

The number of cuts in the DNA of an organism made by a particular restriction enzyme is determined by the number of restriction sites specific for that enzyme in the DNA of that organism.

A fragment of DNA produced by a pair of adjacent slices is called a restriction fragment.

A particular restriction enzyme will typically cut the DNA of an organism into many pieces, from several thousand to more than one million.

There is a great variation in restriction sites even within a species.

Although these variations do not have phenotypic expression beyond the base sequences themselves, the variants can be considered molecular “alleles,” and can be detected by sequencing techniques.

As such, they can be used in mapping studies similar to the way in which true genes with known phenotypic effects can be used, but omitting the steps of reproduction and going directly to the molecules.

These “molecular alleles” are a type of molecular marker, since they can be detected and located with labeled probes.

Sticky ends

The burgeoning field of biotechnology was made possible by the discovery of restriction enzymes in the early 1950s. With them, DNA can be cut in precise locations.

A second enzyme, DNA ligase, can be used to reassemble the pieces in the desired order. Together, these two enzymes allow researchers to assemble custom genomes.

For example, researchers can create designer bacteria that produce insulin or growth hormone or add genes for disease resistance in agricultural plants.

An interesting property of restriction enzymes simplifies this cutting and molecular bonding. Restriction enzymes typically recognize a symmetric sequence of DNA, such as the EcoRI site.

The upper thread is the same as the lower thread, read backwards. When the enzyme cuts the chain between G and A, it leaves protruding chains.

These are called “adhesive ends” because the base pairs formed between the two protruding parts will glue the two pieces together, even if the spine is cut.

Adhesive ends are an essential part of genetic engineering, allowing researchers to cut small pieces of DNA and place them in specific locations, where the sticky ends coincide.