Restriction enzymes (also known as restriction endonucleases) are a group of bacterial enzymes which cut double-stranded DNA (dsDNA) into smaller fragments at specific points. They are a defence mechanism used by bacteria to cleave the DNA of invading viruses, thereby restricting their expression. The exploitation of restriction enzymes ability to cut large pieces of DNA into smaller fragments (called restriction fragments) and the highly specific way in which they do this has played a crucial role in the exponential advancement of biotechnology in recent decades.
Restriction enzymes cut DNA at specific places by recognising short DNA sequences called restriction sites. These sequences are typically 4-8bp long and are specific to each enzyme. Restriction sites can be found on any DNA molecule, whether it be viral DNA, fish DNA, human DNA etc. This is an important aspect in biotechnology as it means genomic DNA from any species of interest can be cut into smaller, more manageable junks. However, this does pose the question: how do bacteria prevent their own DNA form being cleaved by their restriction enzymes?
Bacteria methylate their own restriction sites to overcome this problem. The addition of a methyl group prevents restriction enzymes form binding and cleaving the DNA. It allows the bacteria to identify self from non-self. This is an efficient tactic employed by bacteria as restriction sites are not only found in all types of DNA but they all share a similar design wherever they are found. Most restriction sites are palindromic. They exhibit twofold rotational symmetry. This means that the two strands within the restriction site will have the same sequence if read in a 5’-3’ direction (Diagram 1).
Different restriction enzymes will act upon different palindromic sequences but the nature by which they cut the DNA is always the same. Restriction enzymes cut the DNA backbone by hydrolysis of the phosphodiester bond. This results in a 3’-hydoxyl group being formed at one end and a 5’-phosphate group forming at the other. Restriction enzymes always cut within the short sequence of the restriction site. However, each strand may or may not be cut at the same location. This results in the ends of restriction fragments taking one of two forms.
The cleavage of restriction sites can result in restriction fragments being produced with either “sticky” or “blunt” ends. Some restriction enzymes e. g. Taq? (Diagram 2) cleave the DNA in a staggered way, producing single-stranded DNA (ssDNA). The ssDNA are complimentary to one another and are able to form hydrogen bonds. Therefore, if two restriction fragments which have been cut with the same restriction enzyme (each ssDNA will be unique to each restriction enzyme) they will be capable of joining together, hence the term “sticky”. Other restriction enzymes e. . Hae??? (Diagram 2) cleave the DNA in a way which produces two dsDNA ends. Such fragments are incapable of forming hydrogen bonds with one another and are hence called “blunt”. Restriction enzymes which form sticky ends are particularly useful as they can be used with DNA ligase to join specific pieces of DNA from different sources together, producing recombinant DNA. If two pieces of DNA are cut with the same restriction enzyme they will form complimentary strands which are capable of bonding with one another, a process called hybridisation (Diagram 3).
The result of hybridisation is the formation of a new “recombinant” piece of DNA with a continuous sequence of dsDNA bonded together. However, although the base-pairs have formed hydrogen bonds the sugar-phosphate backbone of the recombinant DNA is not joined together. DNA ligase can be introduced to do this. DNA ligase forms covalent bonds along the sugar-phosphate backbone of the recombinant DNA, sealing it together (Diagram 3). The combination of action by restriction enzymes and DNA ligase can thus stick two separate pieces of DNA together producing recombinant DNA (Diagram 3).
Blunt ends are also capable of producing recombinant DNA with the addition of DNA ligase alone, but this is a very inefficient process. Altering blunt ends can speed up this process and allows blunt ended fragments to more effectively produce recombinant DNA. This can be achieved in a numerous ways. For instance, in PCR, where blunt ends are produce as a product, specialised primers containing a restriction site can be used. If treated with the appropriate restriction enzyme, sticky ends can be produced.
Alternately, dsDNA called oligonucleotides which contain restriction sites can be ligated to blunt ends. Again, if treated with restriction enzymes, sticky ends can be made (Diagram 5). In both cases it is important to ensure that the original DNA sequence does not contain the same restriction site as the added DNA as this will cause the restriction enzyme to also cleave the desired DNA sequence. Alternative restriction sites can be added if this is the case.
Whether by sticky or blunt ends, the combination of restriction enzymes and DNA ligase allows recombinant DNA to be produced. The importance of this to modern genetic analysis cannot be underestimated. Previously, the sheer size of genomic DNA rendered genetic analysis extremely difficult. The use if restriction enzymes to cut large junks of DNA into more manageable sizes was the first important step which permitted huge advancements in analysis of genes and their expression.
For instance, the first step in the human genome project involved using restriction enzymes to fragment the human genome into smaller pieces which in turn could be sequenced by different labs throughout the world and pieced together again. It can therefore be seen that along with the development of other techniques such as cloning and probing, the discovery restriction enzymes and their ability to cut DNA at specific points is the basis of modern recombinant technology. It could be argued that such technology has propelled biology from a mainly theoretical field to one of application.
