Structural Biochemistry/DNA recombinant techniques/Restriction endonucleases

History

Restriction enzymes were first discovered by Werner Arber and Hamilton Smith. Daniel Nathans pioneered their use which led to recombinant DNA technology.

Restriction Endonucleases

Restriction endonucleases, also known as restriction enzymes, are responsible for the phenomenon in bacteria known as host-controlled restriction modification or phenotypic modification. Restriction/Modification enzyme systems are divided into three categories: Type I, Type II, and Type III. The key distinctions between these systems are that Type II enzymes contain separate restriction and methylation systems, while Type I and Type III enzymes carry both restriction and methylation properties in one enzyme, consisting of two or three heterologous subunits. Typical commercial restriction enzymes used in molecular biology are produced by Type II systems. Type II restriction endonucleases recognize specific palindromic sequences (a sequence that reads the same on both strands except one strand is reversed). The restriction enzyme recognizes a particular sequence of base pairs (about 4-8 bp long) with an axis of rotational symmetry. Once this site of recognition is established, it cleaves the phosphodiester bond in each strand of the double helical DNA. The number and size of the fragments produced depends on the frequency of occurrence of the recognition site in the DNA to be cut. The restriction enzyme cuts the DNA into smaller fragments so that they can be analyzed and manipulated easier. Restriction endonucleases can help with the analysis of chromosome structure, sequencing long DNA molecules, isolating genes, and creating new DNA molecules to be cloned.

Cleavage by a restriction enzyme can generate a number of various ends. Often, these ends have 3'-hydroxyl and 5'-phosphate ends. Some cleavages produce single-stranded overhangs, called cohesive ends or sticky ends, while others generate blunt ends. These ends or cleaved sites can be subsequently annealed and ligated to vector DNA or any kind of DNA having compatible ends. Not all cuts may necessarily be symmetrical as BamHI for example, cuts from the ends of DNA sequence in a non-symmetrical fashion.

It is also possible to visualize restriction fragments by gel electrophoresis. There are three different methods that can be used. The first method is a polyacrylamide gel which can separate fragments up to 1000 base pairs. The next one is an agarose gel which can separate up to 20kb. And finally, the Pulsed-Field Gel Electrophoresis (PFGE) which can separate up to millions of nucleotides based on the stretching and relaxation of DNA as the electric field is turned on and off. Autoradiography or staining by ethidium bromide can then be used to visualize the DNA. The gel electrophoresis is run through an electric field which isolates fragments by size, noting that smaller fragments travel farther within the gel and the larger fragments are closer to the start. Compared to a known size standard, the location of where the restriction enzymes cut are known.

Example
Suppose the following segment of DNA is recognized by the restriction enzyme. The red (*) symbolized the axis of symmetry. One major component of these cleavage sites is the presence of twofold rotational symmetry about this axis. The cleavage site is also highlighted on the diagram. Once this site recognition is established, the phosphodiester bond between the highlighted C-G and G-C will be cleaved by the restriction enzyme on the corresponding strands of the double helix. Note that different restriction enzymes have different cleavage sites. Therefore cleavage of phosphodiester bond will not always be between C-G or G-C.

5' C-C-G-C-G-G 3'

*

3' G-G-C-G-C-C 5'

BamHI as an example

BamHI is an example of a restriction enzyme. It cleaves palindromic sequences, 6 bases at a time. For example:

File:BamHI palendromic sequence.jpg

This image shows where the BamHI would cleave the palendromic sequence.

BamHI binds to non-specific DNA and slides down the DNA strands with a dimer enzyme that quickly reads the DNA to find palindromic sequences of 6 bases. If it finds 6 bases that are not palindromic, it will still cut the bases, but will do so poorly. BamHI works best when it finds a palindromic sequence to cut.

File:Catalytic mechanism.jpg This image shows the mechanism that BamHI uses to cleave the phosphodiester bond. In the first step, when reacting with water, the substrate obtains a hydroxide group and gives the phosphate two negative charges. A metal is usually in the transition state because the metal is a positive charge that balances out the two negative charges, and makes the transition state more stable. Magnesium is a good metal to use. Calcium is also sometimes used, but it does not work as well because it hinders the cleaving process. Water is then added to the transition state, which results in the donation of a proton to the leaving group, finally breaking the bond and cleaving the DNA.

Everything in the pre-reactive and post-reactive states, excepting the fact that the phosphodiester bond is now broken, cleaving the DNA, basically remains the same as shown in the following picture.

File:Pre and post reactive states for BamHI.jpg

The red image shows the post-reactive state and the blue shows the pre reactive state. The most obvious difference is shown in the middle, where the phosphodiester bond is now broken (near the A), and the two bases are now separated.

Restriction Enzyme Control

Restriction endonucleases exhibit their high specificity due to two major characteristics. One being that they must not degrade the host DNA that contains the sequence recognized by the restriction enzymes. Second, they must only cleave sites that are specifically recognized. The restriction endonucleases must be able to tell the difference between one specific cleaving sequence and a different sequence. Restriction endonucleases are able to exhibit these properties through the process of methylation. Methylate enzymes present in the organism protect the organisms DNA containing the palindromic sequences from being cleaved by the restrictio endonucleases. Once the methylate enzymes methylate the adenines of the organisms personal bases, the restriction enzymes will not cut at these sites. Every restriction endonuclease results in a specific methylate enzyme in the host cell that will methylate the specific sequence sites in the hosts DNA. This system of self-methylation and restriction enzyme action is known as restriction-modification system. The restriction endonucleases produced by the host organism can only cleave sequences that are not marked with the methylated adenines allowing for restriction enzyme control.

Cleavage Mechanism

As stated above, restriction endonucleases cleave the bond between the oxygen 3' and phosphorous atoms. Restriction endonucleases catalyze the hydrolysis of these phosphodiester bonds in DNA. The mechanism of this reaction is based upon nucleophillic attack of the phosphorous creating a pentacoordinate transition state. This results in a bipyramidal structure.

Type I Type I restriction enzymes were the first to be identified and are characteristic of two different strains of E. coli. The recognition site is asymmetrical and is composed of two portions: one containing 3-4 nucleotides, and another containing 4-5 nucleotides which are separated by a spacer. Several enzyme cofactors, are required for their activity. Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction, HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity) and HsdS is important for specificity of cut site recognition in addition to its methyltransferase activity.

Type II Typical type II restriction enzymes differ from type I restriction enzymes in several ways. They are composed of only one subunit, their recognition sites are usually undivided and palindromic and 4-8 nucleotides in length, they recognize and cleave DNA at the same site, and they do not use cofactors for their activity (except Mg2+). These are the most commonly available and used restriction enzymes.

Type III Type III restriction enzymes recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.

Analyzing Restriction Digests

After a restriction enzyme digest of plasmid DNA was done, the DNA fragments were analysis on an argyrose gel. By examining the pattern of bands obtained on the gel, the size of DNA and vector can be determined. This can be use to confirm if the correct plasmid was isolated or not.

From the Figure above conclusion like following can be made:

Lane 3 and 6 were digested by enzyme twice. Two bands represent two pieces of linear DNA. Bottom band is the size of the gene inserted between the two enzyme sites in the multiple cloning sites and the other is the size of the rest of the plasmid.

Lane 4 and 7 were digested by enzyme once. The band moves slower than other linear DNA indicates that DNA is nicked. This DNA uncoils, but remains circular and usually migrates more slowly than linear DNA of the same size.

Lane 5 and 8 were undigested. Super coiled DNA migrates more rapidly than linear DNA of the same size.