Restriction endonucleases and other enzymes needed in genetic engineering

Restriction Enzyme:- It is a nuclease enzyme that cleaves DNA sequence at a specific recognition sites known as restriction sites. 

Discovery:- In 1970 the first restriction endonuclease enzyme HindII was isolated. For the subsequent discovery and characterization of numerous restriction endonucleases, in 1978 Daniel Nathans, Werner Arber, and Hamilton O. Smith awarded for Nobel Prize for Physiology or Medicine.

Restriction modification system:- In bacteria, modification enzymes methylate the bacterial DNA. Methylation of bacterial DNA at the recognition sequence typically protects the own DNA of the bacteria from being cleaved by restriction enzyme.

Types:- There are two different kinds of restriction enzymes:

(1) Exonucleases:- They catalyses hydrolysis of terminal nucleotides from the end of DNA or RNA molecule either 5’to 3’ direction or 3’ to 5’ direction. Example: exonuclease I, exonuclease II etc.

(2) Endonucleases:- They can recognize specific base sequence (restriction site) within DNA or RNA molecule and cleave internal phosphodiester bonds within a DNA molecule. Example: EcoRI, Hind III, BamHI etc.

Restriction Endonuclease (RE):-

Nomenclature:- Restriction endonucleases are named according to the organism in which they were discovered, using a system of letters and numbers. 

For example, HindIII (pronounced “hindee-three”) was discovered in Haemophilus influenza (strain d). The Roman numerals are used to identify specific enzymes from bacteria that contain multiple restriction enzymes indicating the order in which restriction enzymes were discovered in a particular strain.

Classification of Restriction Endonucleases:- There are three major classes of restriction endonucleases based on the types of sequences recognized, the nature of the cut made in the DNA, and the enzyme structure -

a. Type I restriction enzymes

b. Type II restriction enzymes

c. Type III restriction enzymes

a. Type I restriction enzymes:-

> These enzymes have both restriction and modification activities. Restriction depends upon the methylation status of the target DNA.

> Cleavage occurs approximately 1000 bp away from the recognition site.

> The recognition site is asymmetrical and is composed of two specific portions in which one portion contain 3–4 nucleotides while another portion contain 4–5 nucleotides and both the parts are separated by a non-specific spacer of about 6–8 nucleotides.

> They require S-adenosylmethionine (SAM), ATP, and magnesium ions (Mg2+) for activity.

> These enzymes are composed of mainly three subunits, a specificity subunit that determines the DNA recognition site, a restriction subunit, and a modification subunit.

> Examples:- EcoB, EcoK

b. Type II restriction enzymes:-

> Restriction and modification are mediated by separate enzymes so it is possible to cleave DNA in the absence of modification. Although the two enzymes recognize the same target sequence, they can be purified separately from each other.

> Cleavage of nucleotide sequence occurs at the restriction site.

> These enzymes are used to recognize rotationally symmetrical sequence which is often referred as palindromic sequence.

> These palindromic binding site may either be interrupted (e.g. BstEII recognizes the sequence 5´-GGTNACC-3´, where N can be any nucleotide) or continuous (e.g. KpnI recognizes the sequence 5´-GGTACC-3´).

> They require only Mg2+ as a cofactor and ATP is not needed for their activity.

> Type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize specific sites and cleave just at these sites.

> Examples:- EcoR I, BamH I

c. Type III restriction enzymes:-

> These enzymes recognize and methylate the same DNA sequence but cleave 24–26 bp away.

> They have two different subunits, in which one subunit (M) is responsible for recognition and modification of DNA sequence and other subunit (R) has nuclease action.

> Mg+2 ions, ATP are needed for DNA cleavage and process of cleavage is stimulated by SAM.

> Cleave only one strand. Two recognition sites in opposite orientation are necessary to break the DNA duplex.

> Examples:- EcoP I, Hinf III

Applications:- In various applications related to genetic engineering DNA is cleaved by using these

restriction enzymes.

i. They are used in the process of insertion of genes into plasmid vectors during gene cloning and protein expression experiments.

ii. Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs). This is only possible if a mutation alters the restriction site present in the allele.

iii. Restriction enzymes are used for Restriction Fragment Length Polymorphism (RFLP) analysis for identifying individuals or strains of a particular species.

DNA Ligase:- It is a specific type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. DNA ligase is used in DNA repair, DNA replication and genetic engineering.

Types:-

a. E. coli DNA ligase:- 

> It is encoded by the lig gene. DNA ligase in E. coli, as well as most prokaryotes, uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. 

> It does not ligate blunt-ended DNA and cannot join RNA to DNA efficiently. 

> The activity of E. coli DNA ligase can be enhanced by DNA polymerase - I at the right concentrations. 

b. T4 DNA ligase:- 

> The DNA ligase from bacteriophage T4 (a bacteriophage that infects Escherichia coli bacteria). 

> The T4 ligase is the most-commonly used in genetic engineering.

> It can ligate either cohesive or blunt ends of DNA, oligonucleotides, as well as RNA and RNA-DNA hybrids, but not single-stranded nucleic acids. 

> It can also ligate blunt-ended DNA with much greater efficiency than E. coli DNA ligase. 

> T4 DNA ligase cannot utilize NAD and it has an absolute requirement for ATP as a cofactor. 

> The optimal incubation temperature for T4 DNA ligase is 16 °C.

c. Mammalian DNA ligase:- In mammals, there are four specific types of ligase -

i. DNA ligase I:- It ligates the nascent DNA of the lagging strand after the Ribonuclease H has removed the RNA primer from the Okazaki fragments.

ii. DNA ligase III:- Complexes with DNA repair protein XRCC1 to aid in sealing DNA during the process of nucleotide excision repair and recombinant fragments. Of the all known mammalian DNA ligases, only Lig III has been found to be present in mitochondria.

iii. DNA ligase IV:- Complexes with XRCC4. It catalyzes the final step in the non-homologous end joining DNA double-strand break repair pathway. It is also required for V(D)J recombination, the process that generates diversity in immunoglobulin and T-cell receptor loci during immune system development.

Note:- DNA ligase from eukaryotes and some microbes uses adenosine triphosphate (ATP) rather than NAD.

d. Thermostable DNA ligase:-

> Derived from a thermophilic bacterium, the enzyme is stable and active at much higher temperatures than conventional DNA ligases. 

> Its half-life is 48 hours at 65 °C and greater than 1 hour at 95 °C. 

> Ampligase DNA Ligase has been shown to be active for at least 500 thermal cycles (94 °C/80 °C) or 16 hours of cycling. This exceptional thermostability permits extremely high hybridization stringency and ligation specificity.