Endonucleases

Endonucleases are enzymes that cleave the phosphodiester link that exists between two nucleotide chains inside a polynucleotide chain. The deoxyribonuclease I enzyme is one example of an endonuclease that cuts DNA nonspecifically (without regard to sequence), whereas many others, which are commonly referred to as restriction endonucleases or restriction enzymes, cut DNA exclusively at very precise nucleotide sequences. Endonucleases are distinct from exonucleases in that they cleave the ends of recognition sequences rather than the middle (endo) region of the sequence. A subset of nucleases called “exo-endonucleases,” on the other hand, are not restricted to either nuclease function, exhibiting characteristics that are both endogenous and exogenous in nature. The evidence implies that the activity of endonucleases is delayed when compared to that of exonuclease activity.

Enzymes known as restriction enzymes are endonucleases found in bacteria and archaea that are unique to one DNA sequence.

The restriction site is the nucleotide sequence that is recognised by a restriction enzyme as being suitable for cleavage. Restriction enzymes were discovered and characterised in the late 1960s and early 1970s by molecular biologists Werner Arber, Hamilton O. Smith, and Daniel Nathans. Restriction enzymes are proteins that restrict the movement of molecules. Because of the enzymes’ capacity to cut DNA at exact spots, researchers were able to isolate gene-containing pieces and recombine them with other molecules of DNA, a process known as cloning gene expression. For example, the restriction enzyme EcoRI is generated by the Escherichia coli strain RY13, and its name derives from the genus, species, and strain designations of the bacteria that create it; for this reason, restriction enzyme names are derived from these designations. Genetic recombination and gene amplification are hypothesised to have resulted in the evolution of restriction enzymes from a common ancestral protein that was able to detect specific sequences through these processes.

Restriction Endonuclease

Restriction Endonuclease restriction enzyme, also known as restriction endonuclease, is a protein generated by bacteria that cleaves DNA at certain locations along the molecule. Restriction enzymes in the bacterial cell cut foreign DNA, thereby eradicating the invading organisms from the cell. Recombinant DNA technology is based on the usage of restriction enzymes, which may be obtained from bacterial cells and utilised in the laboratory to modify DNA fragments, such as those containing genes. As a result, restriction enzymes are essential tools in the field of recombinant DNA technology.

An enzyme known as restriction enzyme is used by bacteria to defend themselves against bacterial viruses known as bacteriophages, or phages. It is the DNA of the phage that is introduced into the bacterial cell when it infects the bacteria and allows it to proliferate. By breaking the phage DNA into multiple pieces, the restriction enzyme inhibits the phage DNA from replicating. They were given this term because of their ability to restrict or limit the number of bacteriophage strains that can infect a bacterium when infected with bacteriophage.

In order to function properly, each restriction enzyme must recognise a short, unique sequence of nucleotide bases (the four fundamental chemical subunits of the linear double-stranded DNA molecule: adenine, cytosine, thymine, and guanine) in the presence of a restriction enzyme. These sections are referred to as recognition sequences or recognition sites, and they are found in a random distribution throughout the DNA molecule. Different bacterial species produce restriction enzymes that detect nucleotide sequences that are distinct from one another.

When a restriction endonuclease detects a sequence, it catalyses the hydrolysis (breaking of a chemical bond by the addition of a water molecule) of the bond between neighbouring nucleotides, allowing the DNA molecule to be snipped. Bacteria prevent their own DNA from being destroyed in this manner by masking their recognition sequences with other sequences. Enzymes known as methylases are responsible for adding methyl groups (—CH3) to adenine or cytosine bases inside the recognition sequence, which is then changed and shielded from the endonuclease by the methylases. An individual bacterial species’ restriction-modification system is comprised of a restriction enzyme and its associated methylase enzyme pair. There are four varieties of restriction enzymes acknowledged by the scientific community, denoted by the letters I, II, III, and IV. These enzymes are distinguished principally by their structure, cleavage site, specificity, and cofactors. They are similar in that they perform both restriction and methylase activities as part of a single big enzyme complex, as opposed to the type II system, in which the restriction enzyme is completely independent of the methylase activity. Other distinctions between types II and III restriction enzymes include the fact that they cleave DNA at specified positions within the recognition site, whereas the others cut DNA at random locations within the recognition site, sometimes hundreds of bases away from the recognition sequence. Numerous type II restriction enzymes have been discovered in a range of bacterial species, amounting to several thousand in total. These enzymes are capable of recognising a few hundred different sequences, which are typically four to eight bases in length. Type IV restriction enzymes cut only methylated DNA and have a low sequence specificity, making them useful for a variety of applications.

Endonuclease Structure

Endonucleases of Type I and Type II restriction types are multisubunit complexes that contain endonucleases as well as methylase activity. A random location roughly 1,000 base pairs in length from the recognition sequence can be cleaved by type I restriction enzymes, which are the most common. Type II enzymes, in contrast to Type I enzymes, are simpler and do not require ATP as an energy source. Adenosine triphosphate (ATP) is required for Type III cleavage of the DNA at roughly 25 base pairs from the recognition sequence.

Endonuclease Function

Endonucleases are enzymes that aid in DNA repair. When DNA is incised at AP sites, it is catalysed by AP endonuclease, which prepares the DNA for subsequent excision, repair synthesis, or DNA litigation. In E.coli cells, there are two AP endonucleases, whereas in eukaryotes, there is only one AP endonuclease present. Endonucleases can also be mutated, as has been demonstrated. Xeroderma pigmentosum is a rare autosomal recessive illness caused by a deficiency in a UV-specific endonuclease, which means that DNA damage produced by sunlight cannot be repaired in the affected person. Another type of mutation is Sickle Cell Anaemia, which occurs when the recognition site for the restriction endonuclease that identifies the nucleotide sequences is removed from the gene’s DNA.

Conclusion

It is also known as restriction endonuclease, and it is a protein generated by bacteria that cleaves DNA at certain locations along the molecule’s length. Restriction enzymes in the bacterial cell cut foreign DNA, thereby eradicating the invading organisms from the cell.

The enzymes that restrict DNA entry are found naturally in bacteria, where they cut through foreign DNA. Researchers benefit from the usage of restriction enzymes such as BamHI because they cut DNA at specific recognition sequences, making them helpful tools. When the BamHI enzyme breaks a DNA molecule, one strand on either side of the cut protrudes from the cut site. Endonucleases are enzymes that are involved in DNA repair. AP endonuclease is a unique type of endonuclease that catalyses the incision of DNA only at AP sites, hence preparing DNA for future excision, repair synthesis, and DNA ligation processes.