In biochemistry, nucleases are a diverse class of hydrolytic enzymes that carry out hydrolysis of phosphodiester bonds of nucleic acids — DNA and RNA.
Nucleases attack the hydroxyl group of the phosphodiester bond, performing phosphatase activity by the removal of the phosphate group. There are various types of nucleases like DNases, RNases, and Cas9. Characterization of nucleases is based on their region of action, either at the end of sequence or somewhere in the middle.
Endonuclease is an enzyme that cuts the nucleotide sequence at a specific position within the DNA. Endonuclease activity first involves the inspection of the length of the DNA sequence and once a specific recognition sequence is found, endonuclease binds to it and cuts each of the strands of DNA at specific points.
Endonucleases are also called molecular scissors as they are used in genetic engineering, assaying, cloning, and recombinant protein production.
Whereas, exonuclease functions in contrast to endonuclease. Exonuclease removes nucleotides from the free ends of the DNA and not within the strand. These are present in both eukaryotic and prokaryotic cells. They are also found in venoms of lizards and snakes which work as toxins and cleave DNA encoding essential proteins.
Breaking down the word itself, “exo” means “outside” and nuclease is the name given to an enzyme that digests nucleic acids.
In prokaryotes and eukaryotes, exonucleases are of 3 types:
- Decapping 5’ to 3’ exonuclease
- Independent 5’ to 3’ exonuclease
- Poly A specific 3’ to 5’ exonuclease
The majority of exonucleases act in the 3’-5’ direction of the DNA/ RNA strand and biochemically catalyze the removal of mononucleotides.
As an exonuclease removes nucleotides from terminal positions of nucleic acids one at a time, this creates overhangs called sticky ends.
Exonucleases mainly function in the control of genetic quality, DNA proofreading during replication, homologous combination, and DNA repair. Therefore, they are known to be the protectors of genome stability and thus exonucleases are central to many DNA metabolism processes.
Some examples include:
- Exonuclease I: Used for degrading excess ssDNA primers in vitro from a mixture of double-stranded products. Holds application in PCR.
- Exonuclease III: Involved in unidirectional nested deletion
- Lambda exonuclease: Performs removal of single nucleotides from 5’ ends in double-stranded DNA.
In this article, we will overview the activity, functions, and applications of exonucleases.
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Function & Applications
Exonucleases exhibit a wide range of applications and perform varied functions as shown below:
Exonuclease is a prime enzyme involved in the maintenance of cellular metabolism. All of the cellular components, especially proteins, are a result of transcription and translation of DNA. So, any malformation of DNA will result in protein loss or malfunction which will disrupt the cellular pathways and signaling.
To prevent such defects, DNA undergoes proofreading by these special exonucleases. These exonucleases, through their catalytic action, ensure that they produce error-free DNA so that cellular mechanisms are not disrupted. Exonuclease inhibitors can be used to regulate the activity of exonuclease activity.
Genome Stability & Aging
For evolutionary fitness and long-term health, genome stability is essential. To achieve this,
cells have evolved DNA packaging and highly accurate repair mechanisms in which exonuclease plays a crucial role.
Abnormal DNA structures or defects called lesions result in premature aging of the cell. In order to avoid this, exonucleases come into action and remove these defective sequences from the DNA thereby promoting gene stability and preventing early aging. To enhance polymerase fidelity, exonuclease acts in multiple pathways of DNA repair and maintenance including:
- MMR- Mismatch repair
- BER- Base excision repair
- NER- Nucleotide excision repair
- Recombinational repair
- Telomerase maintenance
In order to maintain genetic stability and fidelity, exonucleases perform DNA editing and proofreading of DNA for errors. This is a highly specific process and requires a high degree of precision.
During DNA polymerization, exonucleases proofread the DNA and look for any unusual structures that might cause problems in DNA replication thereby repairing damaged DNA. Because of this application, they are called the protectors of DNA.
During replication, polymerase δ, ε, and γ proofread DNA from 3’ to 5’ direction. They are intrinsic proofreaders whereas ExoN is an extrinsic one. DNA repair is subjective to the type and extent of lesion on the strand(s) which includes base excision repair.
The nucleotide excision repair pathway (NER) facilitates the removal of bulky lesions like UV-induced pyrimidine dimers. These dimers hinder the progression of the replication fork and transcription elongation.
Even though DNA replication is a highly accurate process, there are chances of incorrect inserts, substrate template mismatch, or base pair mismatch. This is when the exonucleases come into play, they perform proofreading of the DNA strands and remove the errors as the accumulation of these errors can be carcinogenic and may lead to cellular aging.
There are DNA polymerases that have intrinsic proofreading capabilities to excise the incorrectly incorporated bases while carrying out replication. These are the 3’-5’ exonuclease subunits of polymerase.
On the other hand, there are extrinsic exonucleases that work in sync with the DNA polymerases. For example, ExoN 3’-5’ exonuclease removes 3’ mismatched termini from the double-stranded DNA (dsDNA).
Exonuclease Activity of DNA Polymerase
DNA polymerase is an enzyme that is responsible for carrying out DNA replication. It is mainly involved in the addition of DNA primers and in the synthesis of the new DNA strands. But there are DNA polymerases that possess intrinsic exonuclease activity.
For DNA polymerase to perform highly accurate replication, intrinsic proofreading activity is required. To accomplish this, DNA polymerase has a 3’-5’ exonuclease subunit that removes the incorrectly added bases. The new duplex DNA form during replication becomes the active site for 3’-5’ exonuclease proofreading.
For example, in Escherichia coli (E. coli), the DNA pol III holoenzyme consists of several subunits including the 3’-5’ exonuclease.
Differences from Endonuclease
Belonging to the same category of enzymes — restriction enzymes — exonuclease works in contrast to endonuclease. There are numerous differences observed between these types, which are as follows:
Risk of Nuclease Mutations or Loss
As nucleases are the enzymes involved in the degradation of nucleotides, mutagenesis or loss of specific nucleases may result in DNA editing and processing defects. Some of the clinical features associated with nuclease mutation are:
Defects or loss of such exonucleases can cause serious medical ailments and are listed under genetic disorders.
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Exonucleases are a class of enzymes that cleave nucleotides from extreme ends of DNA or RNA strands i.e. 3’ or 5’ end. They hydrolyze phosphodiester bonds and cut specific nucleotides i.e they are exonucleolytic in nature.
With numerous applications in the field of molecular biology, exonucleases are instrumental in precision manipulation and quality control of genetic material. Because of this, there is a high demand for these nucleases commercially. Commercial exonucleases mainly include Exonuclease I, Exonuclease III, and Lambda Exonuclease.
Additional reading about exonucleases and their specifications can be found on websites and journals like PubMed, j.biol.chem, Proc. Natl. Acad. Sci. USA, Nucleic Acids Res, which have numerous papers and citations that provide detailed information on the topic.
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