What Are Restriction Endonucleases and Exonucleases?
The restriction enzyme originated from the research of phage λ, a virus that infects bacteria, and the phenomenon of host-managed restriction and modification of bacteriophage. The restriction enzymes studied through Arber and Meselson were type I restriction enzymes, which cleave DNA randomly far away from the recognition area or sites. In 1970, Hamilton O. Smith, Thomas Kelly, and Kent Wilcox insulated and characterized the primary kind II restriction enzyme, HindII, from the bacterium Haemophilus influenzae. Restriction enzymes of this kind are more beneficial for laboratory work as they cleave DNA on the area in their recognition series and are the most usually used as a molecular biology tool.
What are Restriction Enzymes?
Restriction enzymes also known as molecular scissors, are essential tools of in biotechnology. Their discovery has enabled the recognition and replacement of specific segment of DNA sequence in order to produce recombinant DNA. They are pretty cool, and there are at least 3,000 of them. Each of these enzymes cuts a selected or a specific DNA sequence and does not discriminate in which the DNA comes from — microorganism, fungi, mouse, or human. Based on the position at which these enzymes cut the sequence they recognize, they are classified as endonucleases and exonucleases.
What is Exonuclease?
Exonucleases are enzymes that work through cleaving nucleotides separately from the end of a polynucleotide chain. Exonuclease function involves a hydrolyzing response that breaks phosphodiester bonds at both the 3′ and the 5′ end occurs. Its near relative is the endonuclease, which cleaves phosphodiester bonds within the middle (endo) of a polynucleotide chain. Eukaryotes and prokaryotes have 3 kinds of exonucleases involved within the normal turnover of mRNA: 5′ to 3′ exonuclease (Xrn1), that is a dependent decapping protein; 3′ to 5′ exonuclease, an independent protein; and poly (A)-specific 3′ to 5′ exonuclease.
Examples of Restriction Exonuclease
Snake venom, Exonuclease I, and Xrn1 are the examples of exonuclease enzymes.
What is Endonuclease?
Endonucleases are enzymes that cleave the phosphodiester bond inside a polynucleotide chain. Some, including deoxyribonuclease I, cut DNA pretty nonspecifically, at the same time as many; commonly known as restriction endonucleases, cleave only at very specific nucleotide sequences. Endonucleases vary from exonucleases, which cleave the ends of recognition sequences rather than the middle (endo) portion. Some enzymes called "exo-endonucleases", however, aren't limited to both nuclease functions, showing characteristics which are both endo- and exo-like. Evidence shows that endonuclease activity experiences a lag as compared to exonuclease activity.
Example of restriction endonuclease
BamHI, EcoRV, EcoRI, HindIII, and HaeIII are examples of endonuclease enzymes.
Recombinant DNA Technology
Recombinant DNA technology contains changing genetic material outside an organism to attain improved and preferred traits in dwelling organisms or as their products. This technology includes the insertion of DNA fragments from a lot of sources, having a suitable gene series through a suitable vector. Manipulation in an organism's genome is accomplished both through the introduction of 1 or numerous new genes and regulatory factors or through lowering or blocking the expression of endogenous genes via recombining genes and factors.
Enzymatic cleavage is carried out to attain specific DNA fragments through the usage of restriction endo-nucleases for specific target series DNA sites accompanied by DNA ligase activity to enroll in the fragments to restore the preferred gene in the vector. The vector is then brought into a host organism that is grown to provide more than one copy of the integrated DNA fragment in culture, and finally, clones containing a relevant DNA fragment are selected and harvested.
Endonuclease vs Exonuclease
Interesting Facts
Restriction enzymes are helpful to bacteria.
They are non-discriminating and specific cutters.
Restriction enzymes have methyltransferases which methylate DNA.
The main in endonucleases vs exonucleases
Important Questions
1. What are the similarities between exonuclease and endonuclease?
Ans: Exonuclease and endonuclease both help in DNA repair within the cell. They act on both RNA and DNA.
2. Define restriction enzymes.
Restriction enzyme is a protein isolated from microorganisms that cleave DNA sequences at sequence-specific areas, generating DNA fragments with a recognised series at every end.
Key features of endonuclease and exonuclease
Restriction enzyme is a protein insulated from microorganisms that cleave DNA sequences at sequence-specific areas, generating DNA fragments with a recognised series at every end.
The use of restriction enzymes is essential to laboratory methods, along with recombinant DNA generation and genetic engineering.
Exonuclease enzymes work by cleaving nucleotides separately from the end of a polynucleotide chain.
Endonucleases are enzymes that cleave the phosphodiester bond inside a polynucleotide chain.
These enzymes are involved in recognising and cleaving up foreign DNA getting into the cell; their more possible function is shielding the microorganism from phage infection. The asset which is applicable to us is that those enzymes recognize specific DNA sequences.
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FAQs on Difference Between Restriction Endonuclease and Exonuclease
1. What is the main difference between an exonuclease and an endonuclease enzyme?
The primary difference lies in their site of action on a DNA strand. Exonucleases are enzymes that cleave nucleotides one by one from the free ends (either the 5' or 3' end) of a polynucleotide chain. In contrast, endonucleases cut the phosphodiester bonds at specific sites within the DNA strand, away from the ends.
2. How is a restriction endonuclease like EcoRI different from a general exonuclease?
EcoRI is a specific type of endonuclease that differs from an exonuclease in two key ways:
- Site of Action: EcoRI cuts DNA internally only at a specific recognition sequence (GAATTC). An exonuclease is non-specific in sequence but is specific to the ends of the DNA molecule.
- Function in Biotechnology: EcoRI's ability to create predictable 'sticky ends' is essential for gene cloning. Exonucleases, which degrade DNA from the ends, are not used for inserting genes into plasmids.
3. What are some common examples of exonucleases and endonucleases?
Common examples include:
- Exonucleases: DNA Polymerase I and III have proofreading exonuclease activity. Exonuclease I is another example.
- Endonucleases: The most famous examples are restriction endonucleases used in genetic engineering, such as EcoRI, HindIII, and BamHI. DNase I is an example of a non-specific endonuclease.
4. Why do restriction enzyme names like HindIII include Roman numerals?
The Roman numeral in the name of a restriction enzyme indicates the order of its isolation from a specific bacterial strain, not the order of its discovery. For example, HindIII was the third restriction enzyme to be isolated from the bacterium Haemophilus influenzae, strain Rd. This naming convention helps scientists differentiate between multiple restriction enzymes found in the same organism.
5. Why are endonucleases often just called 'restriction enzymes' in biotechnology?
This is because the endonucleases used in biotechnology are almost exclusively restriction endonucleases. These enzymes earned their name from their natural function in bacteria, where they 'restrict' viral infections by cutting up the foreign DNA of bacteriophages. Due to their critical role as 'molecular scissors' in genetic engineering, the term 'restriction enzyme' has become a common shorthand for restriction endonuclease.
6. What is the functional importance of 3'→5' versus 5'→3' exonuclease activity?
The directionality is crucial for different cellular functions:
- 3'→5' Exonuclease Activity: This is the proofreading function. It allows DNA polymerase to move backward to remove a mismatched nucleotide just added during replication, ensuring the high fidelity of DNA synthesis.
- 5'→3' Exonuclease Activity: This function is used to remove the RNA primers that initiate DNA synthesis. The enzyme moves in the same direction as synthesis, clearing the path ahead by degrading the primer.
7. If exonucleases cut from the ends, why are they ineffective for cutting a circular plasmid in gene cloning?
Exonucleases are ineffective on circular DNA, like a bacterial plasmid, because a circular molecule has no free 5' or 3' ends. The action of an exonuclease is strictly limited to cleaving nucleotides from the termini of a linear DNA or RNA strand. To open a plasmid for gene insertion, an endonuclease is essential to make the first internal cut, which linearises the circular DNA and creates the ends that a gene can be inserted into.
8. Can any endonuclease be used for recombinant DNA technology? Explain.
No, not all endonucleases are suitable. It is crucial to distinguish between a general endonuclease and a restriction endonuclease. A general endonuclease, like DNase I, cuts DNA at random, non-specific sites. This creates unpredictable fragments and is not useful for precise genetic manipulation. In contrast, restriction endonucleases are vital because they recognise and cleave DNA only at specific palindromic sequences, generating predictable fragments with defined ends (sticky or blunt) that are essential for successful ligation in cloning.
9. In genetic engineering, which is generally more advantageous: an endonuclease that creates 'sticky ends' or one that creates 'blunt ends'?
While both can be used, endonucleases that generate sticky ends are generally more advantageous. Sticky ends are short, single-stranded overhangs that are complementary to each other. This complementarity significantly increases the efficiency of ligation, as the overhangs can anneal (base-pair) with a corresponding sticky end on another DNA fragment. This temporarily holds the vector and insert DNA together, making it easier for the enzyme DNA ligase to form a permanent phosphodiester bond.
