Difference between Exonuclease and Endonuclease Enzyme

In this difference between exonuclease and endonuclease enzyme post we have briefly explained about exonuclease, mechanism, endonuclease enzyme and its mechanism. Key endonuclease and exonuclease difference.

The difference between endonuclease and exonuclease is mainly due to the two properties like mode of action and the site of cleavage. A nuclease is a protein that can break phosphodiester links between nucleotides in nucleic acids. Because it hydrolyzes the chemical bonds between nucleotides, it belongs to the hydrolase enzyme family. This enzyme is required for natural DNA repair mechanisms in cells as well as biotechnological procedures including gene cloning, recombinant DNA technology, RFLP, AFLP, gene sequencing, gene therapy, genome mapping, and so on.

Nucleases are divided into two types: ribonuclease and deoxyribonuclease, which operate on and break chemical bonds between RNA and DNA monomers, respectively. Nucleases are divided into two classes based on the site of action: endonuclease and exonuclease. Endonucleases cleave the phosphodiester bonds between nucleotides in the centre of nucleic acids when they detect certain sequence areas in the nucleic acids. Exonucleases are enzymes that cleave phosphodiester links between nucleotides at the ends of nucleic acids.

Endonuclease vs Exonuclease


The word endonuclease can be broken down into endo and nuclease, with the former referring to the interior and the latter to the catalyst for nucleic acid degradation. To generate oligonucleotides, the endonuclease enzyme creates a break in the polynucleotide chain. To make a snip, you don’t need any free 3′ or 5′ ends. These can be either sequence-specific or non-specific. Sequence-specific endonuclease includes restriction enzymes that create nick at the specific site only, whereas the non-specific endonucleases can cut the DNA or RNA from anywhere in the strand.


Endo-nuclease first breaks the phosphodiester bond of the mismatched nucleotide bases. Then, it separates the whole mismatched base pairs. After that, endonuclease forms the DNA hybrid with the help of DNA polymerase and DNA ligase, in which a former polymerized the strand and a latter binds the fragment. At last, the endonuclease creates a nick.


Exonuclease is a nuclease enzyme that breaks chemical bonds between nucleotides at the 3′ and 5′ ends of DNA strands. At the end of the chain, it breaks single nucleotides and creates nucleosides by transferring phosphate groups to water. Archaea, bacteria, and eukaryotes all have exonucleases. 

DNA polymerase 1, 2, and 3 are among the 17 distinct exonucleases found in E. coli. Several DNA polymerases have activity as 3′ to 5′ exonuclease proofreaders. Exonucleases have a role in DNA repair, genetic recombination, mutation prevention, genome stabilization, and other processes.


Exonuclease attacks the phosphodiester link first, and then removes the nucleotides from the polynucleotide chain one by one. Cohesive or sticky ends are those that hang over the end of the strand. After the cohesive ends have been removed, the DNA overlaps to form a whole DNA hybrid or segment.

Difference between Exonuclease and Endonuclease Enzyme

Endonuclease and Exonuclease Difference



An endonuclease is a group of enzymes that cleave the phosphodiester bond present within a polynucleotide chain.

Exonucleases are enzymes that cleave DNA sequences from the 5′ or 3′ end of a polynucleotide chain one at a time.

The nucleotide sequence is cleaved from the middle by endonucleases.

The ends of a nucleotide sequence are cleaved by exonulceases.

The activity of some endonucleases, such as restriction endonucleases, is preceded by a lag phase.

There is no lag time between exonuclease activity and its onset.

In the middle, the endonuclease cuts a DNA part, resulting in oligonucleotides.

Exonulceases are enzymes that cleave DNA sequences into single nucleotides or nucleosides.

Endonucleases might form either sticky or blunt ends.

Exonulceases form sticky ends.

Endonucleases that cleave specific locations within a DNA sequence, commonly known as restriction endonucleases.

Exonuclease is a non-specific enzyme.

Endonuclease has a protective role in which certain pathogens may be blocked from entering.

There are no protective properties of exonuclease against the invasion of pathogenic microbes.

Restriction endonuclease is a restriction endonuclease that may cleave particular locations in circular DNA.

When it comes to circular DNA, exonulceases are less active than when it comes to linear DNA.

Phosphorothioate bonds cannot inhibit endonucleases unless the complete sequence has the bonds between all nucleotides.

By adding five phosphorothioate linkages in a row to a sequence, exonuclease can be blocked.

Endonucleases do not require free 3′ or 5′ ends to function.

Exonucleases should be able to act on the ends.

Examples include Bam HI, EcoRI, Hind III, Hpa I, Sma I,

Examples include Exonuclease I, Exonuclease III, RecBCD (Exonuclease V), RecJ exonuclease, Exonuclease VIII/RecE, Exonuclease IX, Exonuclease T, Exonuclease X etc.


1. Exonuclease cuts the nucleotide chain from the ends, whereas endo-nuclease cuts it in between endonuclease and exonuclease difference.

2. Exonuclease cleaves the polynucleotide chains’ phosphodiester bonds at the 3′ or 5′ ends, while endonuclease cleaves the phosphodiester bonds within the polynucleotide chain.

3. Exonuclease cuts the nucleotide bases one by one from the 3′ or 5′ end, whereas endonuclease cuts the polynucleotide chain directly and produces two or more pieces.

4. Exonuclease removes the single nucleotides off the end of the chain, whereas endo-nuclease generates a single-stranded nick within the chain.


1. Both endonuclease and exonuclease have some features in common, and their functions are comparable.

2. Endonuclease and exonuclease are enzymes that operate on nucleic acids, such as DNA and RNA.

3. Endonuclease and exonuclease are nuclease enzymes that cleave DNA or RNA nucleotide chains.

4. The phosphodiester linkage of DNA and RNA can be hydrolyzed by both endonuclease and exonuclease.

Key Differences

1. Endonuclease divides the nucleotide chain into two or more fragments, whereas exonuclease separates the nucleotides from a polynucleotide chain one at a time.

2. Endonuclease cleaves in the middle of the polynucleotide chain, whereas exonuclease cleaves at the ends of the chain (either 3′ or 5′).

3. The cleavage of endo-nuclease does not need the presence of 3′ or 5′-hydroxyl groups, but the cleavage of exonuclease must.

4. Endonuclease has sequence-specific specificity, whereas exonuclease has non-specific specificity.

5. Exonuclease does not go through a lag period before starting to work, but endonuclease does.

6. Exonuclease produces nucleosides, which are the monomer units of nucleotide that lack a phosphate group. Endonuclease causes the creation of oligonucleotides, which are short nucleotide sequences.

Further Readings