Differences between Endonuclease and Exonuclease(Full Details)

Restriction Endonucleases and exonucleases are enzymes that cut DNA and RNA at specific places. But where they are cut is different for each one.

Nucleases are enzymes that are known to break phosphodiester bonds in nucleic acids. They are an important part of repairing DNA and cloning molecules. Nucleases are split into two groups based on where they work: endonucleases and exonucleases.

The two qualities like mode of action and the site of cleavage are the key reasons why endonuclease and exonuclease differ from one another.

  • Mode of action. By rupturing the phosphodiester backbone of the nucleotide, endonuclease divides the bases of the nucleotide from the middle. Exo-nuclease breaks the phosphodiester backbone of the nucleotide either from the 5′ or 3′ end, cutting the nucleotide bases at the ends.
  • Site of Cleavage. The polynucleotide chain’s interior nucleotides are cut by endonuclease. Exo-nuclease cuts the nucleotides that are present at the exterior ends of the polynucleotide chain.
Differences between Endonuclease and Exonuclease

What is Endonuclease?

A certain enzyme called an endonuclease cleaves the DNA’s internal bond.

The good news is that, largely towards the polynucleotide chain’s end, the enzyme does not require a free 3′ or 5′ hydroxyl group.

No matter where it occurs in the polynucleotide chain, the nuclease enzyme is known to target the 3′ or 5′ hydroxyl linkages.

Another division of the enzyme is into Deoxyribonuclease I and Deoxyribonuclease II. Deoxyribonuclease I is in charge of catalyzing the 3′ links of DNA to produce oligonucleotides, which typically include four nucleotide residues.

On the other hand, Deoxyribonuclease II is in charge of the hydrolysis of 5′ links, which results in a collection of nucleotides.

Deoxyribonuclease I is recovered from the pancreas of a cow, whereas Deoxyribonuclease II is found in the spleen and thymus of specific bacterial species.

What is Exonuclease?

Another kind of nuclease enzyme called an exonuclease is known for cleaving the nucleotides at the ends of DNA molecules.

Since the DNA molecule’s two strands are complementary to one another, the 3′ and 5′ arms are used to represent them.

Later, two groups of the enzyme identified as “a” and “b” attack the phosphodiester bridges of RNA and DNA.

The ester connection between the 3′ carbon and the phosphoric group is hydrolyzed by enzyme “a.” The ester connection between the 5′ carbon and phosphoric groups is hydrolyzed by enzyme “b.”

The exonuclease of group “a” is known to be present in the venom of rattlesnake and Russell’s viper.

Comparison Table (Endonuclease Vs Exonuclease)

MeaningThis enzyme divides the nucleotides and cleaves the polynucleotide chain.This enzyme removes nucleotides one at a time by cleaving the polynucleotide chain at the end.
Mechanism ActionCleave nuclei acid at the middle.Cleave the nuclei acid at the end.
Lag PhaseUndergo a lag period before their activityDoes not undergo lag period before activity
Final ResultResult in oligonucleotidesResult in single nucleotides.
Blunt EndsForm either sticky or blunt endsForm sticky ends only
FunctionBlock entry of pathogens causing diseasesHave no significant role
ExamplesRestriction enzymes, DNases, and S1 nucleaseSpleen phosphodiesterase, polymerase III of 3’ to 5’, and Snake venom

Key Differences Between Endonuclease and Exonuclease

  • While exonuclease removes one nucleotide at a time from a polynucleotide chain, endonucleases work by splitting the nucleotide chain into two or more fragments.
  • In contrast to exonuclease, which cleaves at the ends of the polynucleotide chain (either at 3′ or 5′), endonucleases cleave between the polynucleotide chains.
  • Unlike exonuclease, which needs the 3′ or 5′ hydroxyl groups for the cleavage, endo-nuclease does not need these groups.
  • In contrast to exonuclease, which lacks specificity, endonucleases are sequence-specific.
  • Exonuclease does not experience lag phase, whereas endonuclease does prior to their activation.
  • Nucleoside, which are the monomer portions of nucleotides devoid of phosphate groups, are created as a result of exonuclease. Oligonucleotides, which are the condensed sequences of nucleotides, are produced as a result of endonuclease.


  • Both are types of the enzyme nuclease that cleave nucleic acid.
  • Both influence DNA and RNA.
  • Both are involved in a cell’s DNA repair process.


We have covered the fundamental differences between the endonuclease and exonuclease, as well as the differences in attributes and mechanisms. Therefore, we can say that both endonuclease and exonuclease are enzymes with different cleavage activity sites. Additionally, in addition to their primary job in separating bases with the incorrect or mismatched nucleotides, they are crucial for DNA repair and stabilization.