Difference Between Endonuclease and Exonuclease

The difference between Endonuclease and Exonuclease is mainly due to the following two properties like mode of action and the site of cleavage.

Mode of action: Endo-nuclease cuts the nucleotide bases from the middle by breaking the phosphodiester backbone of nucleotide. Exo-nuclease cuts the nucleotide bases at the ends by breaking the phosphodiester backbone of nucleotide either from the 5’ or 3’ end.

Site of Cleavage: Endo-nuclease cuts the nucleotides that are interior to the polynucleotide chain. Exo-nuclease cuts the nucleotides that are present at the exterior ends of the polynucleotide chain.

Content: Endonuclease Vs Exonuclease

  1. Comparison Chart
  2. Definition
  3. Differences in properties
  4. Similarities in properties
  5. Difference in mechanism
  6. Key Differences
  7. Conclusion

Comparison Chart

Mode of actionIt separates the nucleotides into two or more fragmentIt removes the nucleotides one by one from the fragment
Site of occurrenceCleavage occurs in the middle of the polynucleotide chainCleavage occurs at the end of the polynucleotide chain
3’ or 5’-OH groupsDo not requireIt requires
Lag-phaseUndergo lag period before their activityAbsent
SpecificitySequence specificNon-specific
ResultIt releases oligonucleotidesIt releases nucleosides
RolePlays an important role in DNA repair, blocks the entry of pathogenPlays an important role in DNA repair, stabilization and proofreading but does not blocks the entry of the pathogen


The nuclease is an enzyme that breaks the polynucleotide chain by hydrolysing the phosphodiester bond that holds the nucleotides together. It is mainly of two types namely endonuclease and exonuclease.

Endonuclease: It creates a nick between the polynucleotide chain.

Exonuclease: It creates a nick at the ends of the polynucleotide chain either 3′ or 5′ end.

Differences in properties

  • Endo-nuclease cuts between the nucleotide chain whereas exonuclease cuts from the end of the nucleotide chain.
  • Exonuclease cleaves the phosphodiester bonds of the polynucleotide chains either at 3’ or 5’ end whereas endonuclease cleaves the phosphodiester bond that is present within the polynucleotide chain.
  • Endo-nuclease directly cuts the polynucleotide chain results into two or more fragments whereas exonuclease cuts the nucleotide bases one by one from 3’ or 5’ end.
  • Endo-nuclease creates single-stranded nick within a chain whereas exonuclease removes the single nucleotides from the end.

Similarities in properties

  • Both endonuclease and exonuclease share some similar properties where they show similarities in their function.
  • Endonuclease and exonuclease can act upon the nucleic acids i.e. both DNA and RNA.
  • Both endonuclease and exonuclease are the nuclease enzymes which performs the cleavage of the nucleotide chain of DNA and RNA.
  • Endo-nuclease and exonuclease both can hydrolyse the phosphodiester linkage of DNA and RNA.

Difference in mechanism

  • Exonuclease first attacks on the phosphodiester bond which removes the nucleotides one by one from the polynucleotide chain. The ends which are overhanging from the strand refers to “Cohesive or sticky ends”. After the removal of a cohesive end, both the DNA overlaps which makes the complete DNA hybrid or a segment.

exonuclease activity

  • 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, which undergo polymerization and then binding of fragment where the endonuclease creates the nick.

endonuclease activity

Key Differences Between Endonuclease and Exonuclease

  1. The mode of action of the endonuclease is to separate the nucleotide chain into two or more fragment whereas exonuclease separates the nucleotides at a time from a polynucleotide chain.
  2. The site of cleavage for endonuclease is in between the polynucleotide chain whereas for exonuclease it is at the ends of the chain either at 3′ or 5′.
  3. Endo-nuclease does not require 3′ or 5′-hydroxyl groups for the cleavage whereas exonuclease requires the 3′ or 5′-hydroxyl groups for the cleavage.
  4. The specificity of Endo-nuclease is sequence-specific whereas exonuclease is non-specific.
  5. Endo-nuclease undergoes lag phase before their activity whereas exonuclease does not undergo lag phase.
  6. Exonuclease results in the formation of nucleoside which are the monomer unit of nucleotide that lacks phosphate group. Endo-nuclease results in the formation of oligonucleotides which are the short sequences of nucleotides.


We have discussed the difference in properties, mechanism and the key differences between the endo and exonuclease. Hence we can conclude that both endonuclease and exonuclease is having different activity site for the cleavage but in addition to this, their main function is to separate the mismatched or wrong nucleotides base and play an important role in DNA repair and stabilization.

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