Difference Between Endonuclease and Exonuclease

Main Difference – Endonuclease vs Exonuclease

Nucleases are a class of hydrolases that cleave the phosphodiester bonds between the nucleotides in the nucleic acids, both DNA and RNA. The nucleases can be divided into two types based on the type of the substrate nucleic acid they work on: ribonucleases and deoxyribonucleases. Ribonucleases work on RNA whereas deoxyribonuclease work on DNA. Nucleases can also be divided into two as endonucleases and exonucleases. The main difference between endonucleases and exonucleases is that endonuclease cleaves nucleic acid strand at the middle whereas exonuclease cleaves nucleic acid strands from the ends. The major role of nucleases inside the cell is to take part in the DNA repair mechanisms.

Key Areas Covered

1. What is an Endonuclease
      – Definition, Characteristics, Function
2. What is an Exonuclease
      – Definition, Characteristics, Function
3. What are the Similarities Between Endonuclease and Exonuclease
      – Outline of Common Features
4. What is the Difference Between Endonuclease and Exonuclease
      – Comparison of Key Differences

Key Terms: DNA, DNA repair, Endonuclease, Exonuclease, Restriction Endonucleases, Restriction Site, RNADifference Between Endonuclease and Exonuclease - Comparison Summary

What is an Endonuclease

An endonuclease is a class of hydrolase that cleaves nucleic acids at the middle. The action of endonucleases may result in two or more fragment of nucleic acids. Endonucleases are capable of acting on both DNA and RNA.  The cleavage of some endonucleases such as deoxyribonucleases (DNases) is non-specific. However, many endonucleases cleave the target nucleotide sequences in a specific manner. These type of specific endonucleases are called restriction endonucleases. They are capable of recognizing a specific sequence of the nucleic acid strand. Thus, these restriction endonucleases undergo a lag period prior to their action upon the nucleic acid, scanning for the specific nucleotide sequence. This specific nucleotide sequence is called the restriction site.

Difference Between Endonuclease and Exonuclease

Figure 1: The action of Hind III

A typical restriction site is a palindromic sequence of four to six nucleotides. Many restriction endonucleases cleave DNA strands, leaving single-stranded ends called sticky ends. In genetic engineering, these type of restriction endonucleases are widely used to produce recombinant DNA by ligating different, desired DNA strands together. DNA methylation in higher organisms prevents the action of endonucleases upon their genome. However, prokaryotic DNA lacks methylation. Therefore, prokaryotic DNA within a eukaryotic host can be easily targeted for cleavage. The sticky end formation by the action of the restriction endonuclease, Hind III is shown in figure 1

What is an Exonuclease

An exonuclease is a type of hydrolases that cleave the nucleic acid chain at its end. Exonucleases remove nucleotides one by one from the nucleic acid chain by hydrolyzing the phosphodiester bonds at either 3’ or 5’ ends. Three types of exonucleases can be identified in both prokaryotes and eukaryotes. They are 5’ to 3’ exonuclease, 3’ to 5’ exonuclease, and poly (A)-specific 3’ to 5’ exonucleases. All three types have a role in mRNA turnover. The E.coli DNA polymerase III, which catalyze the addition of nucleotides to the growing strand during prokaryotic DNA replication, consists of 3’ to 5’ exonuclease activity in its ε subunit. The ε subunit removes the mispaired nucleotides from the end of the growing chain.

Main Difference - Endonuclease vs Exonuclease

Figure 2: 3’ to 5’ exonuclease domain of DNA polymerase I

The 3’ to 5’ exonuclease activity of the DNA polymerase III is called the proofreading activity of the enzyme. The DNA polymerase I has both 5’ to 3’ exonuclease activity as well as 3’ to 5’ exonuclease activity. The 5’ to 3’ exonuclease activity can remove up to ten nucleotides at a time. Exonucleases play a vital role in DNA proofreading, DNA repair, and DNA stabilization. The 3’ to 5’ exonuclease domain of the DNA polymerase I is shown in figure 2. 

Similarities Between Endonuclease and Exonuclease

  • Endonuclease and exonuclease are two type of nucleases, which cleave nucleic acids.
  • Both endonucleases and exonucleases act on both DNA and RNA.
  • Both nucleases are involved in the DNA repair inside the cell.

Difference Between Endonuclease and Exonuclease


Endonuclease: Endonuclease refers to an enzyme that cleaves the polynucleotide chain separating nucleotides other than the two end ones.

Exonuclease: Exonuclease refers to an enzyme that cleaves polynucleotide chain from the end of the chain by removing the nucleotides one by one. 

Mechanism of Action

Endonuclease: Endonucleases cleave nucleic acids at the middle of the nucleic acids.

Exonuclease: Exonucleases cleave nucleic acids at the ends.

Lag Phase

Endonuclease: Restriction endonucleases undergo a lag period before their activity.

Exonuclease: Exonuclease does not have a lag period before their activity.


Endonuclease: Endonucleases result in oligonucleotides.

Exonuclease: Exonucleases result in single nucleotides or nucleosides.

Blunt/Sticky Ends

Endonuclease: Endonucleases may form either blunt ends or sticky ends.

Exonuclease: Exonucleases form sticky ends.


Endonuclease: Endonucleases block the entry of pathogens.

Exonuclease: Exonucleases have no significant role in blocking the entry of pathogens.


Endonuclease: DNases, S1 nuclease, and restriction enzymes such as Bam H1, Hind III, and Eco RI are the examples of endonucleases.

Exonuclease: Snake venom, spleen phosphodiesterase, 3’ to 5’ exonuclease domain of the DNA polymerase III, the 5’ to 3’ exonuclease activity and the 3’ to 5’ exonuclease domain of the DNA polymerase I are examples of exonucleases.


Endonucleases and exonucleases are two types of nucleases, which cleave nucleic acids by hydrolyzing the phosphodiester bonds between nucleotides. Endonucleases cleave the polynucleotide chain in the middle whereas exonucleases cleave the polynucleotide chain at the ends. Restriction endonucleases are a type of endonucleases, which cleave the polynucleotide chain at a specific sequence. Exonucleases are important in the proofreading of the replicated DNA. However, the main difference between endonuclease and exonuclease is the place of cleavage of the polynucleotide chain.


1. McMahon, Mary, and O. Wallace. “What is an Endonuclease?” WiseGEEK. Conjecture Corporation, 04 July 2017. Web. 08 Aug. 2017. 
2. “Exonuclease.” Wikipedia. Wikimedia Foundation, 01 Aug. 2017. Web. . 08 Aug. 2017.
3. Lodish, Harvey. “The DNA Replication Machinery.” Molecular Cell Biology. 4th edition.U.S. National Library of Medicine, 01 Jan. 1970. Web. .  08 Aug. 2017. 

Image Courtesy:

1. “HindIII Restriction site and sticky ends vector” By Helixitta – Own work via
2. “Poly I structure” By Christopherrussell – Gunther S, Rother K, Frommel C via

About the Author: Lakna

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things

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