What are exonucleases and their applications?

Learn about exonucleases and some of their common applications.


Exonucleases are enzymes that catalyze the removal of nucleotides in either the 5´ to 3´ or the 3´ to 5´ direction from the ends of single-stranded and/or double-stranded DNA. Removal of nucleotides is achieved by cleavage of phosphodiester bonds via hydrolysis. These important enzymes can have slightly different activities, and therefore can be used for a wide variety of applications. Most exonucleases digest at nicks in the DNA. Some exonucleases remove one base at a time. Lambda Exonuclease is an example of this and transforms double-stranded DNA into single-stranded DNA by chewing from the free ending containing a 5´ phosphate, degrading one strand preferentially but not the other. Other examples are Exo I and Exo III. Other exonucleases, such as T5, ExoV or Exo VII remove short oligos. The products of T5 Exo and Exo V digestion also include individual bases. Exonucleases such as Exo VII and V, digest in both the 5´ to 3´ and 3´ to 5´ direction, while others, such as Exo T and Exo I, only work in one direction. Some exonucleases, such as Exo I and Exo T only digest single-stranded DNA while leaving behind double-stranded DNA. This can be useful to remove PCR primers, for example. Exonucleases such as T7 Exo digest only double-stranded DNA, while others, such as T5 Exo and Exo V, can digest both single and double-stranded DNA. A unique enzyme is Exo V, also known as Rec BCD. This enzyme consists of three subunits. This includes helicase activity, that unwinds double-stranded DNA to expose single-stranded DNA, as well as single-stranded exo- and endonuclease activity, which digests the DNA. The end result is activity like an exonuclease, for example shortening from the end. This enzyme is often used to clean-up plasmid DNA because it selectively degrades linear DNA without touching circular DNA. As you can see, exonucleases work in many different ways and can be used individually or combined to achieve the activity that you require for your specific application.
For more information regarding NEB exonucleases, please visit the following resources at www.neb.com.
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