- Exonuclease III (Exo III) is a bacterial 3′→5′ exonuclease derived from Escherichia coli, known for its ability to sequentially remove mononucleotides from the 3′-hydroxyl termini of double-stranded DNA (dsDNA). It is highly specific for double-stranded regions and exhibits very limited activity on single-stranded DNA or RNA. Exo III is Mg²⁺-dependent and functions optimally at neutral to slightly alkaline pH.
- In addition to its exonucleolytic activity, Exonuclease III possesses several other catalytic functions, including apurinic/apyrimidinic (AP) endonuclease activity, 3′-phosphatase, and 3′-phosphodiesterase activities. These properties make it an important enzyme in DNA repair, recombination, and processing of damaged DNA.
- In molecular biology, Exonuclease III is widely used for progressive unidirectional deletion of DNA sequences. This application is useful in nested deletions for sequencing, promoter analysis, and gene truncation studies. By using time-controlled Exo III digestion followed by S1 nuclease treatment (to remove single-stranded overhangs), researchers can generate a series of overlapping DNA fragments with defined 5′ ends and staggered 3′ ends.
- It is important to note that Exo III is inhibited by 3′ overhangs longer than four nucleotides, and it does not efficiently degrade DNA with 3′-phosphate termini or blocked 3′ ends, providing useful control points in experimental design.
- In summary, Exonuclease III is a versatile enzyme with 3′→5′ exonuclease and DNA repair activities, extensively used in laboratory settings for precise manipulation of double-stranded DNA, and for probing DNA-protein interactions, mapping functional domains, and constructing deletion libraries.
