Exonuclease III is a 3’ → 5’ exonuclease specific for double-stranded DNA. The enzyme catalyzes the stepwise removal of mononucleotides starting from a 3’-OH at nicks, blunt ends, 5’ – overhangs and 3’ – overhangs of less than 4 bases, yielding nucleoside 5’ – phosphates. A limited number of nucleotides are removed during each binding event, resulting in progressive deletions within the population of DNA molecules. Exonuclease III activity depends partially on helical structure and displays sequence dependence (C>A=T>G).Temperature, salt concentration and the ratio of enzyme to DNA greatly affect enzyme activity, requiring reaction conditions to be adjusted to specific applications. Exonuclease III degrades DNA from 3’ – phosphate ends due to its intrinsic 3’ – phosphatase activity. In addition, the enzyme has apurinic endonuclease activity and ribonuclease H activity. Exonuclease III is used in conjunction with S1 nuclease for unidirectional deletion of sequences from the termini of DNA fragments.

Source: Purified from E. coli strain harbouring the plasmid with Exo III gene

Reaction buffer (10x):

• 500 mM Tris-HCl (pH 7.6 at 30 °C)

• 100 mM MgCl₂

Applications:

• Unidirectional nested deletions

• Site-directed mutagenesis

• Preparation of strand-specific probes

• Preparation of single-stranded substrates for dideoxy sequencing

Unit Definition: One unit is defined as the amount of enzyme required to produce 1 nmol of acid soluble nucleotides in 30 minutes at 37°C

Concentration: 40-100 units/μl

Heat inactivation: 70°C for 20 minutes

Storage conditions: -20°C