Exonuclease III

Exonuclease III catalyzes the stepwise removal of mononucleotides from dsDNA starting from a 3´-OH at nicks, blunt ends, recessed ends and 3´-overhangs of less than 4 bases, yielding nucleoside 5´-phosphates. This 3´→5´ exonuclease will also degrade DNA from 3´-phosphate ends due to its intrinsic 3´-phosphatase activity. In addition, the enzyme has apurinic endonuclease activity and ribonuclease H activity. The enzyme is used in conjunction with S1 nuclease for unidirectional deletion of sequences from the termini of DNA fragments. Exonuclease III is provided with a 10X Reaction Buffer consisting of 660mM Tris-HCl (pH 8.0 at 25°C), 6.6mM MgCl₂.

References

1. Weiss, B. (1976) J. Biol. Chem. 251, 1896–901.
2. Rogers, S.G. and Weiss, B. (1980) Meth. Enzymol. 65, 201–11.
3. Henikoff, S. (1984) Gene 28, 351–9.
4. Promega Product Information #9PIM181, Promega Corporation.

Storage Buffer: 20mM Tris-HCl (pH 7.5 at 25°C), 1mM DTT, 100mM KCl, 50% (v/v) glycerol.

Source: Recombinant E. coli strain.

QC Tests: Activity, SDS-PAGE/purity, endonuclease, 3´ overhang protection assay.

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

Applications

• Generating nested sets of deletions with double-stranded, linear DNA.
• Preparation of single-stranded DNA.