Exonuclease III catalyzes the stepwise removal of 5' mononucleotides from
the 3'-hydroxyl termini of double-stranded DNA. Linear double-stranded
DNA and circular DNAs containing nicks or gaps are substrates. The enzyme
is not active on single-stranded DNA or double-stranded DNA with a protruding
3' terminus. It will not degrade double-stranded DNA with 3' overhang
of four bases or longer. The enzyme also carries three other activities:
an endonuclease specific for apurinic DNA, an RNase H activity, and a
3' phosphatase activity.
Recombinant E. coli strain.
¡Ü Generating nested sets
of deletions of the terminal sequences of double-stranded linear DNAs.
¡Ü Site-directed mutagenesis.
¡Ü Preparation of single-stranded
substrates for dideoxy sequencing.
One unit is defined as
the amount of enzyme required to produce 1nmole of acid-soluble
nucleotides in 30 minutes at 37¡É.
STANDARD UNIT ASSAY CONDITIONS
Tris-HCl (pH7.6), 1mM MgCl2, 1mM DTT and 125¥ì g calf thymus DNA.
CONCENTRATION: 150-200 units/¥ìl.
10X REACTION BUFFER
(pH8.0@25¡É), 50mM MgCl2, 10mM 2-mercaptoethanol.
(pH7.9), 1mM DTT. 160mM KCl and 50% glycerol. Store at -20¡É.¡¡
1. Rogers, S.G. and Weiss,
B. (1980) Methods. Enzymol. 65, 201.
2. Weiss, B. (1976) J. Biol.
Chem. 251, 1896.
3. Sambrook, J., Fritsch, E.F. and Maniatis, T.
(1989) Molecular Cloning: A Laboratory Manual, second ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor.