Some of the common applications of DNase I in rDNA technology have been mentioned below:

• Eliminating DNA contamination (e.g. plasmid) from preparations of RNA.
• Analyzing the DNA-protein interactions via DNA footprinting.
• Nicking DNA prior to radio-labeling by nick translation.

2) DeoxyribonucleaseII (DNaseII):

It is a non-specific endonuclease with optimal activity at acidic pH (4.5-5.5) and conserved from human to C.elegans. It does not require any divalent cation for its activity. DNaseII initially introduces multiple single stranded nicks in DNA backbone and finally generates 3' phosphate groups by hydrolyzing phosphodiester linkages.

This enzyme releases 3' phosphate groups by hydrolyzing phosphodiester linkage and creating nicks in the DNA backbone. DNaseII acts by generating multiple single stranded nicks followed by production of acid soluble nucleotides and oligonucleotides. The catalytic site of the enzyme contains three histidine residues which are essential for enzyme activity.

Some of the common applications of DNase II are as follows:

•  DNA fragmentation

•  Molecular weight marker

•  Cell apoptosis assays etc.

3) Exonuclease III:

Exonuclease III is a globular enzyme which has 3'→5' exonuclease activity in a double stranded DNA. The template DNA should be double stranded and the enzyme does not cleave single stranded DNA. The enzyme shows optimal activity with blunt ended sequences or sequences with 5' overhang.

Exonuclease III enzyme has a bound divalent cation which is essential for enzyme activity. The mechanism of the enzyme can be affected by variation in temperature, monovalent ion concentration in the reaction buffer, and structure and concentration of 3' termini. The enzyme shows optimal activity at 37°C at pH 8.0.

Various application of exonuclease III in molecular cloning experiments are:

•  To generate template for DNA sequencing

•  To generate substrate for DNA labeling experiments

•  Directed mutagenesis

•  DNA-protein interaction assays (to find blockage of exonuclease III activity by protein-DNA binding) etc.