2-2.2 Deoxyribonuclease (DNase):

•  A nuclease enzyme that can catalyze the hydrolytic cleavage of phosphodiester bonds in the DNA backbone are known as deoxyribonuclease (DNase).

•  Based on the position of action, these enzymes are broadly classified as endodeoxyribonuclease (cleave DNA sequence internally) and exodeoxyribonuclease (cleave the terminal nucleotides).

•  Unlike restriction enzymes, DNase does not have any specific recognition/restriction site and cleave DNA sequence at random locations.

•  There is a wide variety of deoxyribonucleases known which have different substrate specificities, chemical mechanisms, and biological functions. They are:

1) Deoxyribonuclease I (DNase I):

An endonuclease which cleaves double-stranded DNA or single stranded DNA. The cleavage preferentially occurs adjacent to pyrimidine (C or T) residues. The major products are 5'-phosphorylated bi-, tri- and tetranucleotides. It requires divalent ions (Ca²⁺ and Mn²⁺ /Mg²⁺) for its activity and creates blunt ends or 1-2 overhang sequences.

DNaseI is the most widely used enzyme in cloning experiments to remove DNA contamination from mRNA preparation (to be used for cDNA library preparation, northern hybridization, RT-PCR etc). The mode of action of DNaseI varies according to the divalent cation used.

In the presence of magnesium ions (Mg⁺²), DNaseI hydrolyzes each strand of duplex DNA producing single stranded nicks in the DNA backbone, generating various random cleavages.

On the other hand, in the presence of manganese ions (Mn⁺²), DNaseI cleaves both strands of a double stranded DNA at approximately the same site, producing blunt ended DNA fragments or with 1-2 base overhangs. The two major DNases found in metazoans are: deoxyribonuclease I and deoxyribonuclease II.

 

Fig 2-2.2: Action of DNase I in the presence of Mg⁺² and Mn⁺² ions. (Arrowhead denoting random site of cleavage in double stranded DNA by DNase I)