Nucleases: DNase and RNase

Nucleases: DNase and RNase

Most of the time nucleases are the enemy of the molecular biologist who is trying to preserve the integrity of RNA or DNA samples. However, deoxyribonucleases (DNases) and ribonucleases (RNases) have certain indispensible roles in molecular biology laboratories.

Numerous types of DNase and RNase have been isolated and characterized. They differ among other things in substrate specificity, cofactor requirements, and whether they cleave nucleic acids internally (endonucleases), chew in from the ends (exonucleases) or attack in both of these modes. In many cases, the substrate specificity of a nuclease depends upon the concentration of enzyme used in the reaction, with high concentrations promoting less specific cleavages.

The most widely used nucleases are DNase I and RNase A, both of which are purified from bovine pancreas:

Deoxyribonuclease I cleaves double-stranded or single stranded DNA. Cleavage preferentially occurs adjacent to pyrimidine (C or T) residues, and the enzyme is therefore an endonuclease. Major products are 5′-phosphorylated di, tri and tetranucleotides.

In the presence of magnesium ions, DNase I hydrolyzes each strand of duplex DNA independently, generating random cleavages. In the presence of manganese ions, the enzyme cleaves both strands of DNA at approximately the same site, producing blunt ends or fragments with 1-2 base overhangs. DNase I does not cleave RNA, but crude preparations of the enzyme are contaminated with RNase A; RNase-free DNase I is readily available.

Some of the common applications of DNase I are:

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

Ribonuclease A is an endoribonuclease that cleaves single-stranded RNA at the 3′ end of pyrimidine residues. It degrades the RNA into 3′-phosphorylated mononucleotides and oligonucleotides.

Some of the major use of RNase A are:

  • Eliminating or reducing RNA contamination in preparations of plasmid DNA.
  • Mapping mutations in DNA or RNA by mismatch cleavage. RNase will cleave the RNA in RNA:DNA hybrids at sites of single base mismatches, and the cleavage products can be analyzed.

A number of other nucleases that are used to manipulate DNA and RNA are described in the following table:

Nuclease and Source Substrates, Activity and Uses
Exonuclease III
(E. coli)
Removes mononucleotides from the 3′ termini of duplex DNA. The preferred substrates are DNAs with blunt or 5′ protruding ends. It will also extend nicks in duplex DNA to create single-stranded gaps. In works inefficiently on DNA with 3′ protruding ends, and is inactive on single-stranded DNA.

Used most commonly to prepare a set of nested deletions of the termini of linear DNA fragments.

Mung Bean Nuclease
(Mung bean sprouts)
Digests single-stranded DNA to 5′-phosphorylated mono or oligonucleotides. High concentrations of enzyme will also degrade double-stranded nucleic acids.

Used to remove single-stranded extensions from DNA to produce blunt ends.

Nuclease BAL 31
Functions as an exonuclease to digest both 5′ and 3′ ends of double-stranded DNA. It also acts as a single-stranded endonuclease that cleaves DNA at nicks, gaps and single stranded regions. Does not cleave internally in duplex DNA.

Used for shortening fragments of DNA at both ends.

Nuclease S1
The substrate depends on the amount of enzyme used. Low concentrations of S1 nuclease digests single-stranded DNAs or RNAs, while double-stranded nucleic acids (DNA:DNA, DNA:RNA and RNA:RNA) are degraded by large concentrations of enzyme. Moderate concentrations can be used to digest double-stranded DNA at nicks or small gaps.

Used commonly to analyze the structure of DNA:RNA hybrids (S1 nuclease mapping), and to remove single-stranded extensions from DNA to produce blunt ends.

Ribonuclease T1
An endonuclease that cleaves RNA at 3′ phosphates of guanine residues, producing oligonucleotides terminal guanosine 3′ phosphates.

Used to remove unannealed regions of RNA from DNA:RNA hybrids.

Back to the index of Restriction Endonucleases and DNA Modifying Enzymes

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