117
low efficiency. S1 nuclease remains active in the presence of denaturing agents but
is sensitive to chelating agents like EDTA (Hofstetter et al. 1976). S1 nuclease is
widely used in molecular biology for the specific removal of single-stranded regions
in double-stranded nucleic acid molecules. It is used in DNA-DNA or DNA-RNA
hybridization studies, S1 transcript mapping for finding transcriptional initiation
sites, removal of single-stranded overhangs in sticky ends prior to DNA ligation and
degradation of hairpin loops. Other applications include studies on DNA-binding
molecules, isolation of inserts from plasmid DNA, DNA palindromic analysis, heteroduplex analysis of PCR products, and structural studies on tRNAs and rRNAs
(Rittié and Perbal 2008).
4.8.4.2 P1 Nuclease
P1 nuclease is a zinc-dependent single-strand specific endonuclease isolated from
Penicillium citrinum that has a molecular weight of 42–50 kDa. It has phosphodiesterase and monoesterase activities and can completely hydrolyze single-stranded
DNA and RNA to yield mononucleoside 5′-monophosphates as the product. The
optimum temperature for this enzyme is about 70 °C, but it is preferable to use
below 60 °C for reactions having long incubation time. P1 nuclease does not cleave
double-stranded DNA or RNA-DNA hybrids in native state. It is used to remove
single-stranded overhanging at the end of double-stranded DNA, in eukaryotic
mRNA cap isolation, in the analysis of nucleic acids base composition, and for
nucleic acid degradation during protein purification (Desai and Shankar 2003).
4.8.4.3 Benzonase
Serratia nuclease, commercially known as “Benzonase,” is a nonspecific endonuclease isolated from S. marcescens which hydrolyzes both single- and doublestranded DNA and RNA. It is a highly processive enzyme which requires Mg
2+
for
the cleavage of phosphodiester bond, releasing 5′-phosphorylated oligonucleotides
(Benedik and Strych 1998). Since it lacks proteolytic activity, it is an ideal tool for
removing nucleic acid contamination from purified proteins. It is also used for the
reduction of bacterial lysate viscosity for downstream steps enhancement.
4.8.4.4 BAL 31 Nuclease
BAL 31 nuclease was first isolated from a marine bacterium which was originally
classified as Pseudomonas BAL 31 and later renamed as genus Alteromonas espejiana Bal 31. Two different molecular types of nucleases have been obtained from this
strain, named as fast (F) and slow (S) Bal 31 nucleases. Both of these enzymes show
exonuclease and endonuclease activities. As an exonuclease, it degrades doublestranded DNA and RNA from both 5′-phosphate and 3′-hydroxyl termini. It shows
endonuclease activity on single-stranded DNA and RNA and cleaves at nicks and
gaps. F- and S-Bal 31 enzymes show identical activity on ssDNA, but on dsDNA,
their reaction speed differs. F-Bal 31 has 20 times faster hydrolytic activity on
dsDNA when compared to S-Bal 31, and this reaction rate is dependent on the C/G
content of the substrate DNA (Talmadge et al. 1980; Kilpatrick et al. 1983). Ca
2+
is
an essential cofactor for its exonuclease and endonuclease activities. This enzyme
4 Enzymes as Molecular Tools
low efficiency. S1 nuclease remains active in the presence of denaturing agents but
is sensitive to chelating agents like EDTA (Hofstetter et al. 1976). S1 nuclease is
widely used in molecular biology for the specific removal of single-stranded regions
in double-stranded nucleic acid molecules. It is used in DNA-DNA or DNA-RNA
hybridization studies, S1 transcript mapping for finding transcriptional initiation
sites, removal of single-stranded overhangs in sticky ends prior to DNA ligation and
degradation of hairpin loops. Other applications include studies on DNA-binding
molecules, isolation of inserts from plasmid DNA, DNA palindromic analysis, heteroduplex analysis of PCR products, and structural studies on tRNAs and rRNAs
(Rittié and Perbal 2008).
4.8.4.2 P1 Nuclease
P1 nuclease is a zinc-dependent single-strand specific endonuclease isolated from
Penicillium citrinum that has a molecular weight of 42–50 kDa. It has phosphodiesterase and monoesterase activities and can completely hydrolyze single-stranded
DNA and RNA to yield mononucleoside 5′-monophosphates as the product. The
optimum temperature for this enzyme is about 70 °C, but it is preferable to use
below 60 °C for reactions having long incubation time. P1 nuclease does not cleave
double-stranded DNA or RNA-DNA hybrids in native state. It is used to remove
single-stranded overhanging at the end of double-stranded DNA, in eukaryotic
mRNA cap isolation, in the analysis of nucleic acids base composition, and for
nucleic acid degradation during protein purification (Desai and Shankar 2003).
4.8.4.3 Benzonase
Serratia nuclease, commercially known as “Benzonase,” is a nonspecific endonuclease isolated from S. marcescens which hydrolyzes both single- and doublestranded DNA and RNA. It is a highly processive enzyme which requires Mg
2+
for
the cleavage of phosphodiester bond, releasing 5′-phosphorylated oligonucleotides
(Benedik and Strych 1998). Since it lacks proteolytic activity, it is an ideal tool for
removing nucleic acid contamination from purified proteins. It is also used for the
reduction of bacterial lysate viscosity for downstream steps enhancement.
4.8.4.4 BAL 31 Nuclease
BAL 31 nuclease was first isolated from a marine bacterium which was originally
classified as Pseudomonas BAL 31 and later renamed as genus Alteromonas espejiana Bal 31. Two different molecular types of nucleases have been obtained from this
strain, named as fast (F) and slow (S) Bal 31 nucleases. Both of these enzymes show
exonuclease and endonuclease activities. As an exonuclease, it degrades doublestranded DNA and RNA from both 5′-phosphate and 3′-hydroxyl termini. It shows
endonuclease activity on single-stranded DNA and RNA and cleaves at nicks and
gaps. F- and S-Bal 31 enzymes show identical activity on ssDNA, but on dsDNA,
their reaction speed differs. F-Bal 31 has 20 times faster hydrolytic activity on
dsDNA when compared to S-Bal 31, and this reaction rate is dependent on the C/G
content of the substrate DNA (Talmadge et al. 1980; Kilpatrick et al. 1983). Ca
2+
is
an essential cofactor for its exonuclease and endonuclease activities. This enzyme
4 Enzymes as Molecular Tools