116
4.8.3.2 Ribonuclease H
Ribonuclease H (RNase H) can specifically hydrolyze RNA in RNA-DNA duplex
substrate with 3′-OH and 5′-P-terminated products. This endonuclease does not act
on single-stranded and double-stranded DNA or RNA. RNases H was first isolated
from calf thymus in 1969 (Stein and Hausen 1969). Commercially available RNase
H is produced from E. coli strain that has been cloned and over expressed with
RNase H gene (rnh) from E. coli. It is used in molecular biology to degrade RNA
template from RNA-DNA hybrids produced after first-strand cDNA synthesis by
reverse transcription, for poly(A) tail removal from mRNA linked to oligo(dT), for
selective degradation of noncoding RNA inside or outside the living cell and also in
procedures like nuclease protection assays. RNase H requires Mg
2+
ions or Mn
2+
ions and hence can be inhibited using a chelator like EDTA (Goodwin and Rottman
1992).
4.8.3.3 Ribonuclease T 1
Ribonuclease T 1 (RNase T 1 ) is a 11 kDa monomeric endoribonuclease that specifically cleaves the phosphodiester bond in ssRNA (or deaminated RNA) after guanine
residues. It acts between the 3′ end of guanine residues and 5′-hydroxy residue of
adjacent nucleotides, producing corresponding 2′,3′-cyclic phosphate as an intermediate (Takahashi 1966). It does not require any metal ions for its activity. RNase
T 1 was first discovered in a commercial enzyme mixture from the mold Aspergillus
oryzae called Takadiastase. The RNase T1 is now commercially synthesized by
over expression in E. coli strain cloned with Ribonuclease T1 gene of A. oryzae
(Fujimura et al. 1990). Due to the specificity of RNase T 1 for guanine, it is used to
digest denatured RNA prior to sequencing, in RNA mapping and RNA structure
studies. It can also be used for removing RNA from DNA samples or recombinant
protein preparations, in the synthesis of nucleoside 2′,3′-cyclic phosphates and oligonucleotides (Mohr and Thach 1969).
4.8.4 Sugar Nonspecific Nucleases
As the name implies, they have the ability to hydrolyze nucleic acids with ribose
(RNA) or deoxyribose (DNA) sugars. They do not act on specific sequences, but
can recognize different nucleic acid structures. This property has been utilized in
studying different nucleic acid structures and their interactions with intercalating
molecules. Of about 30 sugar nonspecific nucleases isolated so far, many show
multiple activities and metal ion requirement.
4.8.4.1 S1 Nuclease
S1 nuclease is a single-strand endonuclease of 32 kDa size, isolated from Aspergillus
oryzae. It specifically hydrolyzes ssRNA or ssDNA into 5′-phosphoryl mono- or
oligonucleotides, but does not act on double-stranded nucleic acids (dsDNA,
dsRNA, or RNA-DNA hybrids). This enzyme is stable at 65 °C, and its activity
requires Zn
2+
as a cofactor. Zn
2+
can be replaced by Co
2+
and Hg
2+
, which show only
G. Valsala and S. Sugathan
4.8.3.2 Ribonuclease H
Ribonuclease H (RNase H) can specifically hydrolyze RNA in RNA-DNA duplex
substrate with 3′-OH and 5′-P-terminated products. This endonuclease does not act
on single-stranded and double-stranded DNA or RNA. RNases H was first isolated
from calf thymus in 1969 (Stein and Hausen 1969). Commercially available RNase
H is produced from E. coli strain that has been cloned and over expressed with
RNase H gene (rnh) from E. coli. It is used in molecular biology to degrade RNA
template from RNA-DNA hybrids produced after first-strand cDNA synthesis by
reverse transcription, for poly(A) tail removal from mRNA linked to oligo(dT), for
selective degradation of noncoding RNA inside or outside the living cell and also in
procedures like nuclease protection assays. RNase H requires Mg
2+
ions or Mn
2+
ions and hence can be inhibited using a chelator like EDTA (Goodwin and Rottman
1992).
4.8.3.3 Ribonuclease T 1
Ribonuclease T 1 (RNase T 1 ) is a 11 kDa monomeric endoribonuclease that specifically cleaves the phosphodiester bond in ssRNA (or deaminated RNA) after guanine
residues. It acts between the 3′ end of guanine residues and 5′-hydroxy residue of
adjacent nucleotides, producing corresponding 2′,3′-cyclic phosphate as an intermediate (Takahashi 1966). It does not require any metal ions for its activity. RNase
T 1 was first discovered in a commercial enzyme mixture from the mold Aspergillus
oryzae called Takadiastase. The RNase T1 is now commercially synthesized by
over expression in E. coli strain cloned with Ribonuclease T1 gene of A. oryzae
(Fujimura et al. 1990). Due to the specificity of RNase T 1 for guanine, it is used to
digest denatured RNA prior to sequencing, in RNA mapping and RNA structure
studies. It can also be used for removing RNA from DNA samples or recombinant
protein preparations, in the synthesis of nucleoside 2′,3′-cyclic phosphates and oligonucleotides (Mohr and Thach 1969).
4.8.4 Sugar Nonspecific Nucleases
As the name implies, they have the ability to hydrolyze nucleic acids with ribose
(RNA) or deoxyribose (DNA) sugars. They do not act on specific sequences, but
can recognize different nucleic acid structures. This property has been utilized in
studying different nucleic acid structures and their interactions with intercalating
molecules. Of about 30 sugar nonspecific nucleases isolated so far, many show
multiple activities and metal ion requirement.
4.8.4.1 S1 Nuclease
S1 nuclease is a single-strand endonuclease of 32 kDa size, isolated from Aspergillus
oryzae. It specifically hydrolyzes ssRNA or ssDNA into 5′-phosphoryl mono- or
oligonucleotides, but does not act on double-stranded nucleic acids (dsDNA,
dsRNA, or RNA-DNA hybrids). This enzyme is stable at 65 °C, and its activity
requires Zn
2+
as a cofactor. Zn
2+
can be replaced by Co
2+
and Hg
2+
, which show only
G. Valsala and S. Sugathan