111
4.8.1.1 Deoxyribonuclease I
Deoxyribonuclease I (DNase I) obtained from bovine pancreas was the first DNase
to be isolated. This endonuclease cuts DNA nonspecifically and generates di-, tri-,
and oligonucleotides with 5′-phosphate and 3′-hydroxyl ends as products. Earlier,
this enzyme was obtained directly from pancreatic tissues, but now it is produced by
over expression in E. coli containing the bovine pancreatic DNase I gene. This 31
kDa glycoprotein cleaves ssDNA, dsDNA, chromatin, and RNA- DNA hybrids.
However, lysis of ssDNA requires high concentration of enzyme (Vanecko and
Laskowski 1961). DNase I has been commercially available for more than 25 years,
and is used for the complete degradation of unwanted DNA in protein and RNA
samples. RNA samples can be treated with this enzyme prior to RT-PCR to remove
any contaminant DNA. It is also used in partial hydrolysis of DNA molecule for
nick translation, random fragmentation of DNA for dideoxy sequencing, in DNAprotein interaction analysis for DNase foot printing and to eliminate the DNA template after in vitro transcription for mRNA production. The cleavage action of this
enzyme requires Ca
2+
, and its activation needs Mg
2+
or Mn
2+
ions. DNase I cleaves
each strand of dsDNA separately and randomly when Mg
2+
is present, while both
DNA strands are cleaved roughly at the same site in the presence of Mn
2+
. DNase I
can be eliminated from a reaction mixture, by heating the mixture with 5 mM EGTA
(Ethylene Glycol Tetraacetic Acid) to 75 °C for 5 min (Huang et al. 1996).
4.8.1.2 Endonuclease IV, E. coli
Endonuclease IV isolated from E. coli is a 30 kDa Zn
2+
-dependent endonuclease
that identifies apurinic/apyrimidinic (AP) or abasic sites in dsDNA and hydrolyze
phosphate group present 5′ to the abasic site generating a 3′-OH on one terminus
and a deoxyribose 5′-phosphate at the 5′ end. It also has 3′ → 5′ exonuclease activity and 3′-diesterase activity that can generate 3′-phosphoglycolate or 3′-phosphate
from the 3′ end of dsDNA (Levin et al. 1988). Endonuclease IV is produced by over
expression in E. coli cloned with nfo gene. It has applications in DNA damage and
repair studies, comet assay, analysis of DNA structure, SNP genotyping, and in in
vivo studies on the effects of antitumor drugs on nucleic acids (Hosfield et al. 1999).
4.8.1.3 Endonuclease V, T. maritima
Endonuclease V obtained from the thermophilic bacterium Thermotoga maritima, is a
thermostable endonuclease involved in DNA repair. It can cleave the phosphodiester
bond present on the 3′-side, 1 nucleotide away from a deaminated base lesion and
remove deaminated bases from defective DNA. Inosine (I), a product of adenosine
deamination, xanthosine and oxanosine derived from guanosine deamination, and uridine from cytidine deamination act as substrates for endonuclease V (Feng et al. 2006).
This 24.9 kDa enzyme has an optimal temperature range from 65 to 70 °C and is used
in mutation research, DNA repair studies, mismatch cleavage, and genotyping.
4.8.1.4 Exonuclease I, E. coli
Exonuclease I of E. coli is a 3′ → 5′ exonuclease that acts specifically on singlestranded DNA releasing deoxyribonucleoside 5′-monophosphates (dNMP) sequentially, leaving 5′-terminal dinucleotides unaltered. This enzyme, coded by SbcB
4 Enzymes as Molecular Tools
4.8.1.1 Deoxyribonuclease I
Deoxyribonuclease I (DNase I) obtained from bovine pancreas was the first DNase
to be isolated. This endonuclease cuts DNA nonspecifically and generates di-, tri-,
and oligonucleotides with 5′-phosphate and 3′-hydroxyl ends as products. Earlier,
this enzyme was obtained directly from pancreatic tissues, but now it is produced by
over expression in E. coli containing the bovine pancreatic DNase I gene. This 31
kDa glycoprotein cleaves ssDNA, dsDNA, chromatin, and RNA- DNA hybrids.
However, lysis of ssDNA requires high concentration of enzyme (Vanecko and
Laskowski 1961). DNase I has been commercially available for more than 25 years,
and is used for the complete degradation of unwanted DNA in protein and RNA
samples. RNA samples can be treated with this enzyme prior to RT-PCR to remove
any contaminant DNA. It is also used in partial hydrolysis of DNA molecule for
nick translation, random fragmentation of DNA for dideoxy sequencing, in DNAprotein interaction analysis for DNase foot printing and to eliminate the DNA template after in vitro transcription for mRNA production. The cleavage action of this
enzyme requires Ca
2+
, and its activation needs Mg
2+
or Mn
2+
ions. DNase I cleaves
each strand of dsDNA separately and randomly when Mg
2+
is present, while both
DNA strands are cleaved roughly at the same site in the presence of Mn
2+
. DNase I
can be eliminated from a reaction mixture, by heating the mixture with 5 mM EGTA
(Ethylene Glycol Tetraacetic Acid) to 75 °C for 5 min (Huang et al. 1996).
4.8.1.2 Endonuclease IV, E. coli
Endonuclease IV isolated from E. coli is a 30 kDa Zn
2+
-dependent endonuclease
that identifies apurinic/apyrimidinic (AP) or abasic sites in dsDNA and hydrolyze
phosphate group present 5′ to the abasic site generating a 3′-OH on one terminus
and a deoxyribose 5′-phosphate at the 5′ end. It also has 3′ → 5′ exonuclease activity and 3′-diesterase activity that can generate 3′-phosphoglycolate or 3′-phosphate
from the 3′ end of dsDNA (Levin et al. 1988). Endonuclease IV is produced by over
expression in E. coli cloned with nfo gene. It has applications in DNA damage and
repair studies, comet assay, analysis of DNA structure, SNP genotyping, and in in
vivo studies on the effects of antitumor drugs on nucleic acids (Hosfield et al. 1999).
4.8.1.3 Endonuclease V, T. maritima
Endonuclease V obtained from the thermophilic bacterium Thermotoga maritima, is a
thermostable endonuclease involved in DNA repair. It can cleave the phosphodiester
bond present on the 3′-side, 1 nucleotide away from a deaminated base lesion and
remove deaminated bases from defective DNA. Inosine (I), a product of adenosine
deamination, xanthosine and oxanosine derived from guanosine deamination, and uridine from cytidine deamination act as substrates for endonuclease V (Feng et al. 2006).
This 24.9 kDa enzyme has an optimal temperature range from 65 to 70 °C and is used
in mutation research, DNA repair studies, mismatch cleavage, and genotyping.
4.8.1.4 Exonuclease I, E. coli
Exonuclease I of E. coli is a 3′ → 5′ exonuclease that acts specifically on singlestranded DNA releasing deoxyribonucleoside 5′-monophosphates (dNMP) sequentially, leaving 5′-terminal dinucleotides unaltered. This enzyme, coded by SbcB
4 Enzymes as Molecular Tools