112
gene, requires magnesium and a free 3′-hydroxyl terminus for its action. It cannot
act on DNA strands with terminal 3′-OH containing phosphoryl or acetyl groups.
Inactivation of this enzyme is done by heating it to 80 °C for 15 min. It is mainly
used for the removal of linear ssDNA and oligonucleotides from a heterogeneous
mixture of nucleic acids (Lehman and Nussbaum 1964).
4.8.1.5 Exonuclease III, E. coli
Exonuclease III, from E. coli strain BE 257/pSGR3 is a 28 kDa monomeric enzyme
that has both endonuclease and exonuclease activity on ds DNA. Its endonuclease
activity can create a gap at abasic sites in ds DNA, while as an exonuclease it acts
on blunt end or recessed 3′- recessed termini of ds DNA in 3′ → 5′ direction creating ssDNA segments on the opposite strand. The enzyme is not active on singlestranded DNA, and hence they cannot act on 3′-extensions.
This enzyme also has RNase H activity that degrades RNA strands of DNA-RNA
hybrids (Keller and Crouch 1972) and phosphatase activity that dephosphorylates
DNA strands with 3′- phosphate terminus (Rogers and Weiss 1980). It is relatively
specific for double-stranded DNA and displays sequence dependence (C > A = T >
G) (Demple and Harrison 1994).
Exonuclease III can be used in combination with E. coli Klenow fragment for the
synthesis of strand-specific labeled probes. It can be used along with S1 nuclease in
a step-by-step manner to shorten the length of dsDNA. Other applications include
site-directed mutagenesis and synthesis of ssDNA for dideoxy sequencing.
4.8.1.6 Exonuclease VII, E. coli
Exonuclease VII is an 88-kDa polypeptide, obtained from E. coli K12 strain. It catalyzes the degradation of ssDNA, but cannot act on RNA or DNA-RNA hybrids
(Chase and Richardson 1974). It can degrade ssDNA in both directions and release
5′-phosphomononucleotides in an ATP-independent fashion and hence can be used
to remove single-stranded protruding ends in dsDNA and generate blunt ends. It is
the only ssDNA-specific exonuclease that has bi-directional activity. The degradation occurs progressively with initial release of large, acid-insoluble oligonucleotides, which are subsequently degraded into acid-soluble oligonucleotides.
Exonuclease VII does not require divalent cations for their action and are hence
active even in the presence of chelating agents like EDTA (Ethylenediaminetetraacetic
acid). It can be used in nested PCR after the first PCR reaction to remove singlestranded oligonucleotide primers, prior to the second PCR reaction using another
set of primers. Another application is in exon-intron mapping of genomic DNA.
4.8.1.7 Lambda Exonuclease
Lambda exonuclease is produced by E. coli infected with lambda bacteriophage. It
is an exonuclease that selectively acts on 5′-phosphorylated strand of dsDNA in 5′
→ 3′ direction. The products of its degradation are 5′-mononucleotides and nonhydrolyzed complementary single-stranded DNA (ssDNA). It has only low digestive
activity on ssDNA and nonphosphorylated DNA. Lambda exonuclease is used to
convert dsDNA into single-stranded form for DNA sequencing and single-strand
conformation polymorphism (SSCP). It is used in in-situ 3′-tailing reaction (ISTR)
G. Valsala and S. Sugathan
gene, requires magnesium and a free 3′-hydroxyl terminus for its action. It cannot
act on DNA strands with terminal 3′-OH containing phosphoryl or acetyl groups.
Inactivation of this enzyme is done by heating it to 80 °C for 15 min. It is mainly
used for the removal of linear ssDNA and oligonucleotides from a heterogeneous
mixture of nucleic acids (Lehman and Nussbaum 1964).
4.8.1.5 Exonuclease III, E. coli
Exonuclease III, from E. coli strain BE 257/pSGR3 is a 28 kDa monomeric enzyme
that has both endonuclease and exonuclease activity on ds DNA. Its endonuclease
activity can create a gap at abasic sites in ds DNA, while as an exonuclease it acts
on blunt end or recessed 3′- recessed termini of ds DNA in 3′ → 5′ direction creating ssDNA segments on the opposite strand. The enzyme is not active on singlestranded DNA, and hence they cannot act on 3′-extensions.
This enzyme also has RNase H activity that degrades RNA strands of DNA-RNA
hybrids (Keller and Crouch 1972) and phosphatase activity that dephosphorylates
DNA strands with 3′- phosphate terminus (Rogers and Weiss 1980). It is relatively
specific for double-stranded DNA and displays sequence dependence (C > A = T >
G) (Demple and Harrison 1994).
Exonuclease III can be used in combination with E. coli Klenow fragment for the
synthesis of strand-specific labeled probes. It can be used along with S1 nuclease in
a step-by-step manner to shorten the length of dsDNA. Other applications include
site-directed mutagenesis and synthesis of ssDNA for dideoxy sequencing.
4.8.1.6 Exonuclease VII, E. coli
Exonuclease VII is an 88-kDa polypeptide, obtained from E. coli K12 strain. It catalyzes the degradation of ssDNA, but cannot act on RNA or DNA-RNA hybrids
(Chase and Richardson 1974). It can degrade ssDNA in both directions and release
5′-phosphomononucleotides in an ATP-independent fashion and hence can be used
to remove single-stranded protruding ends in dsDNA and generate blunt ends. It is
the only ssDNA-specific exonuclease that has bi-directional activity. The degradation occurs progressively with initial release of large, acid-insoluble oligonucleotides, which are subsequently degraded into acid-soluble oligonucleotides.
Exonuclease VII does not require divalent cations for their action and are hence
active even in the presence of chelating agents like EDTA (Ethylenediaminetetraacetic
acid). It can be used in nested PCR after the first PCR reaction to remove singlestranded oligonucleotide primers, prior to the second PCR reaction using another
set of primers. Another application is in exon-intron mapping of genomic DNA.
4.8.1.7 Lambda Exonuclease
Lambda exonuclease is produced by E. coli infected with lambda bacteriophage. It
is an exonuclease that selectively acts on 5′-phosphorylated strand of dsDNA in 5′
→ 3′ direction. The products of its degradation are 5′-mononucleotides and nonhydrolyzed complementary single-stranded DNA (ssDNA). It has only low digestive
activity on ssDNA and nonphosphorylated DNA. Lambda exonuclease is used to
convert dsDNA into single-stranded form for DNA sequencing and single-strand
conformation polymorphism (SSCP). It is used in in-situ 3′-tailing reaction (ISTR)
G. Valsala and S. Sugathan