NUCLEASES
by
Sirisha
NUCLEASES
• When nucleic acids need to be cut randomly or
sometimes partially digested into approximate sizes,
endonucleases are not useful.
• There are many nucleases with specific digesting
properties that are found in vivo and also isolated for
in vitro investigations.
1. Nuclease BAL 31
2. Nuclease S1
3. Mung-bean nuclease
4. Ribonucleases
a) Ribonuclease A:
b) Ribonuclease T1:
5. Deoxyribonuclease 1
6. Exonuclease III
1. Nuclease BAL 31
• BAL 31 is predominantly a 3’ exonuclease
that removes mononucleotides from both 3’
termini of the two strands of linear DNA.
• BAL31 is also an endonuclease; thus, the
single-stranded DNA generated by the 3’-
exonuclease activity is degraded by the
endonuclease.
Cont’d…
• The degradation is absolutely dependent on the
presence of calcium, and the reaction can therefore
be stopped at different stages by the addition of the
chelating agent EGTA.
• Because degradation occurs relatively uniformly
from the termini of DNA, digestion with Bal31 can
be used to map restriction sites in small fragments
of DNA.
• Bal 31 can also be used to remove unwanted
sequences from the termini of DNAs before cloning.
2. Nuclease S1
• Nuclease S1 degrades single-stranded DNA or RNA to yield
5’ phosphate mono- or oligonucleotides.
• Double-stranded DNA, double-stranded RNA, and DNA-RNA
hybrids are relatively resistant to the enzyme.
• However, double-stranded nucleic acids are digested
completely by nuclease S1 if they are exposed to the enzyme
at very high concentrations.
• This enzyme is mainly used for analyzing the structure of
DNA-RNA hybrids, for removing single-stranded tails from
DNA fragments to produce blunt ends, and for opening the
hairpin loop generated during synthesis of double-stranded
cDNA.
3. Mung-bean nuclease
• Mung-bean nuclease degrades single-stranded DNA to mono-
or oligonucleotides with phosphate groups at their 5’ termini.
• Double-stranded DNA, double-stranded RNA, and DNA-RNA
hybrids are relatively resistant to the enzyme.
• Although mung-bean nuclease and nuclease S1 are similar to
each other in their physical and catalytic properties, mung-
bean nuclease may be less severe in its action than the S1
nuclease.
• This enzyme is primarily used to convert protruding termini of
DNA to blunt ends.
4. Ribonucleases
(a) Ribonucleases A:
• RNase A is an endo ribonuclease that specifically
attacks single-stranded RNA 3’ to form pyrimidine
residues and cleaves the phosphate linkage to the
adjacent nucleotide.
• The end products are pyrimidine 3’ phosphates. RNase
is primarily used for removing un-hybridized regions of
RNA from DNA-RNA hybrids and for mapping single-
base mutations in DNA or RNA.
• In this method, single-base mismatches in RNA-DNA or
RNA-RNA hybrids are recognized and cleaved by
RNase A.
(b) Ribonuclease T1
• RNase T1 is an endoribonuclease that specifically
attacks the 3’-phosphate groups of guanine
nucleotides and cleaves the 5’ phosphate linkage to
the adjacent nucleotide.
• The end products are guanosine 3’ phosphate and
oligonucleotides with terminal guanosine 3’-
phosphate groups.
Cont’d…
5’pApGp’Gp’CpCp Gp’ApApGp’UpGp’CpApGp’C3’
• Reaction with Rnase T1 gives
5’pApGp + Gp + CpCpGp + ApApGp + UpGp + CpApGp + C3’
• Ribonuclease T1 is also primarily used for
removing un-hybridized regions of RNA from
DNA-RNA hybrids.
5. Deoxyribonuclease 1
• DNase is an endo nuclease that hydrolyses ss- or
dsDNA preferentially at sites adjacent to pyrimidine
nucleotides.
• Products are complex mixtures of 5’-phosphate
mono- and oligonucleotides.
• In the presence of Mg2+, DNase 1 attacks each
strand of DNA independently, and the sites of
cleavage are distributed in a statistically random
fashion.
DNase 1 is used for:
(i) introducing random nicks into closed dsDNAs in
preparation for radio labeling by nick translation;
(ii) introducing a single nick into closed circular DNAs
in preparation for restriction prior to bisulfate-
mediated mutagenesis;
(iii) generating random clones for sequencing in
bacteriophage M13 vectors;
(iv) analysis of protein-DNA complexes (DNase foot-
printing).
6. Exonuclease III
• Exonuclease III, catalyses the stepwise removal of 5’
mononucleotides only from the 3’-hydroxyl termini of
dsDNA.
• Linear double-stranded and circular DNA containing
nicks or gaps are the substrate.
• The activity of the enzyme results in the formation of
long single-stranded regions in dsDNA.
• The enzyme also carries out three activities:
• an endonuclease specific for apurinic DNA, an RNase
H activity, and a 3’phosphatase activity, which
removes 3’ phosphate termini.
cont’d…
• The uses are;
(i) generating partially restricted DNAs that can be
used as substrates for the Klenow fragment of E.
coli DNA polymerase I;
(ii) generating nested sets of deletions of the terminal
sequences of linear dsDNAs.
• This reaction is generally carried out in conjunction
with mung-bean nuclease or nuclease S1 and is an
alternative to using BAL 31.
Cont’d…
• Some methods of site-specific mutagenesis use
thiophosphate derivatives of the dNTPs for second-
strand synthesis primed by the mutagenic primer.
• The parental template strand can be preferentially
degraded with exonuclease III, increasing the
frequency of mutants obtained upon transformation
of E. coli, since exonuclease III will not cleave
thioester bonds.
