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NUCLEASES

The document describes several nucleases that are used to cut or digest nucleic acids, including endonucleases that cut internally such as Bal31, exonucleases that remove nucleotides from ends like exonuclease III, and ribonucleases that act on RNA such as RNase A. These nucleases have specific activities and produce defined products that allow them to be used for applications like mapping restriction sites, removing unwanted DNA sequences, and analyzing nucleic acid structure.

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Ravi Sankar
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27 views

NUCLEASES

The document describes several nucleases that are used to cut or digest nucleic acids, including endonucleases that cut internally such as Bal31, exonucleases that remove nucleotides from ends like exonuclease III, and ribonucleases that act on RNA such as RNase A. These nucleases have specific activities and produce defined products that allow them to be used for applications like mapping restriction sites, removing unwanted DNA sequences, and analyzing nucleic acid structure.

Uploaded by

Ravi Sankar
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPT, PDF, TXT or read online on Scribd
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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.

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