Biotechnology Engineering

1. RESTRICTION
ENDONUCLEASES
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Lecture- 9

2. RESTRICTION ENDONUCLEASES
Prokaryotic nucleases that recognize
specific DNA sequences and cleave
sugar-phosphate backbone, either
within or close to recognition site.
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3. 3
•These enzymes are produced by bacteria and archaea for defending themselves
against the foreign DNA molecules.
•Restriction enzymes recognize specific sequences and make cuts in the DNA
molecules. Stuart Linn and Werner Arber in 1968 showed in vitro restriction of fd
phage DNA by an E. coli cell extract.
•
•Soon after this discovery, Hamilton Smith and Kent Wilcox in the year 1970 isolated
the first restriction enzyme, endonuclease R (later renamed as HindII), from
Hemophilus influenzae.
• In the subsequent year Kathleen Dana and Daniel Nathans pioneered the
applications of restriction enzymes by showing specific cleavage of SV40 DNA by
HindII.
•Werner Arber, Hamilton Smith and Daniel Nathans were awarded Nobel Prize in
Physiology or Medicine in 1978 for the discovery and applications of restriction
enzymes.

4. 4
•After the isolation of HindII in 1970, many other
restriction enzymes have been isolated from several
bacterial and archaeal sources.
•Comprehensive information about all the
characterized restriction enzymes is available in the
database known as REBASE given by R. J. Roberts
and his colleagues in 2007.
•As per this database, 3805 restriction enzymes
have been biochemically or genetically
characterized till now.

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•Restriction enzymes have been classified into four major
types known as Type I, Type II, Type III and Type IV,
depending on their composition, cofactor requirement,
target sequence, position of cleavage site, etc. Among
these, Type II enzymes have separate enzymes for
endonuclease and methylase activities.
•A Type II enzyme cuts at or near its recognition
sequence. Hence, Type II enzymes have been found to be
most useful in recombinant DNA technology.
•Type IV recognize modified, typically methylated DNA
•As per the REBASE database, 611 Type II restriction
enzymes are commercially available.

6. Werner Arber
(Jun 3, 1929 - )
Nobel Prize in Physiology or Medicine 1978
showed
in vitro restriction of
fd phage DNA
by E. coli extract
1968
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7. 1970
Isolated and characterized
the first restriction enzyme
Endonuclease R
( later renamed HindII )
Hamilton O. Smith
(Aug 23, 1931-)
Nobel Prize in Physiology or Medicine 1978
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8. 1971
Daniel Nathans
(Oct 30, 1928 – Nov 16, 1999)
Pioneered the application of
restriction enzymes :
Specific cleavage of SV 40 DNA
Nobel Prize in Physiology or Medicine 1978
Kathleen Janet Danna
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9. TYPES OF RESTRICTION ENDONUCLEASES
Type I : EcoK , EcoB
Type II: EcoRI , EcoRV
Type III: EcoPI , EcoP15
Type IV: McrBC, Mrr
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• On cutting DNA at specific sequences, some of the Type II
enzymes produce blunt ends. The cut sites for Type II enzyme SmaI
have been shown below:
5′ C C C↓G G G 3′
3′ G G G↑C C C 5′
• Some of the Type II enzymes produce cohesive ends. The
cohesive ends may have 5′ overhangs as has been shown below in
case of BamHI enzyme.
5′ G↓G A T C C 3′
3′ C C T A G↑G 5′
• The cohesive ends produced by some of the Type II restriction
enzymes have 3′ overhangs as given below in case of PstI enzyme:
5′ C T G C A↓G 3′
3′ G↑A C G T C 5′

11. PROPERTY TYPEI TYPEII TYPEIII
Enzyme activities and
structure
Single multisubunit,
having both
endonuclease and
methylase functions,
Large size
Separate
endonuclease and
methylase enzymes,
Small size
Separate subunits
for restriction and
methylation
Recognition
Sequence
Assymetric
Bipartite
Palindromic with
dyad symmetry
Assymetric
Unipartite
Recognition site
length
Long, 8-16bp Short, 4-9bp Short, 5-7bp
Methylation site Recognition Site Recognition Site Recognition Site
Requirements for
Restriction
ATP , Mg2+ , SAM Mg2+ ATP , Mg2+ ,
SAM (optional)
Cleavage Site Random
1000bp away from
Recognition Site
At or Near
Recognition Site
≈25bp downstream
of Recognition Site
Examples EcoK , EcoB EcoRI , EcoRV EcoPI , EcoP15
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12. TYPES OF RESTRICTION SITES
PALINDROMIC RECOGNITION SEQUENCES
Sequences having two-fold axis of dyad symmetry.
i.e. PHRASES THAT READ THE SAME BACKWARDS AS
FORWARD ,
Madam I’m Adam
Continuous : two half-sites of recognition sequence are
adjacent.
e.g. 5’ G AATTC 3’
Discontinuous : two half-sites are seperated or interrupted.
e.g. 5’ GCCNNNN NGGC 3’
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13. Uni / Bipartite Recognition Sequence
An interrupted, non-palindromic recognition sequence
5’ ATGCCNNNNNNNNNTAGCG 3’
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14. CLASSIFICATION ON THE BASIS OF LENGTH OF RECOGNITION
SEQUENCE or FRAGMENT SIZE
• Frequency of occurrence of any recognition sequence in
the random sequence of DNA is given by – 1/4n
where, n= length (in bp) of recognition sequence.
• for e.g. a 4 base cutter that recognizes a tetranucleotide
recognition sequence, would cleave DNA every 44=256bp.
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15. CLASSIFICATION ON THE BASIS OF TYPES OF ENDS GENERATED
BLUNT-END CUTTERS- Endonucleases cut straight across the
axis of symmetry of restriction site.
e.g. SmaI
5’
3’
3’
5’
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16. STICKY-END CUTTERS- Endonucleases cut DNA off the center
of restriction site, but between two same bases on opposite
strands.
5’ Overhang – e.g. EcoRI 3’ Overhang – e.g. PstI
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