Restriction endonucleases

1. FSQA 234.17
Restriction Enzymes
Dr. VIMALA BEERA
Assistant Professor
Department of Food Safety & Quality Assurance
Dr.NTR College of Food Science & Technology-Bapatla
Acharya N.G. Ranga Agricultural University
FOOD BIOTECHNOLOGY

2. CONTEN
TSRestriction enzymes
Nomenclature of enzymes
Nature of cutting ends
 Blunt ends
Sticky ends
Iso-schizomers
Recognition sites
 Neo-schizomers
Cleavage
Mechanism of action
Applications of restriction enzymes

3. Term was coined by Lederberg and Meselson (1964)to describe the
nuclease enzymes that destroy any foreign DNA entering the host
cell.
These enzymes are classified into 3 different types
Type I
Type II
Type III
In gene manipulation technology, restriction endonucleases are called
as molecular knives or molecular scissors or molecular scalpels
RESTRICTION ENDONUCLEASES

4. Type I restriction endonuclease recognize of specific nucleoside
sequence and double strand cut in molecule some where near by but
without any specificity as to the nucleotide that is cut.
Type II restriction endonuclease recognize a specific sequence and
make a double bond cut at a fixed point within that sequence have
symmetric recognition sites.
Type III restriction endonucleases recognize a specific sequence
and make a double strand cut at fixed point. Some number of
nucleotide pairs to the side of that sequence have symmetric
recognition sites.

5.  A system based on proposal of Smith and Nathans has been
followed for the most part. This includes:
 Each enzyme is named by three-letter code
 First letter of this code is derived from the first epithet of the
genus name
 The second and third letters are from species name.
 This is followed by strain name.If particular strain has more than
one restriction enzyme,these will be identified by roman numerals as
I,II,III,etc.
A general name endonuclease R may be added.
For example ECORI
E=genera; CO=species; RI=strain designation
NOMENCLATURE OF RESTRICTION
ENDONUCLEASES

7. The restriction enzymes cut DNA molecule by cleavage. This action
occurs in two styles.
1.Blunt-end style
2.Sticky or cohesive end style
Blunt-end style
Certain restriction endonucleases (eg.Hae III,Sma I) make cuts
across both strands of DNA at same position.
The utility of this style during the joining of DNA fragments is that
any pair of ends may be joined together irrespective of sequence.
This is especially useful for those researchers who are interested in
join two defined sequences without introducing any additional
material between them.
NATURE OF CUT ENDS

8. •In this style restriction enzymes (eg.EcoRI,Bam HI,Hind III) make
single strand cut.
Some nucleotide pairs apart in the opposite strands of DNA, and
so generate fragments with protrubing termini(ends).
These DNA fragments can associate by hydrogen bonding
between overlapping termini,or the fragments can circularize by
intramolecular reaction and for this reason the fragments are said to
have sticky ends
Sticky or cohesive end style

9. Isoschizomers are the restriction enzymes which are isolated
from different organism but recognize identical base sequences in
DNA.
Example:Asp 718 &Kpnn I have identical recognition.
G G T A C C
C C A T G G
Source of Asp 718 is achromobacter species.
Source of Kpn I is Klebsiella pneumonia
Isoschizomers
Neoschizomers
A restriction enzyme that recognizes the same sequence but cuts
it differently.
Example:Sma I, Xam I

10. Each restriction enzyme recognizes a unique sequence of 4 or 6
groups of nucleotide and some even recognize group of 18
nucleotides
Recognized sequence may be continuous or interrupted
Recognition segment present in one DNA can be absent in other
DNA
ECOR reconition site is GAATTC,this occur only once in SV
40(SV 40 genome 1 has 5243bp)
Hac 111 recognition site is GGCC and occurs 18 times
RECOGNITION SITES

12. Type II restriction enzymes has site of cleavage within the
recognition site cut can be
(i)Blunt end or flash end
(ii)Staggered ends or sticky ends
Blunt end
Hpal GTT ACC GTT + AAC
CAA TTG CAA TTG
Staggered end
Bam N1 CGA TCC G+ GATTCG
CAA TTG CCTAG
Cleavage

13. 1. Restriction enzymes are dimmers and bind to both the strands
at recognition sites.
2. Polypeptide subunits of ECOR,wraps itself on the double
helix making complement attachment at major groove.
3. The central kink unwinds on binding of ECOR.
4. Unwinding is by 25º and bends dna 12º.The process result in
enlargement of major groove.
MECHANISM OF ACTION

14. Fig. 17.1 Steps in formation of recombinant DNA by the action of
restriction endonuclease enzyme - ECoRI

15. In recombinant DNA technology studies enzymes are used
For sequence analysis of DNA
For restriction mapping
For making chimeric DNA
Short gun experiment
Chromosome walking
Site specific mutagenesis
Uses of restriction enzymes