Restriction enzymes are proteins isolated from bacteria that cleave DNA into fragments at specific recognition sites. They recognize short palindromic nucleotide sequences, usually 4-8 bases long, and cut through both strands of the DNA double helix. There are four main types of restriction enzymes that differ in their recognition sites and cleavage activities. Restriction enzymes are indispensable tools in recombinant DNA technology, as they allow DNA fragments to be cut and recombined in new configurations.

1. Restriction Enzymes
Presented by
Devi Lekshmi S
M.Sc Plant Biotechnology

2. Restriction Enzymes
A protein isolated from bacteria
Also called restriction endonuclease/ restrictase
Cleaves DNA into fragments at or near specific recognition sites with a
known sequence at each end
Recognition and cleavage site may be same or different
Cut through both strands of double DNA helix
Indispensible tool of recombinant DNA technology
Evolved from a common ancestor, and wide spread via horizontal gene
transfer

4. Recognition site
A specific sequence of nucleotide, usually 4 to 8 bases
Randomly distributed throughout the DNA
Many of them are palindromic in sequences
(read same both backward and forward)
1. Mirror like palindrome
eg. GTAATG (Similar from backward and forward in a
single strand)
2. Inverted palindrome
eg.
Same forward and backward in a complimentary strand
Have greater biological importance than mirror like
palindrome

5. Restriction digestion
After recognizes the sequence, restriction enzymes snips
through the DNA
Catalyzing the hydrolysis of the bond b/t adjacent
nucleotides
Hydrolysis – splitting of a chemical bond by addition of a
water molecule
Bacteria - Restriction modification system exists
Methylase enzyme add methyl group (-CH3) to adenine or
cytosine bases within the recognition sequences, which
modifies and protects the host DNA

6. Types of cut made by restriction enzymes
 Sticky ends (overhanging) are more desired than blunt
ends
 Sticky ends eg. EcoR1, HindIII
 Blunt ends eg. EcoRV, SmaI

7. Types of recognition enzymes
Type I
• First to be identified
• Cut at a site that differs, a random distance of at least 1000 bp
away
• Cleavage site remote from recognition site
• Requires both ATP and S-adenosyl-L-methionine
• Multifunctional (Restriction digestion and methylase activity)
Type II
• Cleave within or short specific distance from restriction sites
• Requires Mg
• Only restriction digestion no methylase activity

8. Types of recognition enzymes….
Type III
Type I and III are similar both restriction and methylase
activities
Type IV
Enzyme targeted modified DNA
Cleave only methylated DNA
Show weak sequence specificity
Eg. Methylated, Hydroxy methylated, Glucosyl-hydroxy
methylated
Type V
Utilize guide RNAs (gRNAs)

9. Nomenclature of Restriction enzymes
EcoRI (pronouncd “eco R one”)
E- Escherichia (Genus)
co- coli (Species)
R- RY13 (Strain)
I- First identified (Order of identification in the bacterium)

10. History
Restriction enzymes were discovered and characterized in
the late 1960s and early 1970s by molecular biologists
1. Werner Arber
2. Hamilton O Smith
3. Daniel Nathans

11. Applications
To assist insertion of genes into plasmid vectors during gene
cloning, protein production
To distinguish gene allels by specifically recognizing single
base changes in DNA known as single nucleotide
polymorphisms
Used to digest genomic DNA for gene analysis by southern
blot

12. Enzyme Source
Recognition
Sequence
Cut
EcoRI Escherichia coli
5'GAATTC
3'CTTAAG
5'---G AATTC---3'
3'---CTTAA G---5'
EcoRII Escherichia coli
5'CCWGG
3'GGWCC
5'--- CCWGG---3‘
3'---GGWCC ---5'
BamHI
Bacillus
amyloliquefaciens
5'GGATCC
3'CCTAGG
5'---G GATCC---3'
3'---CCTAG G---5'
HindIII
Haemophilus
influenzae
5'AAGCTT
3'TTCGAA
5'---A AGCTT---3'
3'---TTCGA A---5'
TaqI Thermus aquaticus
5'TCGA
3'AGCT
5'---T CGA---3'
3'---AGC T--- 5‘

13. Some extra points…
• Neoschizomers – different recognition enzymes identify same
sequence but cleave different locii
• Isoschizomers – different enzymes cleave and recognize same
locii
• Most commonly used artificial restriction enzymes used in
genetically engineered applications – Zinc finger nucleases

14. Thank
you….