MODULE 12 : Restriction Endonucleases
Restriction Enzymes are endonucleases that recognize specific base sequences and break or
restrict the DNA polymer at the sugar-phosphate backbone. These enzymes were originally
isolated from bacteria and functions as part of a primitive defense system to cleave foreign
DNA that enters the cell of the bacteria. Recognized foreign DNA depends on both DNA
sequence recognition and methylation.
- Restriction enzymes are named for the organism from which they were isolated.
- REs are DNA-cutting enzymes and recognizes one or a few target sequences and cuts
DNA at or near those sequences.
- It cleavesDNA into fragments at or near specific sites recognized within molecules and
these are known as restriction sites
Biological Characteristics of Restriction Enzymes
1. Produce by bacteria to cut foreign DNA that enters the bacterial cell
a. Bacteriophage DNA (transduction)
b. DNA from other bacteria (transformation)
2. Each type enzyme cuts a single DNA sequence and precisely does it
3. Since DNA isdouble stranded, complementary strand is also cut by the same enzyme
(symmetrical DNA sequence)
4. The enzyme does not cut the self DNA since this is methylated particularly the G & C
5. Cutting the DNA by restriction enzymes can result in forming sticky ends or blunt end
in the DNA
a. Sticky ends = 5’ overhang or 3’ overhang
5’ overhang (e.i. EcoRI) 3’ overhang (e.i. PstI-Providencia stuartii)
5’…G AATTC…3’ 5’…CTGCA G …3’
3’…CTTAA G…5’ 3’…G ACGTC…5’
5’…CTGCA 3’ 5’ G…3’
5’…G 3’ 5’ AATTC….3’ 3’…G 5’ 3’…ACGTC…5’
3’…CTTAA…5’ 3’ G…5’
b. Blunt ended
XmaI (Xanthomonas malvacearum)
↓
5’…CCC GGG…3’
3’…GGG CCC…5’
↑
5’…CCC 3’ 5’ GGG…3’
3’…GGG 5’ 3’ CCC…5’
6. Restriction
enzymes are named using 3 letters from the scientific name of the bacteria
and a roman numeral number to indicate the sequence of discovery
Example: EcoRI ; Escherichia coli = Eco R= strain type (RY13)
I= first discovery
7. RE usually recognize DNA sequence of 4 – 8 nucleotides
8. Many of the enzymes are palindromic; base sequences read at the same forward and
backward in the double stranded DNA
e.i: Smal 5’…CCCGGG…3’
3’…GGGCCC…5’
9. There are at least 4 types of restriction enzymes
Types of Restriction Enzymes
Type I Restriction Enzymes
- Have both nuclease and methylase activity in a single enzyme
- Bind to host-specific DNA sites of 4 – 6 bp separated by 6 – 8 bp and containing
methylated adenines
- Site of cleavage of the DNA substrate can be over 1000 bp from the binding site
Type II Restriction Enzymes
- Used most frequently in the laboratory
- Do not have inherent methylation activity
- Bind as simple dimers to symmetrical DNA recognition sites – palindromic in nature –
read as the same 5’ to 3’ on both strands of the DNA – bilateral symmetry
- Cleave the DNA directly at the binding site, producing fragments of predictable size
- Found in almost all prokaryotes
Type III Restriction Enzymes
- Resemble type I enzymes in ability to both methylate and restrict DNA.
- Are complex enzymes with 2 sub-units
- Recognition sites are asymmetrical, cleavage of the substrate DNA occurs 24 – 26 bp
from the site to the 3’ side.
Type IV Restriction Enzymes
- Have similar sub-unit structures and enzyme requirements
- Have cutting and methyltransferase functions
RE type Co-factor Cleavage Site Example
I ATP, Adomet, Cleaves at sites away from recognition site EcoB. EcoR
Mg++ Possess restriction and methylase activity
II Mg++ Cleave within or at short specific distance EcoRI, BamHI
from the recognition site SphI
III ATP, MG++ Cleave at sites 25 – 27 bp from recognition stie EcoPI, HindIII
IV Mg++ Cleave close to or within recognition sequence Mrr
Target modified DNA; methylated;
hydroxymethylated; glycosyl
hydroxymethylated
Examples of Restriction Enzymes
EcoRI G AATTC KpnI GGTAC C SphI GCATG C
CTTAA G C CATGG C GTACG
Nael GCG CGC HindIII A AGCTT AluI AG CT
CGC GCG TTCGA A TC GA
SphI or CGTAC G BamHI G GATCC Sau3AI GATC
Bbul G CATGC CCTAG G CTAG
MaeII A CGT PstI CTGCA G EcoRV GAT ATC
TGC A G ACGTC CTA TAG
Uses of Restriction Enzymes
1. Assist in the insertion of a gene into a plasmid vector (gene cloning)
2. Distinguish gene alleles by specifically recognizing single base changes in DNA known
as single nucleotide polymorphism
3. Used to digest genomic DNA for gene analysis (genotyping)
Related links:
Restriction Enzymes:
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