Restriction enzymes were discovered in 1970 and cut DNA molecules at specific recognition sequences. There are four main types of restriction enzymes that differ in their composition, cofactor requirements, target sequences, and position of DNA cleavage. Type II restriction enzymes cleave within or near their recognition sequences and are the most commonly used enzymes in molecular cloning. They recognize palindromic sequences ranging from 4-8 base pairs and cut the DNA, producing sticky or blunt ends.

1. Restriction Enzymes
◘ Cutting DNA Molecules ◘
• Before 1970 there was no method of cleaving DNA at
discrete points.) ‫منفصلة‬ ‫نقاط‬ (
• All the available methods for fragmenting DNA were
non-specific .
• The available endonucleases had little site specificity
and chemical methods produced very small fragments of
DNA .
• The only method where any degree of control could be
exercised was the use of mechanical shearing )‫.(قص‬
• The long , thin threads which constitute duple DNA
molecules are sufficiently rigid to be very easily broken
by shear forces in solution .
• Intense sonication with ultrasound can reduce the length
to about 300 nucleotide pairs .
- Sonication is the act of applying sound energy to
agitate particles in a sample, for various purposes

2. • The much sought – after breakthrough
finally came in 1970 with the discovery
in Heamophilus influenza of an enzyme
that behave more simply .
Heamophilus influenza ) ‫(بكتيريا‬
• That is , the enzyme recognizes a
particular target sequence in a duplex
DNA molecule and breaks the poly
nucleotide chain within that sequence to
give rise to discrete ‫منفصلة‬ fragments of
defined length and sequence .

3. ◘ A restriction enzyme ( Restriction Endonuclease ) ◘
• Enzyme that cuts DNA at or near specific recognition nucleotide
sequence Known as Restriction sites.
• Restriction Enzymes are commonly classified into three types ,
which differ in their structure and whether they cut their DNA
substrate at their recognition site , or if the recognition and cleavage
sites are separate from one another .
• To cut DNA all restriction enzymes make two incisions ‫شقوق‬ , once
through sugar-phosphate backbone (i.e each strand ) of the DNA
double helix .

4. ◘ Restriction Modification System ◘
• these enzymes are found in bacteria and archaea and
provide a defence mechanism against anvading viruses
• Inside a prokaryotic , the restriction enzymes
selectively cut up foreign DNA in a process called
restriction .
• while host DNA is protected by modification enzyme (
a methyltransferase ) that modifies the prokaryotic
DNA and blocks cleavage .
• Together , these two processes from the restriction
modification system .

6. ◘ Recognition site ◘
• Restriction enzymes recognize a specific sequence of
nucleotides and produce a double-stranded cut in the DNA .
• The recognition sequences can also be classified by the
number or bases in its recognition site , usually between 4 and
8 bases .
• The amount of bases in the sequence will determine how
often the site will appear by chance in any given genome .
e.g : ○ a 4 base pair sequence would theoretically occur once
every 44
or 256 bp
○ 6 bases , 46
or 4,096 bp
○ and 8 bases would be 48
or 65,536 bp

7. • Many of them are palindromic, meaning the base sequence
reads the same backwards and forwards .
-In theory, there are two types of palindromic sequences that
can be possible in DNA.
• The mirror-like palindrome is similar to those found in
ordinary text, in which a sequence reads the same forward and
backward on a single strand of DNA, as in GTAATG.
• The inverted repeat palindrome is also a sequence that
reads the same forward and backward, but the forward and
backward sequences are found in complementary DNA
strands (i.e., of double-stranded DNA), as in GTATAC
(GTATAC being complementary to CATATG)

8. ◘ Types of ends produced using restriction enzymes :-
• EcoRI digestion produces "sticky" ends,
• whereas SmaI restriction enzyme cleavage
produces "blunt" ends:

11. • Recognition sequences in DNA differ for each
restriction enzyme, producing differences in the
length, sequence and strand orientation (5'
end or 3' end) of a sticky-end "overhang" of an
enzyme restriction.
• Different restriction enzymes that recognize
the same sequence are known as neoschizomers.
These often cleave in different locales of the
sequence.
• Different enzymes that recognize and cleave in
the same location are known as isoschizomers.
○ Neoschizomers are restriction enzymes that recognize the
same nucleotide sequence but cleave at a different site
○ Isoschizomers are pairs of restriction enzymes specific to the
same recognition sequence

12. ◘ Types of restriction enzymes ◘
• Naturally occurring restriction endonucleases are
categorized into four groups (Types I, II III, and IV)
based on
• their composition and enzyme
cofactor requirements, the nature of their target
sequence, and the position of their DNA cleavage
site relative to the target sequence
◘ Type I enzymes
○ cleave at sites remote ‫عن‬ ‫بعيد‬ from a recognition
site
○require both ATP and S-adenosyl-L-
methionine to function
○ multifunctional protein with both restriction
and methylase activities.
• one enzyme with different subunits for recognition
,cleavage ,and methylation ,Recognizes and
methylates a single sequence but cleaves DNA up to
1000 bp away .

13. ◘ Type II enzymes
• cleave within or at short specific distances from
a recognition site; most require magnesium; single
function (restriction) enzymes independent of
methylase.
• Two different enzymes which both recognize the
same target sequence , which is symmetrical the
two enzymes either cleave or modify the
recognition sequence .
◘ Type III enzymes
• cleave at sites a short distance from a recognition
site; require ATP (but do not hydrolyse it);
S-adenosyl-L-methionine stimulates the reaction
but is not required; exist as part of a complex with
a modification methylase .
• one enzyme with two different subunits , one for
recognition and modification and one for cleavage
, recognition and methylates same sequence but
cleaves 24-26 bp away .

14. ◘ Type IV
• Enzymes target modified DNA, e.g.
methylated, hydroxymethylated and glucosyl-
hydroxymethylated DNA
• Two different enzymes but recognition
sequence is symmetric , cleavage occurs in one
side of recognition sequence up to 20 bp away .
◘ Nomenclature
• Each enzyme is named after the bacterium
from which it was isolated, using a naming
system based on
bacterial genus, species and strain.
•
For example, the name of
the EcoRI restriction enzyme was derived as
shown in the box.

15. Derivation of the EcoRI name
Abbreviation Meaning Description
E Escherichia genus
co coli specific epithet
R RY13 strain
I First identified
order of identification
in the bacterium

16. ◘ Mechanism of Action of restriction Enzymes ◘
• The process is one of recognition of the binding site , binding of
the enzyme dimer to the DNA , cleavage of the DNA , and
enzyme release .
• To begin , all restriction endonucleases will bind DNA
specifically and , with much less strength , non-specifically .
• It is probable that even non-specific DNA binding will induce a
conformational change in the restriction enzyme dimer that will
result in the protein adapting to the surface of the DNA strands .
• The Homodimer will either bind directly to the recognition site
( specific binding ) or nearby ( non- specific binding )
• In the case of non-specific binding , if the recognition site is not
too far away the enzyme will move along the DNA strand until it
hits the recognition site .
• Once the enzyme locates the recognition site it will couple and
then hydrolyze the sugar phosphate bonds of the DNA .
• Finally , the enzyme will release leaving the cleaved DNA
molecule behind .

17. • In general , intimate contact is held by 15 – 20 hydrogen bonds
that form between the protein and the DNA bases in the
recognition site .
• These bonds are shown to be mediated through specific amino
acids , primarily ASP and GLU , held in a proper three-
dimensional configuration .
• This requires significant conformational changes in both the
protein and the DNA as well as expulsion of water molecules
from the protein / DNA interface so that more intimate contacts
can be established .