Restriction Enzymes

Restriction Enzymes

1. Restriction Enzymes
◘ Cutting DNA Molecules ◘
• Before 1970 there was no method of cleaving DNA at
discrete points. ) ةلصفنم طاقن (
• 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 )صق(.
• 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 ) ايريتكب(

• 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 ةلصفنم 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 قوقش, 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 4
4
or 256 bp
○ 6 bases , 4
6
or 4,096 bp
○ and 8 bases would be 4
8
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 نع ديعب 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 .