Restriction
Enzymes
M.Abdul Sattar
Enzymes
Enzymes are biological catalysts – found in
all cells.
They are molecules (usually proteins) that
speed up chemical reactions.
Enzymes have unique chemical structures
that mean they only act on specific substrates
Enzymes
For a particular enzyme to work
optimally, it must be under specific
conditions (temperature, pH), otherwise
its unique chemical structure will be
disrupted.
Enzymes are critical for a range of
cellular processes including digestion,
DNA replication, protein synthesis.
RESTRICTION ENZYME
Molecular
scissors that cut double stranded
DNA molecules at specific points.
Found naturally in a wide variety of
prokaryotes
An important tool for manipulating DNA.
Restriction Enzymes
• 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 .
Restriction Enzymes
These enzymes were discovered in bacteria.
They help the bacteria destroy viral DNA
(bacteria get viruses too!)
They cut between specific bases (letters) of
the double stranded DNA molecule
ROLE
Most bacteria use Restriction Enzymes as a
defense against bacteriophages.
Restriction enzymes prevent the replication of
the phage by cleaving its DNA at specific sites.
The host DNA is protected by Methylases which
add methyl groups to adenine or cytosine bases
within the recognition site thereby modifying the
site and protecting the DNA.
MECHANISM OF ACTION
Restriction Endonuclease scan the length of the
DNA.
Binds to the DNA molecule when it recognizes
a specific sequence.
Makes one cut in each of the sugar phosphate
backbones of the double helix – by
hydrolyzing the phoshphodiester bond.
Specifically, the bond between the 3’ O atom
and the P atom is broken.
Direct hydrolysis by nucleophilic
attack at the phosphorous atom
3’OH and 5’ PO43- is produced. Mg2+ is required
for the catalytic activity of the enzyme. It
holds the water molecule in a position where it
can attack the phosphoryl group and also helps
polarize the water molecule towards
deprotonation .
• To cut DNA all restriction enzymes make two
incisions, once through sugar-phosphate backbone
(i.e each strand ) of the DNA double helix .
Describe the process of cutting DNA
using restriction enzymes
enzymes are proteins that catalyze specific
chemical reactions under specific conditions
Recognize how restriction enzymes are
used to make recombinant DNA.
Identify restriction sites in double-strand
This is because…
DNA technologies are used in a range of
industries; your body and your food are
made of cells with DNA and protein.
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 .
◘ Recognition site ◘
4 base pair sequence would theoretically occur once
every 44 or 256 bp
6 bases , 46 or 4,096 bp
8 bases would be 48 or 65,536 bp
Restriction Enzymes Sites
Specific restriction enzymes cut at specific DNA
sequences.
For example:EcoRI is an enzyme that cuts at the
following sequence: GAATTC
EcoRI was discovered in E. coli bacteria.
The resulting pieces of DNA are called “restriction
fragments.”
HindIII was discovered in H. influenza
PstI was discovered in P. stuartii
EcoRV was discovered in E. coli
Palindrome Sequences
• The mirror like palindrome in which the
same forward and backwards are on a single
strand of DNA strand, as in GTAATG
• The Inverted repeat palindromes is also a
sequence that reads the same forward and
backwards, but the forward and backward
sequences are found in complementary DNA
strands (GTATAC being complementary to
CATATG)
• Inverted repeat palindromes are more
common and have greater biological
importance than mirror- like palindromes .
Ends Of Restriction Fragments
Blunt ends
Sticky ends
Blunt Ends
• Some restriction enzymes cut DNA at
opposite base
• They leave blunt ended DNA fragments
• These blunt ended fragments can be joined to
any other DNA fragment with blunt ends.
• Enzymes useful for certain types of DNA
cloning experiment.
Sticky ends
• Most restriction enzymes make staggered
cuts
• Staggered cuts produce single stranded
“sticky-ends”
“Sticky Ends” Are Useful
DNA fragments with complimentary
sticky ends can be combined to
create new molecules which allows
the creation and manipulation of
DNA sequences from different
sources.
◘Types of ends produced using restriction
enzymes :-
•EcoRI digestion produces "sticky" ends,
•whereas SmaI restriction enzyme cleavage
produces "blunt" ends:
Purposes
Researchers rely on restriction enzymes to
assist with many processes in laboratories
around the world:
1. Making recombinant DNA and appraising
success
For research, medicine and agriculture
2. DNA profile analysis
For disease diagnosis, paternity/family
relationship testing, and forensics
Nomenclature
• Each enzyme is named after the bacterium
from which it was isolated, using a
naming system based on
bacterial genus, species and strain.
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
Derivation of the EcoRI name
Abbreviation Meaning Description
E Escherichia genus
co coli specific epithet
R RY13 strain
First identified order of identification
I in the bacterium
TYPES OF RESTRICTION ENZYMES
DEPENDING UPON
FUNCTIONALITY
UTILIZING
SOURCE
Neoschizomers:
•Different restriction enzymes that
recognize the same sequence are known as
neoschizomers. These often cleave in
different locales of the sequence and form
different fragments.
Isoschizomers: v
Different enzymes that recognize and
cleave in the same location, are
known as isoschizomers,but isolated
from different strains
Isocaudomers:
•Differentenzymes that recognize
and cleave in the different location
are known as isoschizomers, but
fragments are same
Thank You
ANY QUESTION???
