WHAT ARE RESTRICTION

ENZYMES?
Restriction Enyzmes
molecular scissors
able to cut DNA

HOW DO RESTRICTION
ENZYMES WORK?
Usually cut DNA at a palindrome such as
GAATTC.
Palindrome word or phrase when spelled
backwords, spells the same word or phrase
Ex.
BOB
MADAM IM ADAM
A Toyota! Race fast, safe car. A Toyota

HOW DO RESTRICTION
ENZYMES WORK?

GAATTC
| | | | | |
3 CTTAAG 5

Restriction site
or
Recognition Sequence

HOW DO RESTRICTION
ENZYMES WORK?
REs cut DNAs
phosphodiester bonds
and hydrogen bonds.

HOW DO RESTRICTION
ENZYMES WORK?
- REs generate two
different types of
cuts
- Sticky ends
- Blunt cuts.

WHERE DO RES COME FROM?

Bacteria
Immune system to
protect against
bacteriophages
(bacteria-infecting
viruses like Lambda).

HOW ARE RES NAMED?

After bacteria which produces them.
EcoRI

HindIII

Genus

Escherichia

Haemophilus Bacillus

Species

coli

influenzae

amylo.

Strain

Order Isolated

III

Recognition Site

G^AATTC

A^AGCTT

BamHI

G^GATCC

Types of Restriction Enzymes

Cleavage

Location of
methylase

Examples

site

Type I

Random
Around 1000bp
away from
recognition site

Endonuclease
and methylase
located on a
single protein
molecule

EcoK I
EcoA I
CfrA I

Type II

Specific
Within the
recognition site

Endonuclease
and methylase
are separate
entities

EcoR I
BamH I
Hind III

Type III

Random
24-26 bp away

Endonuclease
and methylase

EcoP I
Hinf III

Isoschizomers and Neoschizomers

Restriction enzymes that have the same recognition sequence as
well as the same cleavage site are Isoschizomers.
SphI (CGTAC/G) and BbuI (CGTAC/G)
Restriction enzymes that have the same recognition sequence but
cleave the DNA at a different site within that sequence are
Neoschizomers. Eg:SmaI and XmaI

CCCGGG
GGGCCC
Xma I

CCCGGG
GGGCCC
Sma I

HOW DO RESTRICTION
ENZYMES WORK?
Must provide correct temperature and
buffer (salt, pH) for enzyme to work.
Mimics cellular conditions of bacteria they
come from.

WHAT ARE RES USED FOR?

Genetic engineering
pasting together DNA
from two different
organisms.

HOW DO RESTRICTION
ENZYMES WORK?
Which are more useful in genetic
engineering? REs that generate sticky ends
or ones that produce blunt cuts?

STICKY ENDS

HOW IS DNA PASTED

TOGETHER?
Ligase another enzyme which
reconnects phosphodiester bonds.

WHAT ELSE ARE RES USED

FOR?
Forensics DNA Fingerprinting for crime
scene investigation and paternity testing.
Everyones DNA has a different sequence
even though only 0.1% different.
How frequently would EcoRI cut DNA?
6

4 = once every 4096 bp

Lambda (48,514 bp) would expect about 12 EcoRI sites

THOUGHT QUESTION
Bacteria are prokaryotes.
Prokaryotes do not have a nucleus.
Both DNA and REs are in cytoplasm.
Why isnt bacterial DNA cut by REs?

Biological Role
Most bacteria use Restriction Enzymes as a
defence 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.

Methylation
In humans, methyl groups are used to tag
genes to turn them on or off.