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CHAPTER 2

RESTRICTION ENZYMES

AT THE END OF THE LECTURE,


STUDENTS SHOULD BE ABLE TO:

Describe the discovery of


restriction enzyme usage
Explain the origin of restriction
endonuclease and the
nomenclature
Differentiate the ends produced by
restriction enzymes
Explain the R-M protection system

RESTRICTION ENZYMES

classified as endonucleases
(cutting within).
Their biochemical activity is the
hydrolysis ("digestion") of the
phosphodiester backbone at
specific sites in a DNA
sequence.
Specific" means that an enzyme
will only digest a DNA molecule
after locating a particular
sequence.
are

COHEN AND BOYER EXPERIMENT

AN EXPERIMENT USING
RESTRICTION ENDONUCLEASE:
BOYER AND COHEN
This early experiment used
EcoRI to cut 2 plasmids, small
circular pieces of DNA
independent of the host
chromosome
Each plasmid had 1 EcoRI site
Cutting converted circular
plasmids into linear DNA with
the same sticky ends
The ends base pair
Some ends re-close
Others joint together
DNA ligase joins 2 pieces with
covalent bonds

RESTRICTION ENZYMES
Restriction endonuclease EcoRI cleaves double-stranded
DNA.
The recognition site for EcoRI is the hexameric sequence
GAATTC:
5' . .NNNNGAATTCNNNN ... 3'
3' . .NNNNCTTAAGNNNN ... 5'

Cleavage occurs at the G residue on each strand so that


the DNA is cut in a staggered fashion, leaving 5'overhanging single-stranded ends (sticky ends)

THE ROLE OF RESTRICTION


ENDONUCLEASES
Restriction endonuclease, first discovered in the
late 1960s by Linn and Arber in E. coli, (named
EcoB) are discovered because it prevented
invasion by foreign DNA (such as virus) from
cutting E.coli DNA into pieces
These enzymes cut at sites within the foreign DNA
instead of chewing from the ends (cutting
within..right?)
By cutting DNA at specific sites they function as
finely honed molecular knives

NAMING RESTRICTION
ENDONUCLEASES
Restriction endonucleases are named using the 1st three
letters of their name from the Latin name of their source
microorganism eg Hind III
First letter is from the genus H from Haemophilus
Next two letters are the 1st two letters of the species name
in from influenzae
Sometimes the strain designation is included
d from strain Rd
If microorganism produces only 1 restriction enzyme, end
the name with Roman numeral I Hind I
If more than one restriction enzyme is produced, the others
are numbered sequentially II, III, IV, etc.

NOMENCLATURE OF RE
RE names are based on a species-of-origin.
Taq polymerase (Thermophilus aquaticus, or Thermus
aquaticus).
Eg. EcoR1 (from Escherichia coli)
BamHI (from Bacillus amyloliquifaciens)
Sma I (from Serratia marcescens)
Mlu I (from Micrococcus luteus)
Hpa I (from Haemophilus parainfluenzae)

RE RECOGNITION SEQUENCE
AND TYPE OF ENDS IN PRODUCT

BamHI

G^GATCC

5' overhang

SacI

GAGCT^C

3' overhang

SmaI

CCC^GGG

blunt

COHESIVE ENDS" OR "STICKY


ENDS"

meaning
that
they
could
hydrogen bond
to
other
compatible
complementary strands

COHESIVE ENDS" OR "STICKY ENDS"

SOME RE LEAVE A BLUNT END


What

do we call a DNA molecule


that has ends that line up
evenly with each other (i.e.
neither end is overhanging)?

These

are "blunt" (meaning "not


sharp") or "flush" (meaning "level
or even") DNA ends.

BLUNT ENDS

BLUNT-END LIGATION USING PHAGE T4 DNA LIGASE , WHICH


CATALYZES THE ATP-DEPENDENT LIGATION OF DNA
MOLECULES. AMP AND PPI ARE BYPRODUCTS.

RESTRICTION ENDONUCLEASE
SPECIFICITY
Restriction endonucleases
recognize a specific DNA
sequence, cutting ONLY
at that sequence
They

recognize 4-bp, 6bp, 8-bp palindromic


sequences

They

cut DNA
reproducibly in the
same place

WHAT ARE PALINDROMES?

RESTRICTION ENDONUCLEASE
SPECIFICITY

Palindromic sequences are sequences that


read the same in the forward and reverse
direction.
Four base cutter (44= 256 bp) are more
frequently found than six base cutter (46=

4096 bp) . Some 8 base cutter eg NotI (48=

65,000 bp)
The frequency of cuts lessens as
the recognition sequence is
longer

RESTRICTION-MODIFICATION
SYSTEM
What

prevents these
enzymes from cutting up
the host DNA?
They are paired with
methylases
Theses enzymes recognize,
methylate the same site

Together

they are called a


restriction-modification
system, R-M system
Methylation protects
DNA, after replication the
parental strand is already
methylated

WHY DO YOU USE


RESTRICTION ENZYMES?
DNAs from 2 sources with the
same restriction enzyme
Restriction
enzymes leave all cut
fragments with staggered cuts (sticky
ends); some make blunt cuts
Sticky
ends
are
single-stranded
regions that pair with sticky ends on
other DNAs cut by same enzyme >
restores double-stranded molecule.
Blunt ends also ligated with ligase
treat

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