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Four broad categories of the enzymes:

DNA synthesis enzymes (DNA


polymerases)
Nucleases (Restriction enzymes)
Ligation enzymes (DNA Ligases)
End-modification enzymes

The types of nucleases used in research


Restriction endonuclease
Deoxyribonuclease I (DNase I)
Ribonuclease A (RNase A)

Lecture 5:
Restriction Endonucleases

History

Originated from the studies of phage and the


phenomenon of host-controlled restriction and
modification

RE are found in bacteria and archaea and provide a


defense mechanism against invading viruses

Restriction: a process of selectively cut up foreign


DNA

Modification: a process where host DNA is protected


by a modification enzyme (a methylase) that modifies
their DNA and prevent cleavage

Restriction Modification System


When bacterial invaded by
phage

Methylationa methyl group is


added to the cytosine or adenine,
protect the DNA

Definition
Enzyme that binds to a DNA molecule at a
specific sequence and makes a doublestranded cut at or near that sequence
Sequence specific, the position of cutting
within a DNA molecule can be predicted

Types of RE
Type I: complex, multisubunit, combination restrictionand-modification enzymes that cut DNA at random far
from their recognition sequences
Type II: cut DNA at defined positions close to or within
their recognition sequences
Type III: cleave outside of their recognition sequences
and require two such sequences in opposite orientations
within the same DNA molecule to accomplish cleavage; they
rarely give complete digests
Type IV: recognize modified, typically methylated DNA

The cuts are made in slightly


different positions relative to the
recognition sequenceresulting
fragments have different lengths

Each molecule is cut at exactly the


same position to give exactly the
same pair of fragments

Nomenclature
Based upon the name of the organism from which they
were isolated
PstI:

Indicates a specific enzyme obtained from the bacterium


Providencia stuartii

The letter one (I) indicates the first RE isolated from P.


stuartii

HaeI, HaeII and HaeIII:

Indicates three RE with different specificity isolated from


Haemophilus aegyptius

The letter I, II and III indicate the first, second and third RE
isolated from H. aegyptius respectively

Patterns of DNA Cutting by RE: sticky ends


or cohesive ends
5' overhangs: The enzyme cuts asymmetrically within the
recognition site such that a short single-stranded segment
extends from the 5' ends

3' overhangs: asymmetrical cutting within the recognition


site, but the result is a single-stranded overhang from the two
3' ends

Patterns of DNA Cutting by RE: blunt ends


Blunts: Enzymes that cut at precisely opposite sites in the
two strands of DNA generate blunt ends without overhangs

The Cutting Frequency

The length of the recognition site will affect the frequency of


cutting site

The frequency of recognition sequences in a DNA molecule:

a tetranucleotide sequence (e.g. GATC) should occur once


every 44 = 256 nucleotides (4 cutter) and

a hexanucleotide (e.g. GGATCC) once every 46 = 4096


nucleotides (6 cutter).

Enzymes such as AluI and Sau3A would cut DNA on average


every 44 bases to generate a set of fragments with an average
size of 256 bp

Enzymes such as EcoRI, BamHI would cut DNA on average


every 46 bases to generate a set of fragments with an average
size of 4096 bp

The assumptions in this calculation are not


necessarily valid:
The percentage of G + C and A + T not always
equal.
E. coli 50% GC
Staphylococcus aureus 37% GC
Streptomyces coelicolor 72% GC

The sequence of bases is not random


The ability of restriction endonucleases to cut
DNA is block by DNA methylation.

Mode of Action
1.

Restriction enzymes hydrolyze the phosphodiester backbone once on


each strand (we say the strand is "nicked,")
Nick

Hydrogen
bonds
Nick
2.

The bonds being broken by the enzyme are covalent. The hydrogen bonds
responsible for base pairing are not broken by the restriction enzyme

3.

Thermal energy is high enough at room temperature to separate EcoRI


fragments (for example)

Isoschizomers

Isoschizomers (Greek iso, equal: skhizo, to split):


1. RE that recognize the same sequence of bases
and are derived from different organisms.
2. Some isoschizomers not only have the same
recognition site, but cut in the same place within
that recognition sequence.
3. Some isoschizomers recognize the same
sequence but cut in a different position within
that sequence.

What does a restriction enzyme need in order to do


its duty?

A double-stranded DNA sequence containing the


recognition sequence

Suitable conditions for digestion


BamHI has the recognition
sequence: GGATCC and requires
conditions as below:

SmaI has the recognition


sequence: CCCGGG and requires
conditions as below:

10 mM Tris-Cl (pH 8.0)

33 mM Tris-acetate (pH 7.9)

5 mM Magnesium chloride

10 mM Magnesium acetate

100 mM NaCl

66 mM Potassium acetate

1 mM 2-mercaptoethanol

0.5 mM Dithiothreitol

Reaction conditions: 37 C

Reaction conditions: 25 C

Star Activity
It has been demonstrated that under extreme
non-standard conditions, restriction
endonucleases are capable of cleaving sequences
which are similar but not identical to their
defined recognition sequence
The altered or relaxed specificity has been
termed ""star"" activity

Internet Teaching Resources


Replacing 1 hour lecture on Tuesday

Class Activity

GG|CC and CC|GG

Class Activity

Class Activity

End of Lecture 5
Thank You

Lecture 6:
Gene Cloning and Analysis