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
