RESTRICTION ENZYMES ( NUCLEASES LIGASE )

Restriction Enzymes

1. •A restriction enzyme is a
protein that recognizes a
specific, short nucleotide
sequence and cuts the DNA
only at that specific site, which
is known as restriction site or
target sequence.

2. •Restriction enzymes are found in
bacteria (and other prokaryotes).
They recognize and bind to
specific sequences of DNA,
called restriction sites.
•Each restriction enzyme
recognizes just one or a few
restriction sites

3. •When it finds its target sequence, a
restriction enzyme will make a
double-stranded cut in the DNA molecule.
•These enzymes, which can be purified
from bacteria, cut the DNA double helix at
specific sites defined by the local
nucleotide sequence, thereby cleaving a
long double-stranded DNA molecule into
fragments of strictly defined sizes.
•Typically, the cut is at or near the restriction
site and occurs in a tidy, predictable pattern.

4. •Different species of bacteria make different
restriction nucleases, which protect them from
viruses by degrading incoming viral DNA.
•Each nuclease recognizes a specific
sequence of four to eight nucleotides in
DNA. These sequences, where they occur
in the genome of the bacterium itself, are
protected from cleavage by methylation
at an A or a C residue

5. •the sequences in foreign DNA are
generally not methylated and so are
cleaved by the restriction nucleases.
•Large numbers of restriction
nucleases have been purified from
various species of bacteria; several
hundred, most of which recognize
different nucleotide sequences, are
now available commercially.

6. The DNA nucleotide
sequences recognized by
four widely used
restriction nucleases
such sequences are often six
base pairs long and
“palindromic” (that is, the
nucleotide sequence is the
same if the helix is turned by
180 degrees around the center
of the short region of helix that
is recognized)
The enzymes cut the two
strands of DNA at or near the
recognition sequence.

7. •For some enzymes, such as HpaI,
the cleavage leaves blunt ends
• EcoRI, HindIII, and PstI, the
cleavage is staggered and creates
cohesive ends.
• Restriction nucleases are
obtained from various species of
bacteria: HpaI is from Hemophilus
parainfluenzae, EcoRI is from
Escherichia coli, HindIII is from
Hemophilus influenzae, and PstI is
from Providencia stuartii.

8. •Some restriction nucleases produce
staggered cuts, which leave short
single-stranded tails at the two ends of
each fragment.
• Ends of this type are known as cohesive
ends, as each tail can form
complementary base pairs with the tail at
any other end produced by the same
enzyme

10. How Do Restriction Enzymes Cut DNA Sequences?
•Restriction enzymes dismantle foreign DNA by
cutting it into fragments. This disassembling
process is called restriction.
•Recombinant DNA technology relies on restriction
enzymes to produce new combinations of genes.
•The cell protects its own DNA from disassembly by
adding methyl groups in a process called
modification.
•DNA ligase is a very important enzyme that helps
to join DNA strands together via covalent bonds.

11. •In a typical cell, methyl groups (CH
3
)
are added to the bases in the
sequence to prevent recognition by
the restriction enzymes.
• This process is carried out by
complementary enzymes that
recognize the same sequence of
nucleotide bases as restriction
enzymes.

12. •The methylation of DNA is known
as modification.
•With the processes of
modification and restriction, cells
can both cut up foreign DNA that
pose a danger to the cell while
preserving the important DNA of
the cell.

13. •The 5' end has a phosphate group
attached while the other 3' end has a
hydroxyl group attached.
•Since the DNA is cut on both strands,
there will be complementary ends that
can hydrogen bond to one another.
These ends are often called "sticky
ends."

15. What Is DNA Ligase?
•The sticky ends of the fragments
produced by restriction enzymes are
useful in a laboratory setting.
•They can be used to join DNA
fragments from both different
sources and different organisms. The
fragments are held together by
hydrogen bonds.

16. •From a chemical perspective,
hydrogen bonds are weak attractions
and are not permanent. Using another
type of enzyme however, the bonds
can be made permanent.
•DNA ligase is a very important
enzyme that functions in both the
replication and repair of a cell's DNA.

17. •It functions by helping the joining
of DNA strands together. It works
by catalyzing a phosphodiester
bond.
•This bond is a covalent bond,
much stronger than the
hydrogen bond and able to hold
the different fragments together.

18. •Restriction Enzyme Types :
•There are four broad categories of restriction enzymes:
Type I enzymes, Type II enzymes, Type III enzymes,
and Type IV enzymes
•All have the same basic function, but
the different types are classified
based on their subunit composition,
cleavage position, sequence
specificity and cofactor requirements.

19. •Type I enzymes cut DNA at
locations distant to the
recognition sequence
•they have little practical value since
they do not produce discrete restriction
fragments or distinct gel-banding
patterns.

20. •Type II cut DNA within or close to
the recognition sequence
• They produce discrete restriction
fragments and distinct gel banding
patterns, and they are the only class
used in the laboratory for routine DNA
analysis and gene cloning.

21. •Type II enzymes frequently differ
so completely in amino acid sequence from
one another, and indeed from every other
known protein, that they exemplify the class
of rapidly evolving proteins that are often
indicative of involvement in host-parasite
interactions.
•The most common Type II enzymes are
those like HhaI , HindIII and NotI , that cleave
DNA within their recognition sequences

22. •Type III cut DNA near recognition sequences
•They 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 cleave methylated DNA. exemplified
by the McrBC and Mrr systems of E. coli.

23. Products of Restriction Digestion
Restriction digestion of
double-stranded DNA produces
two kinds of ends: Sticky ends
and Blunt ends.
Blunt ends possess a
5’-phosphate group that
promotes ligation. They are
universally compatible with
other blunt-ended DNA.
Blunt ends generated by EcoR V

24. •The digestion product of DNA strands may
result in a fragment with either blunt ends or
cohesive (sticky) ends at both the ends.

•Cohesive 5' ends generated by EcoR I

25. •Sticky ends are small
stretches of single-stranded
DNA capable of self-ligation
or ligation with a
complementary region from
another DNA molecule. The
sticky ends possess 3’- or
5’-overhangs of 1–4
nucleotides.
•5’ Cohesive end
generated by Bln I

Bln I (Avr II)
from Brevibacterium
linens

26. •3’ Cohesive
end generated
by Kpn I
• Kpn I from
Klebsiella
pneumoniae

29. Factors affecting the activity of restriction
enzymes
•Depending on the substrate DNA and the reaction conditions,
restriction enzymes show a wide variation of cleavage and
possible star activity. In order to obtain the desired cleavage, it
becomes important to control the following factors:
•Star activity: Under sub-optimal reaction conditions, some
restriction enzymes cleave base sequences at sites different from
the defined recognition sequence. In other words, they cleave at
non-specific sites.
• This phenomenon is called star activity. Some of the factors that
induce star activity are high salt and glycerol concentration,
presence of impurities, excessive enzyme compared to substrate
DNA, increased incubation time, or incompatible buffer and
cofactor.

30. •Methylated DNA: Several DNA molecules
are methylated at the recognition site,
making them resistant to cleavage by
certain restriction enzymes.
•Temperature: Most endonucleases
optimally digest the target DNA at 37 °C.
However, there are some exceptions with
lower or higher optimal temperatures. For
example, Taq I optimally digests at 65 °C
and Apa I digests at 25 °C.

31. •The first major application was as a tool for
cutting DNA into fragments in ways that
would make it easier to study and, in
particular, identify and characterize genes.
• A second major use was as a device for
recombining, or joining, DNA molecules from
different genomes, usually with the goal of
identifying and characterizing a gene or
studying gene expression and regulation
(Heinrichs, 2007).