Explains the Types, Properties and Uses of Restriction Enzymes and DNA Ligases - Molecular Biology

1. Dr. Deepa M.A
Assistant Professor of Botany
Government Arts College (Autonomous)
Coimbatore – 641 018
https://www.youtube.com/channel/UCXqG
lEjNmKrTmWVqMIngbDA

2. Restriction enzyme
 An Enzyme that can cut double stranded DNA
 Makes incision in the Phosphate backbone of double
helix
 Cuts/Incisions are made without causing damage to the
bases
 Discovered from E. Coli
 Involved in restricting the entry/infection by
bacteriophages
 Part of Defense Mechanism

3. Sites of cleavage
 Restriction enzyme cuts only double-helical segments that
contain a particular nucleotide sequence.
 Makes incisions only within the sequence
 Restriction site – Recognition site
 Palindromic in nature
 that is, the sequence on one strand reads the same in the
opposite direction on the complementary strand.
 Recognition sequences typically are only four to twelve
nucleotides long.

4. Two types
1. Make nicks eactly on the same position in both the
strands – Blunt end fragments
2. Make Staggered end cuts. i.e after the first strand is
cut it moves further on the second strand and make
a nick – staggered insicion – sticky ends

5.  Fragment complementarity and splicing:
 Each enzyme makes a cut at specific recognition sites
 The DNA sequences cut by same enzymes will result in
similar/complementary hanging sequences.
 These can be Attached wasily
 DNA ligases – can stick any compatible sticky end

6. Restriction enzymes as tools
 Restriction enzymes specific to hundreds of distinct
sequences have been identified and synthesized and are
available commercially.
 Potential "restriction sites" are present in alomost all the
genes and chromosomes
 They are also present in the Plasmid DNA
 Linker DNA sequences with multiple Recognition sites are
also added during construction of plamids
 This enable the scientist to do the process of Cloning

7. Types of restriction enzymes
Restriction enzymes are classified biochemically into three
types.
Type I, Type II and Type III.

8. Type I and III:
Both the methylase and restriction activities are carried
out by a single large enzyme complex.
These enzymes recognize specific DNA sequences at
one position and cleave at variable distances from these
recognition sites, and can be hundreds of bases away.

9. Type II:
 The restriction enzyme is independent of its methylase
activity
 Makes nick at the site of recognition
 Most useful tools in molecular biology lab.
 Vast number of Type II enzymes are identified and
used

10. Naming
E
Escherichia (genus)
c
o
coli (species)
R RY13 (strain)
I First identified
Order ID'd
in
bacterium

11. Recognition Sequence of different enzymes
EcoRI Escherichia coli
5'---G AATTC---3'
3'---CTTAA G---5'
BamHI Bacillus amyloliquefaciens
5'---G GATCC---3'
3'---CCTAG G--- 5'

12. HindIII Haemophilus influenzae
5'---AAGCTT---3'
3'---TTCGAA---5'
TaqI Thermus aquaticus
5'---T CGA---3'
3'---AGC T---5'

14.  Links the cut DNA fragments together
 Needs double stranded break
 Alternative, Single stranded breaks will be sealed by DNA
polymerase
 Involves in DNA repair and DNA Replication
 Used extensively in molecular biology labbs for
recombination experiments

15. Ligase mechanism
Forms Covalent phosphodiester bonds between 3’hydroxyl
end and 5’ phosphate end of another – sticky ends
5'-CTGATCTGACT GATGCGTATGCTAGT-3'
3'-GACTAGACTGACTACG CATACGATCA-5'
becomes
5'-CTGATCTGACTGATGCGTATGCTAGT-3'
3'-GACTAGACTGACTACGCATACGATCA-5'
Blunt ends require higher enzyme concentrations and different
reaction conditions are required.

16. Mertz and Davis (1972) for the first time demonstrated
that cohesive termini of cleaved DNA molecules should
be covalently sealed with E. coli DNA ligase and were
able to produce recombinant molecules.
There are two enzymes which are extensively used for
covalently joining restriction fragments: the ligase from
E. coli and that encoded by T4 phage.
The main source of DNA ligase is T4 phage, hence the
enzyme is known as T4 DNA ligase.

17. Co-Factor
The E. coli DNA ligase - nicotinamide adenine
dinucleaotide (NAD+)
T4 DNA ligase - ATP

18. Both the enzymes contain a –NH2 group on lysine residue.
In both the cases, cofactor breaks into AMP (adenosine
monophosphate), which in turn adenylate the enzyme to
form enzyme – AMP complex.
EAC binds to nick containing 3’ – OH and 5’ – PO4 ends
on a double stranded DNA molecule.
The 5’- phosphoryl terminus of the nick is adenylated by
the EAC with 3’-OH Terminus resulting in formation of
phosphodiester and liberation of AMP (Lehman, 1974).

19. After formation of phosphodiester, nick is sealed. T4 enzyme
has the ability to join the blunt ends of DNA fragments,
whereas E. coli DNA ligase joins the cohesive ends produced
by restriction enzymes.
Additional advantage with T4 enzymes is that it can quickly
join and produce the full base pairs but it would be difficult to
retrieve the inserted DNA from vector. However, cohesive end
ligation proceeds about 100 times faster than the blunt end
ligation.