UNIT 3.

14 DNA Ligases
DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5
phosphate and a 3-hydroxyl terminus in duplex DNA. This activity can repair single-
stranded nicks in duplex DNA (Fig. 3.14.1) and join duplex DNA restriction fragments
having either blunt ends (Fig. 3.14.2) or homologous cohesive ends (Fig. 3.14.3).Two
ligases are used for nucleic acid researchE. coli ligase and T4 ligase. These enzymes
differ in two important properties. One is the source of energy: T4 ligase uses ATP, while
E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends;
under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.

3 5
Example: 5 P P P OH P P P P 3

C T A G C T A G

G A T C G A T C

3 5
5 3 P P P P P P P
3 5 3 5
ATP NAD ATP NAD
(T4, T7 (E. coli (T4, T7 ligase) (E. coli ligase)
ligase) ligase) AMP + PP i AMP + NMN
5 3
3 5 5 P P P P P P P 3

C T A G C T A G

G A T C G A T C

P P P P P P P
3 5

Figure 3.14.1 DNA ligase activity at a nick.

ENZYME T4 DNA LIGASE

T4 DNA ligase, the product of gene 30 of phage T4, was originally purified from
phage-infected cells of E. coli. The phage T4 gene 30 has been cloned, and the enzyme
is now prepared from overproducing strains. Using ATP as a cofactor, T4 DNA ligase
catalyzes the repair of single-stranded nicks in duplex DNA and joins duplex DNA
restriction fragments having either blunt or cohesive ends. It is the only ligase that
efficiently joins blunt-end termini under normal reaction conditions. It appears that T4
DNA ligase activity may be stimulated by T4 RNA ligase (UNIT 3.15). See UNIT 3.16 for a
detailed ligation protocol.

DNA Ligases

3.14.1 Contributed by Stanley Tabor

Current Protocols in Molecular Biology (1987) 3.14.1-3.14.4
Supplement 8 Copyright 2000 by John Wiley & Sons, Inc.
Reaction Conditions
For 50-l reaction:
40 mM TrisCl, pH 7.5
10 mM MgCl2
10 mM DTT
1 g DNA
0.5 mM ATP
50 g/ml BSA
1 Weiss U T4 DNA ligase
Incubate at 12 to 30C for 1 to 16 hr. Stop reaction by adding 2 l of 0.5 M EDTA or by
heating to 75C for 10 min. The volume of reaction, concentration of DNA, and the
temperature and time of the reaction will vary, depending upon the individual application.
One Weiss unit is equivalent to 60 cohesive-end units.
Ligation of cohesive ends is usually carried out at 12 to 15C to maintain a good balance
between annealing of the ends and activity of the enzyme. Higher temperatures make it
difficult for the ends to anneal, whereas lower temperatures diminish ligase activity.
Blunt-end ligations are typically performed at room temperature since annealing is not a
factor (the enzyme is not particularly stable above 30C). Blunt-end ligations require
about 10 to 100 times more enzyme than cohesive-end ligations to achieve an equal
efficiency. T4 DNA ligase is not inhibited by tRNA, but it is strongly inhibited by NaCl
concentrations >150 mM. Macromolecular exclusion molecules (e.g., PEG 8000) have
been shown to greatly increase the rate of both cohesive-end and blunt-end joining by T4
DNA ligase (Pfeiffer and Zimmerman, 1983). An inherent consequence of macromolecu-
lar crowding is that all ligations are intermolecular; thus, this technique is not suitable for
the ligation and circularization of inserts and vectors that are required for most cloning
experiments.

3 5
5 P P P OH P P P P 3
Example:

C T A G C T A G

G A T C G A T C

3 5
5 3 P P P P OH P P P
3 5 3 5
5 3 5 3
ATP ATP
(T4 ligase)
(T4 ligase) AMP
+PP i

5 3 5 3
P P P P P P P
3 5

C T A G C T A G

G A T C G A T C

P P P P P P P
3 5
Enzymatic
Manipulation
of DNA and RNA
Figure 3.14.2 DNA ligase activity at blunt ends.
3.14.2
Current Protocols in Molecular Biology
 Applications
T4 DNA ligase is by far the most commonly used DNA ligase. It can be used for virtually
any application requiring a DNA ligase. Importantly, it efficiently ligates blunt-end
termini, a reaction that other ligases do not carry out in the absence of macromolecular
exclusion molecules.

ENZYME ESCHERICHIA COLI DNA LIGASE

DNA ligase from E. coli is the product of the lig gene. The lig gene has been cloned, and
the enzyme is obtained from an overproducing strain. E. coli DNA ligase catalyzes the
repair of single-stranded nicks in duplex DNA and joins restriction fragments having
homologous cohesive ends. E. coli DNA ligase does not join termini with blunt ends under
normal reaction conditions. Unlike the other ligases, it uses NAD as a cofactor.

Example: 3 5
5 P OH P P P P P P 3

C T A G C T A G

G A T C G A T C
3 5
5 3
3 5
5 3 P P P P P OH P
P
(T4, T7 ligase) (E. coli ligase) 3 5
ATP NA ATP NAD5 3
(T4, T7 ligase) (E. coli ligase)
D AMP AMP
+PP i + NMN
5 3
3 5 5 3
P P P P P P P

C T A G C T A G

G A T C G A T C

P P P P P P P
3 5

Figure 3.14.3 DNA ligase activity at cohesive ends.

Reaction Conditions
For 50-l reaction:
40 mM TrisCl, pH 8
10 mM MgCl2
5 mM DTT
1 g DNA
0.1 mM NAD
50 g/ml BSA
10 Modrich-Lehman U E. coli DNA ligase

Incubate at 10 to 25C for 2 to 16 hr. Stop reaction by adding 2 l of 0.5 M EDTA or by

heating to 75C for 10 min. The volume of reaction, concentration of DNA, and
temperature and time of reaction will vary, depending upon the individual application.

DNA Ligases E. coli DNA ligase, in contrast to T4 DNA ligase, does not require reducing agents

3.14.3
Current Protocols in Molecular Biology
(e.g., DTT) in the reaction. PEG 8000 greatly increases the rate of cohesive end joining
by E. coli DNA ligase (Harrison and Zimmerman, 1983). Interestingly, the presence of
macromolecular exclusion molecules also enables E. coli DNA ligase to efficiently join
blunt-end termini, a reaction it is unable to carry out in their absence.
Modrich-Lehman units measure the ability to form poly d(A-T) circles. One Modrich-
Lehman unit is equivalent to 6 Weiss units (Modrich and Lehman, 1975).

Applications
E. coli DNA ligase can be used as an alternative to T4 DNA ligase when blunt-end
ligations are not required. Transformation using DNA ligated with E. coli DNA ligase
has a lower background that results from aberrant ligations compared with T4 DNA ligase,
since T4 DNA ligase has a much lower specificity for the structure of the termini.

LITERATURE CITED
Harrison, B. and Zimmerman, S.B. 1983. Macromolecular crowding allows blunt-end ligation by DNA
ligases from rat liver or Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 80:5852-5856.
Modrich, T. and Lehman, I.R. 1975. Enzymatic joining of polynucleotides. J. Biol. Chem. 245:3626-3631.
Pfeiffer, B.H. and Zimmerman, S.B. 1983. Polymer-stimulated ligation: Enhanced blunt- or cohesive-end
ligation of DNA or deoxyribonucleotides by T4 DNA ligase in polymer solutions. Nucl. Acids Res. 11:
7853-7871.

KEY REFERENCE
Engler, M.J. and Richardson, D.C. 1982. DNA ligases. In The Enzymes, Vol. 15B (P.D. Boyer, ed.) pp. 3-30.
Academic Press, San Diego.

Contributed by Stanley Tabor

Harvard Medical School
Boston, Massachusetts

Enzymatic
Manipulation
of DNA and RNA

3.14.4
Current Protocols in Molecular Biology