UNIT 3.14 DNA LigasesDNA 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 fragmentshaving either blunt ends (Fig. 3.14.2) or homologous cohesive ends (Fig. 3.14.3).Twoligases are used for nucleic acid research—E. coli ligase and T4 ligase. These enzymesdiffer in two important properties. One is the source of energy: T4 ligase uses ATP, whileE. 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.

ENZYME T4 DNA LIGASE

T4 DNA ligase, the product of gene 30 of phage T4, was originally purified fromphage-infected cells of E. coli. The phage T4 gene 30 has been cloned, and the enzymeis now prepared from overproducing strains. Using ATP as a cofactor, T4 DNA ligasecatalyzes the repair of single-stranded nicks in duplex DNA and joins duplex DNArestriction fragments having either blunt or cohesive ends. It is the only ligase thatefficiently joins blunt-end termini under normal reaction conditions. It appears that T4DNA ligase activity may be stimulated by T4 RNA ligase (UNIT 3.15). See UNIT 3.16 for adetailed ligation protocol.

PPPP 3′PPP5′

P P P P P P P3′ 5′

G A T C G A T C

PPPP

A G C T

3′P PP

A G

P

C T

5′

P

G

P

A

P

T

P

C

P

G A

P

T C

3′ 5′

A G C T A GC T

(E. coli ligase)NADATP

AMP + PP i AMP + NMN(T4, T7 ligase)

5′3′

Example:

(E. coliligase)

(T4, T7ligase)

NADATP

5′3′ 5′

3′

5′3′ 5′

3′5′3′

OHP

P

Figure 3.14.1 DNA ligase activity at a nick.

Contributed by Stanley TaborCurrent Protocols in Molecular Biology (1987) 3.14.1-3.14.4Copyright © 2000 by John Wiley & Sons, Inc.Supplement 8

3.14.1

DNA Ligases

Reaction Conditions

For 50-�l reaction:40 mM Tris⋅Cl, pH 7.510 mM MgCl2

10 mM DTT1 µg DNA0.5 mM ATP50 µg/ml BSA1 “Weiss” U T4 DNA ligase

Incubate at 12° to 30°C for 1 to 16 hr. Stop reaction by adding 2 µl of 0.5 M EDTA or byheating to 75°C for 10 min. The volume of reaction, concentration of DNA, and thetemperature 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 15°C to maintain a good balancebetween annealing of the ends and activity of the enzyme. Higher temperatures make itdifficult for the ends to anneal, whereas lower temperatures diminish ligase activity.Blunt-end ligations are typically performed at room temperature since annealing is not afactor (the enzyme is not particularly stable above 30°C). Blunt-end ligations requireabout 10 to 100 times more enzyme than cohesive-end ligations to achieve an equalefficiency. T4 DNA ligase is not inhibited by tRNA, but it is strongly inhibited by NaClconcentrations >150 mM. Macromolecular exclusion molecules (e.g., PEG 8000) havebeen shown to greatly increase the rate of both cohesive-end and blunt-end joining by T4DNA ligase (Pfeiffer and Zimmerman, 1983). An inherent consequence of macromolecu-lar crowding is that all ligations are intermolecular; thus, this technique is not suitable forthe ligation and circularization of inserts and vectors that are required for most cloningexperiments.

PPP 3′PPP5′

P P P P P P P3′ 5′

G A T C G A T C

PPPP

A G C T

3′P PP

A G

P

C T

5′

P

G

P

A

P

T

P

C

P

G A

P

T C

3′ 5′

A G C T A GC T

ATP

AMP+PP i

(T4 ligase)

5′3′

5′ 3′

Example:

(T4 ligase)

ATP

5′3′ 5′

3′

5′3′ 5′

3′5′3′

3′5′OH

OH PP

P

Figure 3.14.2 DNA ligase activity at blunt ends.

Current Protocols in Molecular Biology

3.14.2

EnzymaticManipulationof DNA and RNA

Applications

T4 DNA ligase is by far the most commonly used DNA ligase. It can be used for virtuallyany application requiring a DNA ligase. Importantly, it efficiently ligates blunt-endtermini, a reaction that other ligases do not carry out in the absence of macromolecularexclusion molecules.

ENZYME ESCHERICHIA COLI DNA LIGASE

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

Reaction Conditions

For 50-�l reaction:40 mM Tris⋅Cl, pH 810 mM MgCl2

5 mM DTT1 µg DNA0.1 mM NAD50 µg/ml BSA10 “Modrich-Lehman” U E. coli DNA ligase

Incubate at 10° to 25°C for 2 to 16 hr. Stop reaction by adding 2 µl of 0.5 M EDTA or byheating to 75°C for 10 min. The volume of reaction, concentration of DNA, andtemperature and time of reaction will vary, depending upon the individual application.

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

PPPP 3′PPP5′

P P P P P P P3′ 5′

G A T C G A T C

PPPP

A G C T

3′P PP

A G

P

C T

5′

P

G

P

A

P

T

P

C

P

G

P

A

P

T C

3′ 5′

A G C T A GC T

(E. coli ligase)NADATP

AMP+PP i

AMP+ NMN

(T4, T7 ligase)

5′P

OH

3′

3′5′

Example:

(E. coli ligase)(T4, T7 ligase)

NAD

ATP

5′3′ 5′

3′

5′3′ 5′

3′5′3′

3′5′

OH

Figure 3.14.3 DNA ligase activity at cohesive ends.

Current Protocols in Molecular Biology

3.14.3

DNA Ligases

(e.g., DTT) in the reaction. PEG 8000 greatly increases the rate of cohesive end joiningby E. coli DNA ligase (Harrison and Zimmerman, 1983). Interestingly, the presence ofmacromolecular exclusion molecules also enables E. coli DNA ligase to efficiently joinblunt-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-endligations are not required. Transformation using DNA ligated with E. coli DNA ligasehas 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 CITEDHarrison, 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-endligation of DNA or deoxyribonucleotides by T4 DNA ligase in polymer solutions. Nucl. Acids Res. 11:7853-7871.

KEY REFERENCEEngler, 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 TaborHarvard Medical SchoolBoston, Massachusetts

Current Protocols in Molecular Biology

3.14.4

EnzymaticManipulationof DNA and RNA