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DNA Ligases

Gregory J.S. Lohman¹, Stanley Tabor², Nicole M. Nichols¹

¹New England Biolabs, Ipswich, Massachusetts
²Harvard Medical School, Boston, Massachusetts

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 3.14

DOI:

10.1002/0471142727.mb0314s94

Online Posting Date:

April, 2011

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Abstract

The DNA ligase enzyme family catalyzes the formation of a phosphodiester bond between juxtaposed 5¢-phosphate and 3¢-hydroxyl termini in duplex DNA. This activity can seal nicks in duplex DNA or join double-stranded DNA fragments having either blunt or cohesive ends. DNA ligases are central enzymes in molecular biology, nucleic acid research, and in next-generation sequencing applications. Reaction conditions and applications for T4 DNA ligase, E. coli DNA ligase, and thermostable DNA ligases are described in this unit. These enzymes differ in their cofactor requirements, substrate specificity, and thermal stability. Curr. Protoc. Mol. Biol. 94:3.14.1-3.14.7. © 2011 by John Wiley & Sons, Inc.

Keywords: T4 DNA ligase; Taq DNA ligase; DNA ligation; blunt-end ligation; cloning

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Introduction
Enzyme: T4 DNA Ligase
Enzyme: Escherichia coli DNA Ligase
Enzyme: Taq and Other Thermostable DNA Ligases
Literature Cited
Figures

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Figures

Figure 3.14.1

Ligation substrates: (A) nicked dsDNA, (B) cohesive ends, and (C) blunt ends.

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Figure 3.14.2

The mechanism of DNA ligase.

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Figure 3.14.3

RNA/DNA hybrid helices accepted by T4 DNA ligase.

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Literature Cited

Literature Cited
	Bang, D. and Church, G.M. 2008. Gene synthesis by circular assembly amplification. Nat. Methods 5:37-39.
	Barany, F. 1991. The ligase chain reaction in a PCR world. PCR Methods Appl. 1:5-16.
	Barany, F. and Gelfand, D.H. 1991. Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene. Gene 109:1-11.
	Bullard, D.R. and Bowater, R.P. 2006. Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem. J. 398:135-144.
	Cherepanov, A.V. and de Vries, S. 2003. Kinetics and thermodynamics of nick sealing by T4 DNA ligase. Eur. J. Biochem. 270:4315-4325.
	Eun, H.-M. 1996. "DNA ligases". In Enzymology Primer for Recombinant DNA Technology, pp. 109-133. Academic Press, San Diego, Calif.
	Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., and Smith, H.O. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6:343-345.
	Hayashi, K., Nakazawa, M., Ishizaki, Y., Hiraoka, N., and Obayashi, A. 1985. Stimulation of intermolecular ligation with E. coli DNA ligase by high concentrations of monovalent cations in polyethylene glycol solutions. Nucleic Acids Res. 13:7979-7992.
	Hayashi, K., Nakazawa, M., Ishizaki, Y., Hiraoka, N., and Obayashi, A. 1986. Regulation of inter- and intramolecular ligation with T4 DNA ligase in the presence of polyethylene glycol. Nucleic Acids Res. 14:7617-7631.
	Kim, J. and Mrksich, M. 2010. Profiling the selectivity of DNA ligases in an array format with mass spectrometry. Nucleic Acids Res. 38:e2.
	Okayama, H. and Berg, P. 1982. High-efficiency cloning of full-length cDNA. Mol. Cell Biol. 2:161-170.
	Panasenko, S.M., Alazard, R.J., and Lehman, I.R. 1978. A simple, three-step procedure for the large scale purification of DNA ligase from a hybrid lambda lysogen constructed in vitro. J. Biol. Chem. 253:4590-4592.
	Pascal, J.M. 2008. DNA and RNA ligases: Structural variations and shared mechanisms. Curr. Opin. Struct. Biol. 18:96-105.
	Patel, M.P., Baum, D.A., and Silverman, S.K. 2008. Improvement of DNA adenylation using T4 DNA ligase with a template strand and a strategically mismatched acceptor strand. Bioorg. Chem. 36:46-56.
	Pheiffer, B.H. and Zimmerman, S.B. 1983. Polymer-stimulated ligation: Enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions. Nucleic Acids Res. 11:7853-7871.
	Raae, A.J., Kleppe, R.K., and Kleppe, K. 1975. Kinetics and effect of salts and polyamines on T4 polynucleotide ligase. Eur. J. Biochem. 60:437-443.
	Shuman, S. 2009. DNA ligases: Progress and prospects. J. Biol. Chem. 284:17365-17369.
	Weiss, B. and Richardson, C.C. 1967. Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage. Proc. Natl. Acad. Sci. U.S.A. 57:1021-1028.
	Weiss, B., Jacquemin-Sablon, A., Live, T.R., Fareed, G.C., and Richardson, C.C. 1968. Enzymatic breakage and joining of deoxyribonucleic acid. VI. Further purification and properties of polynucleotide ligase from Escherichia coli infected with bacteriophage T4. J. Biol. Chem. 243:4543-4555.
Key Reference
	Eun, H.-M., 1996. See above.
Provides a broader description of the ligation mechanism and an application-focused perspective.