PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of narLink to Publisher's site
 
Nucleic Acids Res. 1997 June 1; 25(11): 2106–2113.
PMCID: PMC146716

Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action.

Abstract

ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes. Here we report a functional characterization of the T4 DNA ligase. One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins. An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding. All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation. From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction. The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase. The formation of an S. complex seems to be relevant during both blunt-end ligation and DNA relaxation. Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.

Full Text

The Full Text of this article is available as a PDF (162K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Lindahl T, Barnes DE. Mammalian DNA ligases. Annu Rev Biochem. 1992;61:251–281. [PubMed]
  • Lehman IR. DNA ligase: structure, mechanism, and function. Science. 1974 Nov 29;186(4166):790–797. [PubMed]
  • Ciarrocchi G, Lestingi M, Wright G, Montecucco A. Bacteriophage T4 and human type I DNA ligases relax DNA under joining conditions. Nucleic Acids Res. 1993 Dec 25;21(25):5934–5939. [PMC free article] [PubMed]
  • Montecucco A, Ciarrocchi G. AMP-dependent DNA relaxation catalyzed by DNA ligase occurs by a nicking-closing mechanism. Nucleic Acids Res. 1988 Aug 11;16(15):7369–7381. [PMC free article] [PubMed]
  • Wei YF, Robins P, Carter K, Caldecott K, Pappin DJ, Yu GL, Wang RP, Shell BK, Nash RA, Schär P, et al. Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination. Mol Cell Biol. 1995 Jun;15(6):3206–3216. [PMC free article] [PubMed]
  • Prigent C, Lasko DD, Kodama K, Woodgett JR, Lindahl T. Activation of mammalian DNA ligase I through phosphorylation by casein kinase II. EMBO J. 1992 Aug;11(8):2925–2933. [PubMed]
  • Savini E, Biamonti G, Ciarrocchi G, Montecucco A. Cloning and sequence analysis of a cDNA coding for the murine DNA ligase I enzyme. Gene. 1994 Jul 8;144(2):253–257. [PubMed]
  • Montecucco A, Savini E, Weighardt F, Rossi R, Ciarrocchi G, Villa A, Biamonti G. The N-terminal domain of human DNA ligase I contains the nuclear localization signal and directs the enzyme to sites of DNA replication. EMBO J. 1995 Nov 1;14(21):5379–5386. [PubMed]
  • Kletzin A. Molecular characterisation of a DNA ligase gene of the extremely thermophilic archaeon Desulfurolobus ambivalens shows close phylogenetic relationship to eukaryotic ligases. Nucleic Acids Res. 1992 Oct 25;20(20):5389–5396. [PMC free article] [PubMed]
  • Shuman S, Schwer B. RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Mol Microbiol. 1995 Aug;17(3):405–410. [PubMed]
  • Subramanya HS, Doherty AJ, Ashford SR, Wigley DB. Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7. Cell. 1996 May 17;85(4):607–615. [PubMed]
  • Weiss B, Jacquemin-Sablon A, Live TR, Fareed GC, Richardson CC. 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. 1968 Sep 10;243(17):4543–4555. [PubMed]
  • Armstrong J, Brown RS, Tsugita A. Primary structure and genetic organization of phage T4 DNA ligase. Nucleic Acids Res. 1983 Oct 25;11(20):7145–7156. [PMC free article] [PubMed]
  • Diviacco S, Norio P, Zentilin L, Menzo S, Clementi M, Biamonti G, Riva S, Falaschi A, Giacca M. A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates. Gene. 1992 Dec 15;122(2):313–320. [PubMed]
  • Elder RH, Montecucco A, Ciarrocchi G, Rossignol JM. Rat liver DNA ligases. Catalytic properties of a novel form of DNA ligase. Eur J Biochem. 1992 Jan 15;203(1-2):53–58. [PubMed]
  • Gassmann M, Thömmes P, Weiser T, Hübscher U. Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J. 1990 May;4(8):2528–2532. [PubMed]
  • Kodama K, Barnes DE, Lindahl T. In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I. Nucleic Acids Res. 1991 Nov 25;19(22):6093–6099. [PMC free article] [PubMed]
  • Shuman S, Ru XM. Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis. Virology. 1995 Aug 1;211(1):73–83. [PubMed]
  • Montecucco A, Lestingi M, Pedrali-Noy G, Spadari S, Ciarrocchi G. Use of ATP, dATP and their alpha-thio derivatives to study DNA ligase adenylation. Biochem J. 1990 Oct 1;271(1):265–268. [PubMed]
  • Tomkinson AE, Totty NF, Ginsburg M, Lindahl T. Location of the active site for enzyme-adenylate formation in DNA ligases. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):400–404. [PubMed]
  • Doherty AJ, Ashford SR, Wigley DB. Characterization of proteolytic fragments of bacteriophage T7 DNA ligase. Nucleic Acids Res. 1996 Jun 15;24(12):2281–2287. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press