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. 1997 Jun 1;25(11):2106–2113. doi: 10.1093/nar/25.11.2106

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

R Rossi 1, A Montecucco 1, G Ciarrocchi 1, G Biamonti 1
PMCID: PMC146716  PMID: 9153309

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.

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Selected References

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  1. Armstrong J., Brown R. S., Tsugita A. Primary structure and genetic organization of phage T4 DNA ligase. Nucleic Acids Res. 1983 Oct 25;11(20):7145–7156. doi: 10.1093/nar/11.20.7145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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. doi: 10.1093/nar/21.25.5934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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. doi: 10.1016/0378-1119(92)90220-j. [DOI] [PubMed] [Google Scholar]
  4. Doherty A. J., Ashford S. R., Wigley D. B. Characterization of proteolytic fragments of bacteriophage T7 DNA ligase. Nucleic Acids Res. 1996 Jun 15;24(12):2281–2287. doi: 10.1093/nar/24.12.2281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Elder R. H., Montecucco A., Ciarrocchi G., Rossignol J. M. Rat liver DNA ligases. Catalytic properties of a novel form of DNA ligase. Eur J Biochem. 1992 Jan 15;203(1-2):53–58. doi: 10.1111/j.1432-1033.1992.tb19826.x. [DOI] [PubMed] [Google Scholar]
  6. 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. doi: 10.1096/fasebj.4.8.1970792. [DOI] [PubMed] [Google Scholar]
  7. 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. doi: 10.1093/nar/20.20.5389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kodama K., Barnes D. E., 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. doi: 10.1093/nar/19.22.6093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lehman I. R. DNA ligase: structure, mechanism, and function. Science. 1974 Nov 29;186(4166):790–797. doi: 10.1126/science.186.4166.790. [DOI] [PubMed] [Google Scholar]
  10. Lindahl T., Barnes D. E. Mammalian DNA ligases. Annu Rev Biochem. 1992;61:251–281. doi: 10.1146/annurev.bi.61.070192.001343. [DOI] [PubMed] [Google Scholar]
  11. 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. doi: 10.1093/nar/16.15.7369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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. doi: 10.1042/bj2710265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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. doi: 10.1002/j.1460-2075.1995.tb00222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Prigent C., Lasko D. D., Kodama K., Woodgett J. R., Lindahl T. Activation of mammalian DNA ligase I through phosphorylation by casein kinase II. EMBO J. 1992 Aug;11(8):2925–2933. doi: 10.1002/j.1460-2075.1992.tb05362.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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. doi: 10.1016/0378-1119(94)90386-7. [DOI] [PubMed] [Google Scholar]
  16. Shuman S., Ru X. M. Mutational analysis of vaccinia DNA ligase defines residues essential for covalent catalysis. Virology. 1995 Aug 1;211(1):73–83. doi: 10.1006/viro.1995.1380. [DOI] [PubMed] [Google Scholar]
  17. Shuman S., Schwer B. RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Mol Microbiol. 1995 Aug;17(3):405–410. doi: 10.1111/j.1365-2958.1995.mmi_17030405.x. [DOI] [PubMed] [Google Scholar]
  18. Subramanya H. S., Doherty A. J., Ashford S. R., Wigley D. B. Crystal structure of an ATP-dependent DNA ligase from bacteriophage T7. Cell. 1996 May 17;85(4):607–615. doi: 10.1016/s0092-8674(00)81260-x. [DOI] [PubMed] [Google Scholar]
  19. Tomkinson A. E., Totty N. F., 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. doi: 10.1073/pnas.88.2.400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wei Y. F., Robins P., Carter K., Caldecott K., Pappin D. J., Yu G. L., Wang R. P., Shell B. K., Nash R. A., Schär P. 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. doi: 10.1128/mcb.15.6.3206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Weiss B., Jacquemin-Sablon A., Live T. R., Fareed G. C., Richardson C. C. 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] [Google Scholar]

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