Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1996 Aug 1;24(15):3079–3085. doi: 10.1093/nar/24.15.3079

Identification of essential residues in Thermus thermophilus DNA ligase.

J Luo 1, F Barany 1
PMCID: PMC146043  PMID: 8760897

Abstract

DNA ligases play a pivotal role in DNA replication, repair and recombination. Reactions catalyzed by DNA ligases consist of three steps: adenylation of the ligase in the presence of ATP or NAD+, transferring the adenylate moiety to the 5'-phosphate of the nicked DNA substrate (deadenylation) and sealing the nick through the formation of a phosphodiester bond. Thermus thermophilus HB8 DNA ligase (Tth DNA ligase) differs from mesophilic ATP-dependent DNA ligases in three ways: (i) it is NAD+ dependent; (ii) its optimal temperature is 65 instead of 37 degrees C; (iii) it has higher fidelity than T4 DNA ligase. In order to understand the structural basis underlying the reaction mechanism of Tth DNA ligase, we performed site-directed mutagenesis studies on nine selected amino acid residues that are highly conserved in bacterial DNA ligases. Examination of these site-specific mutants revealed that: residue K118 plays an essential role in the adenylation step; residue D120 may facilitate the deadenylation step; residues G339 and C433 may be involved in formation of the phosphodiester bond. This evidence indicates that a previously identified KXDG motif for adenylation of eukaryotic DNA ligases [Tomkinson, A.E., Totty, N.F., Ginsburg, M. and Lindahl, T. (1991) Proc. Natl. Acad. Sci. USA, 88, 400-404] is also the adenylation site for NAD+-dependent bacterial DNA ligases. In a companion paper, we demonstrate that mutations at a different Lys residue, K294, may modulate the fidelity of Tth DNA ligase.

Full Text

The Full Text of this article is available as a PDF (119.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  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. Barany F., Gelfand D. H. Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene. Gene. 1991 Dec 20;109(1):1–11. doi: 10.1016/0378-1119(91)90582-v. [DOI] [PubMed] [Google Scholar]
  3. Barany F. Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):189–193. doi: 10.1073/pnas.88.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barany F. The ligase chain reaction in a PCR world. PCR Methods Appl. 1991 Aug;1(1):5–16. doi: 10.1101/gr.1.1.5. [DOI] [PubMed] [Google Scholar]
  5. Barker D. G., Johnson A. L., Johnston L. H. An improved assay for DNA ligase reveals temperature-sensitive activity in cdc9 mutants of Saccharomyces cerevisiae. Mol Gen Genet. 1985;200(3):458–462. doi: 10.1007/BF00425731. [DOI] [PubMed] [Google Scholar]
  6. Barker D. G., White J. H., Johnston L. H. Molecular characterisation of the DNA ligase gene, CDC17, from the fission yeast Schizosaccharomyces pombe. Eur J Biochem. 1987 Feb 2;162(3):659–667. doi: 10.1111/j.1432-1033.1987.tb10688.x. [DOI] [PubMed] [Google Scholar]
  7. Barnes D. E., Johnston L. H., Kodama K., Tomkinson A. E., Lasko D. D., Lindahl T. Human DNA ligase I cDNA: cloning and functional expression in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6679–6683. doi: 10.1073/pnas.87.17.6679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Becker A., Lyn G., Gefter M., Hurwitz J. The enzymatic repair of DNA, II. Characterization of phage-induced sealase. Proc Natl Acad Sci U S A. 1967 Nov;58(5):1996–2003. doi: 10.1073/pnas.58.5.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen J., Tomkinson A. E., Ramos W., Mackey Z. B., Danehower S., Walter C. A., Schultz R. A., Besterman J. M., Husain I. Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination. Mol Cell Biol. 1995 Oct;15(10):5412–5422. doi: 10.1128/mcb.15.10.5412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cong P., Shuman S. Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases. J Biol Chem. 1993 Apr 5;268(10):7256–7260. [PubMed] [Google Scholar]
  11. Cong P., Shuman S. Mutational analysis of mRNA capping enzyme identifies amino acids involved in GTP binding, enzyme-guanylate formation, and GMP transfer to RNA. Mol Cell Biol. 1995 Nov;15(11):6222–6231. doi: 10.1128/mcb.15.11.6222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dunn J. J., Studier F. W. Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4. J Mol Biol. 1981 Jun 5;148(4):303–330. doi: 10.1016/0022-2836(81)90178-9. [DOI] [PubMed] [Google Scholar]
  13. Fresco L. D., Buratowski S. Active site of the mRNA-capping enzyme guanylyltransferase from Saccharomyces cerevisiae: similarity to the nucleotidyl attachment motif of DNA and RNA ligases. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6624–6628. doi: 10.1073/pnas.91.14.6624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gumport R. I., Lehman I. R. Structure of the DNA ligase-adenylate intermediate: lysine (epsilon-amino)-linked adenosine monophosphoramidate. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2559–2563. doi: 10.1073/pnas.68.10.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hammond J. M., Kerr S. M., Smith G. L., Dixon L. K. An African swine fever virus gene with homology to DNA ligases. Nucleic Acids Res. 1992 Jun 11;20(11):2667–2671. doi: 10.1093/nar/20.11.2667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heaphy S., Singh M., Gait M. J. Effect of single amino acid changes in the region of the adenylylation site of T4 RNA ligase. Biochemistry. 1987 Mar 24;26(6):1688–1696. doi: 10.1021/bi00380a030. [DOI] [PubMed] [Google Scholar]
  17. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  18. Ishino Y., Shinagawa H., Makino K., Tsunasawa S., Sakiyama F., Nakata A. Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli. Mol Gen Genet. 1986 Jul;204(1):1–7. doi: 10.1007/BF00330179. [DOI] [PubMed] [Google Scholar]
  19. Jónsson Z. O., Thorbjarnardóttir S. H., Eggertsson G., Palsdottir A. Sequence of the DNA ligase-encoding gene from Thermus scotoductus and conserved motifs in DNA ligases. Gene. 1994 Dec 30;151(1-2):177–180. doi: 10.1016/0378-1119(94)90652-1. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Lauer G., Rudd E. A., McKay D. L., Ally A., Ally D., Backman K. C. Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase. J Bacteriol. 1991 Aug;173(16):5047–5053. doi: 10.1128/jb.173.16.5047-5053.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Modorich P., Lehman I. R. Deoxyribonucleic acid ligase. A steady state kinetic analysis of the reaction catalyzed by the enzyme from Escherichia coli. J Biol Chem. 1973 Nov 10;248(21):7502–7511. [PubMed] [Google Scholar]
  25. Modrich P., Anraku Y., Lehman I. R. Deoxyribonucleic acid ligase. Isolation and physical characterization of the homogeneous enzyme from Escherichia coli. J Biol Chem. 1973 Nov 10;248(21):7495–7501. [PubMed] [Google Scholar]
  26. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parks R. J., Lichty B. D., Karakis C., Evans D. H. Characterization of the Shope fibroma virus DNA ligase gene. Virology. 1994 Aug 1;202(2):642–650. doi: 10.1006/viro.1994.1385. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Schmitt M. P., Beck P. J., Kearney C. A., Spence J. L., DiGiovanni D., Condreay J. P., Molineux I. J. Sequence of a conditionally essential region of bacteriophage T3, including the primary origin of DNA replication. J Mol Biol. 1987 Feb 5;193(3):479–495. doi: 10.1016/0022-2836(87)90261-0. [DOI] [PubMed] [Google Scholar]
  30. Schwer B., Shuman S. Mutational analysis of yeast mRNA capping enzyme. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4328–4332. doi: 10.1073/pnas.91.10.4328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Shark K. B., Conway T. Cloning and molecular characterization of the DNA ligase gene (lig) from Zymomonas mobilis. FEMS Microbiol Lett. 1992 Sep 1;75(1):19–26. doi: 10.1016/0378-1097(92)90450-3. [DOI] [PubMed] [Google Scholar]
  32. Shuman S., Liu Y., Schwer B. Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):12046–12050. doi: 10.1073/pnas.91.25.12046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Smith G. L., Chan Y. S., Kerr S. M. Transcriptional mapping and nucleotide sequence of a vaccinia virus gene encoding a polypeptide with extensive homology to DNA ligases. Nucleic Acids Res. 1989 Nov 25;17(22):9051–9062. doi: 10.1093/nar/17.22.9051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Takahashi M., Yamaguchi E., Uchida T. Thermophilic DNA ligase. Purification and properties of the enzyme from Thermus thermophilus HB8. J Biol Chem. 1984 Aug 25;259(16):10041–10047. [PubMed] [Google Scholar]
  37. Thøgersen H. C., Morris H. R., Rand K. N., Gait M. J. Location of the adenylylation site in T4 RNA ligase. Eur J Biochem. 1985 Mar 1;147(2):325–329. doi: 10.1111/j.1432-1033.1985.tb08753.x. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Walter R. B., Rolig R. L., Kozak K. A., McEntire B., Morizot D. C., Nairn R. S. Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene. Mol Biol Evol. 1993 Nov;10(6):1227–1238. doi: 10.1093/oxfordjournals.molbev.a040066. [DOI] [PubMed] [Google Scholar]
  40. Wang Y. C., Burkhart W. A., Mackey Z. B., Moyer M. B., Ramos W., Husain I., Chen J., Besterman J. M., Tomkinson A. E. Mammalian DNA ligase II is highly homologous with vaccinia DNA ligase. Identification of the DNA ligase II active site for enzyme-adenylate formation. J Biol Chem. 1994 Dec 16;269(50):31923–31928. [PubMed] [Google Scholar]
  41. 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]
  42. Weiss B., Richardson C. C. Enzymatic breadage and joining of deoxyribonucleic acid. 3. An enzyme-adenylate intermediate in the polynucleotide ligase reaction. J Biol Chem. 1967 Sep 25;242(18):4270–4272. [PubMed] [Google Scholar]
  43. Weiss B., Thompson A., Richardson C. C. Ezymatic breakage and joining of deoxyribonucleic acid. VII. Properties of the enzyme-adenylate intermediate in the polynucleotide ligase reaction. J Biol Chem. 1968 Sep 10;243(17):4556–4563. [PubMed] [Google Scholar]
  44. Wiedmann M., Wilson W. J., Czajka J., Luo J., Barany F., Batt C. A. Ligase chain reaction (LCR)--overview and applications. PCR Methods Appl. 1994 Feb;3(4):S51–S64. doi: 10.1101/gr.3.4.s51. [DOI] [PubMed] [Google Scholar]
  45. Xu Q., Teplow D., Lee T. D., Abelson J. Domain structure in yeast tRNA ligase. Biochemistry. 1990 Jul 3;29(26):6132–6138. doi: 10.1021/bi00478a004. [DOI] [PubMed] [Google Scholar]
  46. Yudelevich A., Ginsberg B., Hurwitz J. Discontinuous synthesis of DNA during replication. Proc Natl Acad Sci U S A. 1968 Nov;61(3):1129–1136. doi: 10.1073/pnas.61.3.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zimmerman S. B., Little J. W., Oshinsky C. K., Gellert M. Enzymatic joining of DNA strands: a novel reaction of diphosphopyridine nucleotide. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1841–1848. doi: 10.1073/pnas.57.6.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zimmerman S. B., Oshinsky C. K. Enzymatic joining of deoxyribonucleic acid strands. 3. Further purification of the deoxyribonucleic acid ligase from Escherichia coli and multiple forms of the purified enzyme. J Biol Chem. 1969 Sep 10;244(17):4689–4695. [PubMed] [Google Scholar]

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

RESOURCES