DNA ligases are essential enzymes in cells due to their ability to join DNA strand breaks formed during DNA replication. Several temperature-sensitive mutant strains of , including strain GR501, have been described which can be complemented by functional DNA ligases. Here, it is shown that the mutation in GR501 strain is a cytosine to thymine transition at base 43, which results in a substitution of leucine by phenylalanine at residue 15. The protein product of this gene (LigA251) is accumulated to a similar level at permissive and non-permissive temperatures. Compared to wild-type LigA, at 20 °C purified LigA251 has 20-fold lower ligation activity , and its activity is reduced further at 42 °C, resulting in 60-fold lower ligation activity than wild-type LigA. It is proposed that the mutation in LigA251 affects the structure of the N-terminal region of LigA. The resulting decrease in DNA ligase activity at the non-permissive temperature is likely to occur as the result of a conformational change that reduces the rate of adenylation of the ligase.


Article metrics loading...

Loading full text...

Full text loading...



  1. Barany, F. & Gelfand, D. H.(1991). Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene. Gene 109, 1–11.[CrossRef] [Google Scholar]
  2. Brotz-Oesterhelt, H., Knezevic, I., Bartel, S., Lampe, T., Warnecke-Eberz, U., Ziegelbauer, K., Habich, D. & Labischinski, H.(2003). Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones. J Biol Chem 278, 39435–39442.[CrossRef] [Google Scholar]
  3. Dermody, J. J., Robinson, G. T. & Sternglanz, R.(1979). Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli. J Bacteriol 139, 701–704. [Google Scholar]
  4. Doherty, A. J. & Suh, S. W.(2000). Structural and mechanistic conservation in DNA ligases. Nucleic Acids Res 28, 4051–4058.[CrossRef] [Google Scholar]
  5. Doherty, A. J., Ashford, S. R., Subramanya, H. S. & Wigley, D. B.(1996). Bacteriophage T7 DNA ligase – overexpression, purification, crystallization, and characterization. J Biol Chem 271, 11083–11089.[CrossRef] [Google Scholar]
  6. Gellert, M. & Bullock, M. L.(1970). DNA ligase mutants of Escherichia coli. Proc Natl Acad Sci U S A 67, 1580–1587.[CrossRef] [Google Scholar]
  7. Georlette, D., Blaise, V., Dohmen, C., Bouillenne, F., Damien, B., Depiereux, E., Gerday, C., Uversky, V. N. & Feller, G.(2003). Cofactor binding modulates the conformational stabilities and unfolding patterns of NAD(+)-dependent DNA ligases from Escherichia coli and Thermus scotoductus. J Biol Chem 278, 49945–49953.[CrossRef] [Google Scholar]
  8. Gong, C., Martins, A., Bongiorno, P., Glickman, M. & Shuman, S.(2004). Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J Biol Chem 279, 20594–20606.[CrossRef] [Google Scholar]
  9. Guex, N. & Peitsch, M. C.(1997).swiss-model and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723.[CrossRef] [Google Scholar]
  10. Ishino, Y., Shinagawa, H., Makino, K., Tsunasawa, S., Sakiyama, F. & Nakata, A.(1986). Nucleotide sequence of the lig gene and primary structure of DNA ligase of Escherichia coli. Mol Gen Genet 204, 1–7.[CrossRef] [Google Scholar]
  11. Kaczmarek, F. S., Zaniewski, R. P., Gootz, T. D. & 12 other authors(2001). Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus. J Bacteriol 183, 3016–3024.[CrossRef] [Google Scholar]
  12. Karam, J. D., Leach, M. & Heere, L. J.(1979). Functional interactions beween the DNA ligase of Escherichia coli and components of the DNA metabolic apparatus of T4 bacteriophage. Genetics 91, 177–189. [Google Scholar]
  13. Kodama, K.-I., Barnes, D. E. & Lindahl, T.(1991).In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I. Nucleic Acids Res 19, 6093–6099.[CrossRef] [Google Scholar]
  14. Konrad, E. B., Modrich, P. & Lehman, I. R.(1973). Genetic and enzymatic characterization of a conditional lethal mutant of Escherichia coli K12 with a temperature-sensitive DNA ligase. J Mol Biol 77, 519–529.[CrossRef] [Google Scholar]
  15. Lee, J. Y., Chang, C., Song, H. K., Moon, J., Yang, J. K., Kim, H. K., Kwon, S. T. & Suh, S. W.(2000). Crystal structure of NAD(+)-dependent DNA ligase: modular architecture and functional implications. EMBO J 19, 1119–1129.[CrossRef] [Google Scholar]
  16. Lehman, I. R.(1974). DNA ligase: structure, mechanism, and function. Science 186, 790–797.[CrossRef] [Google Scholar]
  17. Modrich, P. & Lehman, I. R.(1971). Enzymatic characterization of a mutant of Escherichia coli with an altered DNA ligase. Proc Natl Acad Sci U S A 68, 1002–1005.[CrossRef] [Google Scholar]
  18. Petit, M. A. & Ehrlich, S. D.(2000). The NAD-dependent ligase encoded by yerG is an essential gene of Bacillus subtilis. Nucleic Acids Res 28, 4642–4648.[CrossRef] [Google Scholar]
  19. Ren, Z. J., Baumann, R. G. & Black, L. W.(1997). Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector. Gene 195, 303–311.[CrossRef] [Google Scholar]
  20. Sambrook, J. & Russell, D.(2001).Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  21. Sassetti, C. M., Boyd, D. H. & Rubin, E. J.(2003). Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48, 77–84.[CrossRef] [Google Scholar]
  22. Singleton, M. R., Hakansson, K., Timson, D. J. & Wigley, D. B.(1999). Structure of the adenylation domain of an NAD+-dependent DNA ligase. Structure 7, 35–42.[CrossRef] [Google Scholar]
  23. Sriskanda, V. & Shuman, S.(2002). Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+. J Biol Chem 277, 9695–9700.[CrossRef] [Google Scholar]
  24. Sriskanda, V., Schwer, B., Ho, C. K. & Shuman, S.(1999). Mutational analysis of Escherichia coli DNA ligase identifies amino acids required for nick-ligation in vitro and for in vivo complementation of the growth of yeast cells deleted for CDC9 and LIG4. Nucleic Acids Res 27, 3953–3963.[CrossRef] [Google Scholar]
  25. Sriskanda, V., Moyer, R. W. & Shuman, S.(2001). NAD+-dependent DNA ligase encoded by a eukaryotic virus. J Biol Chem 276, 36100–36109.[CrossRef] [Google Scholar]
  26. Timson, D. J. & Wigley, D. B.(1999). Functional domains of an NAD+-dependent DNA ligase. J Mol Biol 285, 73–83.[CrossRef] [Google Scholar]
  27. Timson, D. J., Singleton, M. R. & Wigley, D. B.(2000). DNA ligases in the repair and replication of DNA. Mutat Res 460, 301–318.[CrossRef] [Google Scholar]
  28. Tong, J., Barany, F. & Cao, W.(2000). Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus. Nucleic Acids Res 28, 1447–1454.[CrossRef] [Google Scholar]
  29. Wallis, R., Moore, G. R., James, R. & Kleanthous, C.(1995). Protein–protein interactions in colicin E9 DNase-immunity protein complexes. 1. Diffusion-controlled association and femtomolar binding for the cognate complex. Biochemistry 34, 13743–13750.[CrossRef] [Google Scholar]
  30. Wilkinson, A., Day, J. & Bowater, R.(2001). Bacterial DNA ligases. Mol Microbiol 40, 1241–1248.[CrossRef] [Google Scholar]
  31. Wilkinson, A., Sayer, H., Bullard, D., Smith, A., Day, J., Kieser, T. & Bowater, R.(2003). NAD+-dependent DNA ligases of Mycobacterium tuberculosis and Streptomyces coelicolor. Proteins Struct Funct Genet 51, 321–326.[CrossRef] [Google Scholar]

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error