Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Apr 15;25(8):1485–1492. doi: 10.1093/nar/25.8.1485

Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

W Ramos 1, N Tappe 1, J Talamantez 1, E C Friedberg 1, A E Tomkinson 1
PMCID: PMC146610  PMID: 9092653

Abstract

Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.

Full Text

The Full Text of this article is available as a PDF (145.6 KB).

Selected References

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

  1. Arrand J. E., Willis A. E., Goldsmith I., Lindahl T. Different substrate specificities of the two DNA ligases of mammalian cells. J Biol Chem. 1986 Jul 15;261(20):9079–9082. [PubMed] [Google Scholar]
  2. Banks G. R., Barker D. G. DNA ligase-AMP adducts: identification of yeast DNA ligase polypeptides. Biochim Biophys Acta. 1985 Dec 18;826(4):180–185. doi: 10.1016/0167-4781(85)90004-1. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Barnes D. E., Tomkinson A. E., Lehmann A. R., Webster A. D., Lindahl T. Mutations in the DNA ligase I gene of an individual with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. Cell. 1992 May 1;69(3):495–503. doi: 10.1016/0092-8674(92)90450-q. [DOI] [PubMed] [Google Scholar]
  5. Boulet A., Simon M., Faye G., Bauer G. A., Burgers P. M. Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase III. EMBO J. 1989 Jun;8(6):1849–1854. doi: 10.1002/j.1460-2075.1989.tb03580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Brown G. W., Ray D. S. Purification and characterization of DNA ligase I from the trypanosomatid Crithidia fasciculata. Nucleic Acids Res. 1992 Aug 11;20(15):3905–3910. doi: 10.1093/nar/20.15.3905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Caldecott K. W., McKeown C. K., Tucker J. D., Ljungquist S., Thompson L. H. An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol Cell Biol. 1994 Jan;14(1):68–76. doi: 10.1128/mcb.14.1.68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Caldecott K. W., Tucker J. D., Stanker L. H., Thompson L. H. Characterization of the XRCC1-DNA ligase III complex in vitro and its absence from mutant hamster cells. Nucleic Acids Res. 1995 Dec 11;23(23):4836–4843. doi: 10.1093/nar/23.23.4836. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Hardy S., Aoufouchi S., Thiebaud P., Prigent C. DNA ligase I from Xenopus laevis eggs. Nucleic Acids Res. 1991 Feb 25;19(4):701–705. doi: 10.1093/nar/19.4.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Husain I., Tomkinson A. E., Burkhart W. A., Moyer M. B., Ramos W., Mackey Z. B., Besterman J. M., Chen J. Purification and characterization of DNA ligase III from bovine testes. Homology with DNA ligase II and vaccinia DNA ligase. J Biol Chem. 1995 Apr 21;270(16):9683–9690. doi: 10.1074/jbc.270.16.9683. [DOI] [PubMed] [Google Scholar]
  13. Johnson L. M., Snyder M., Chang L. M., Davis R. W., Campbell J. L. Isolation of the gene encoding yeast DNA polymerase I. Cell. 1985 Nov;43(1):369–377. doi: 10.1016/0092-8674(85)90042-x. [DOI] [PubMed] [Google Scholar]
  14. Johnston L. H., Nasmyth K. A. Saccharomyces cerevisiae cell cycle mutant cdc9 is defective in DNA ligase. Nature. 1978 Aug 31;274(5674):891–893. doi: 10.1038/274891a0. [DOI] [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Li C., Goodchild J., Baril E. F. DNA ligase I is associated with the 21 S complex of enzymes for DNA synthesis in HeLa cells. Nucleic Acids Res. 1994 Feb 25;22(4):632–638. doi: 10.1093/nar/22.4.632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ljungquist S., Kenne K., Olsson L., Sandström M. Altered DNA ligase III activity in the CHO EM9 mutant. Mutat Res. 1994 Mar;314(2):177–186. doi: 10.1016/0921-8777(94)90081-7. [DOI] [PubMed] [Google Scholar]
  18. Mackey Z. B., Ramos W., Levin D. S., Walter C. A., McCarrey J. R., Tomkinson A. E. An alternative splicing event which occurs in mouse pachytene spermatocytes generates a form of DNA ligase III with distinct biochemical properties that may function in meiotic recombination. Mol Cell Biol. 1997 Feb;17(2):989–998. doi: 10.1128/mcb.17.2.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Morrison A., Araki H., Clark A. B., Hamatake R. K., Sugino A. A third essential DNA polymerase in S. cerevisiae. Cell. 1990 Sep 21;62(6):1143–1151. doi: 10.1016/0092-8674(90)90391-q. [DOI] [PubMed] [Google Scholar]
  20. Nasmyth K. A. Temperature-sensitive lethal mutants in the structural gene for DNA ligase in the yeast Schizosaccharomyces pombe. Cell. 1977 Dec;12(4):1109–1120. doi: 10.1016/0092-8674(77)90173-8. [DOI] [PubMed] [Google Scholar]
  21. Petrini J. H., Xiao Y., Weaver D. T. DNA ligase I mediates essential functions in mammalian cells. Mol Cell Biol. 1995 Aug;15(8):4303–4308. doi: 10.1128/mcb.15.8.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Prasad R., Singhal R. K., Srivastava D. K., Molina J. T., Tomkinson A. E., Wilson S. H. Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis. J Biol Chem. 1996 Jul 5;271(27):16000–16007. doi: 10.1074/jbc.271.27.16000. [DOI] [PubMed] [Google Scholar]
  23. Prasad R., Widen S. G., Singhal R. K., Watkins J., Prakash L., Wilson S. H. Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme. Nucleic Acids Res. 1993 Nov 25;21(23):5301–5307. doi: 10.1093/nar/21.23.5301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Prigent C., Satoh M. S., Daly G., Barnes D. E., Lindahl T. Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I. Mol Cell Biol. 1994 Jan;14(1):310–317. doi: 10.1128/mcb.14.1.310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rabin B. A., Hawley R. S., Chase J. W. DNA ligase from Drosophila melanogaster embryos. Purification and physical characterization. J Biol Chem. 1986 Aug 15;261(23):10637–10645. [PubMed] [Google Scholar]
  26. Robins P., Lindahl T. DNA ligase IV from HeLa cell nuclei. J Biol Chem. 1996 Sep 27;271(39):24257–24261. doi: 10.1074/jbc.271.39.24257. [DOI] [PubMed] [Google Scholar]
  27. Shimizu K., Santocanale C., Ropp P. A., Longhese M. P., Plevani P., Lucchini G., Sugino A. Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta. J Biol Chem. 1993 Dec 25;268(36):27148–27153. [PubMed] [Google Scholar]
  28. Söderhäll S. DNA ligases during rat liver regeneration. Nature. 1976 Apr 15;260(5552):640–642. doi: 10.1038/260640a0. [DOI] [PubMed] [Google Scholar]
  29. Söderhäll S., Lindahl T. Mammalian DNA ligases. Serological evidence for two separate enzymes. J Biol Chem. 1975 Nov 10;250(21):8438–8444. [PubMed] [Google Scholar]
  30. Söderhäll S., Lindahl T. Two DNA ligase activities from calf thymus. Biochem Biophys Res Commun. 1973 Aug 6;53(3):910–916. doi: 10.1016/0006-291x(73)90178-2. [DOI] [PubMed] [Google Scholar]
  31. Takahashi M., Senshu M. Two distinct DNA ligases from Drosophila melanogaster embryos. FEBS Lett. 1987 Mar 23;213(2):345–352. doi: 10.1016/0014-5793(87)81520-x. [DOI] [PubMed] [Google Scholar]
  32. Takahashi M., Tomizawa K. Purification and characterization of DNA ligase II from Drosophila melanogaster. Eur J Biochem. 1990 Sep 24;192(3):735–740. doi: 10.1111/j.1432-1033.1990.tb19284.x. [DOI] [PubMed] [Google Scholar]
  33. Teraoka H., Sumikawa T., Tsukada K. Purification of DNA ligase II from calf thymus and preparation of rabbit antibody against calf thymus DNA ligase II. J Biol Chem. 1986 May 25;261(15):6888–6892. [PubMed] [Google Scholar]
  34. Tomkinson A. E., Bardwell A. J., Tappe N., Ramos W., Friedberg E. C. Purification of Rad1 protein from Saccharomyces cerevisiae and further characterization of the Rad1/Rad10 endonuclease complex. Biochemistry. 1994 May 3;33(17):5305–5311. doi: 10.1021/bi00183a038. [DOI] [PubMed] [Google Scholar]
  35. Tomkinson A. E., Lasko D. D., Daly G., Lindahl T. Mammalian DNA ligases. Catalytic domain and size of DNA ligase I. J Biol Chem. 1990 Jul 25;265(21):12611–12617. [PubMed] [Google Scholar]
  36. Tomkinson A. E., Roberts E., Daly G., Totty N. F., Lindahl T. Three distinct DNA ligases in mammalian cells. J Biol Chem. 1991 Nov 15;266(32):21728–21735. [PubMed] [Google Scholar]
  37. Tomkinson A. E., Tappe N. J., Friedberg E. C. DNA ligase I from Saccharomyces cerevisiae: physical and biochemical characterization of the CDC9 gene product. Biochemistry. 1992 Dec 1;31(47):11762–11771. doi: 10.1021/bi00162a013. [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. Waga S., Bauer G., Stillman B. Reconstitution of complete SV40 DNA replication with purified replication factors. J Biol Chem. 1994 Apr 8;269(14):10923–10934. [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. 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]

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

RESOURCES