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. 1995 Jun;15(6):3206–3216. doi: 10.1128/mcb.15.6.3206

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.

Y F Wei 1, P Robins 1, K Carter 1, K Caldecott 1, D J Pappin 1, G L Yu 1, R P Wang 1, B K Shell 1, R A Nash 1, P Schär 1, et al.
PMCID: PMC230553  PMID: 7760816

Abstract

Three distinct DNA ligases, I to III, have been found previously in mammalian cells, but a cloned cDNA has been identified only for DNA ligase I, an essential enzyme active in DNA replication. A short peptide sequence conserved close to the C terminus of all known eukaryotic DNA ligases was used to search for additional homologous sequences in human cDNA libraries. Two different incomplete cDNA clones that showed partial homology to the conserved peptide were identified. Full-length cDNAs were obtained and expressed by in vitro transcription and translation. The 103-kDa product of one cDNA clone formed a characteristic complex with the XRCC1 DNA repair protein and was identical with the previously described DNA ligase III. DNA ligase III appears closely related to the smaller DNA ligase II. The 96-kDa in vitro translation product of the second cDNA clone was also shown to be an ATP-dependent DNA ligase. A fourth DNA ligase (DNA ligase IV) has been purified from human cells and shown to be identical to the 96-kDa DNA ligase by unique agreement between mass spectrometry data on tryptic peptides from the purified enzyme and the predicted open reading frame of the cloned cDNA. The amino acid sequences of DNA ligases III and IV share a related active-site motif and several short regions of homology with DNA ligase I, other DNA ligases, and RNA capping enzymes. DNA ligases III and IV are encoded by distinct genes located on human chromosomes 17q11.2-12 and 13q33-34, respectively.

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Selected References

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  1. Adams M. D., Kelley J. M., Gocayne J. D., Dubnick M., Polymeropoulos M. H., Xiao H., Merril C. R., Wu A., Olde B., Moreno R. F. Complementary DNA sequencing: expressed sequence tags and human genome project. Science. 1991 Jun 21;252(5013):1651–1656. doi: 10.1126/science.2047873. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Berger R., Le Coniat M., Derré J., Vecchione D. Secondary nonrandom chromosomal abnormalities of band 13q34 in Burkitt lymphoma-leukemia. Genes Chromosomes Cancer. 1989 Nov;1(2):115–118. doi: 10.1002/gcc.2870010202. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Dianov G., Lindahl T. Reconstitution of the DNA base excision-repair pathway. Curr Biol. 1994 Dec 1;4(12):1069–1076. doi: 10.1016/s0960-9822(00)00245-1. [DOI] [PubMed] [Google Scholar]
  7. Gatti R. A., Berkel I., Boder E., Braedt G., Charmley P., Concannon P., Ersoy F., Foroud T., Jaspers N. G., Lange K. Localization of an ataxia-telangiectasia gene to chromosome 11q22-23. Nature. 1988 Dec 8;336(6199):577–580. doi: 10.1038/336577a0. [DOI] [PubMed] [Google Scholar]
  8. German J., Roe A. M., Leppert M. F., Ellis N. A. Bloom syndrome: an analysis of consanguineous families assigns the locus mutated to chromosome band 15q26.1. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6669–6673. doi: 10.1073/pnas.91.14.6669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Higashitani A., Tabata S., Endo H., Hotta Y. Purification of DNA ligases from mouse testis and their behavior during meiosis. Cell Struct Funct. 1990 Feb;15(1):67–72. doi: 10.1247/csf.15.67. [DOI] [PubMed] [Google Scholar]
  10. Ikejima M., Noguchi S., Yamashita R., Ogura T., Sugimura T., Gill D. M., Miwa M. The zinc fingers of human poly(ADP-ribose) polymerase are differentially required for the recognition of DNA breaks and nicks and the consequent enzyme activation. Other structures recognize intact DNA. J Biol Chem. 1990 Dec 15;265(35):21907–21913. [PubMed] [Google Scholar]
  11. Jessberger R., Podust V., Hübscher U., Berg P. A mammalian protein complex that repairs double-strand breaks and deletions by recombination. J Biol Chem. 1993 Jul 15;268(20):15070–15079. [PubMed] [Google Scholar]
  12. Johnson C. V., McNeil J. A., Carter K. C., Lawrence J. B. A simple, rapid technique for precise mapping of multiple sequences in two colors using a single optical filter set. Genet Anal Tech Appl. 1991 Apr;8(2):75–76. doi: 10.1016/1050-3862(91)90052-s. [DOI] [PubMed] [Google Scholar]
  13. Johnson C. V., Singer R. H., Lawrence J. B. Fluorescent detection of nuclear RNA and DNA: implications for genome organization. Methods Cell Biol. 1991;35:73–99. [PubMed] [Google Scholar]
  14. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lawrence J. B., Villnave C. A., Singer R. H. Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell. 1988 Jan 15;52(1):51–61. doi: 10.1016/0092-8674(88)90530-2. [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. 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]
  18. 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]
  19. Masson N., Hurst H. C., Lee K. A. Identification of proteins that interact with CREB during differentiation of F9 embryonal carcinoma cells. Nucleic Acids Res. 1993 Jun 11;21(11):1163–1169. doi: 10.1093/nar/21.5.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Masutani C., Sugasawa K., Yanagisawa J., Sonoyama T., Ui M., Enomoto T., Takio K., Tanaka K., van der Spek P. J., Bootsma D. Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23. EMBO J. 1994 Apr 15;13(8):1831–1843. doi: 10.1002/j.1460-2075.1994.tb06452.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Modrich P. Mismatch repair, genetic stability and tumour avoidance. Philos Trans R Soc Lond B Biol Sci. 1995 Jan 30;347(1319):89–95. doi: 10.1098/rstb.1995.0014. [DOI] [PubMed] [Google Scholar]
  22. Nelson W. G., Kastan M. B. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol. 1994 Mar;14(3):1815–1823. doi: 10.1128/mcb.14.3.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Noguiez P., Barnes D. E., Mohrenweiser H. W., Lindahl T. Structure of the human DNA ligase I gene. Nucleic Acids Res. 1992 Aug 11;20(15):3845–3850. doi: 10.1093/nar/20.15.3845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Op het Veld C. W., Zdzienicka M. Z., Vrieling H., Lohman P. H., van Zeeland A. A. Molecular analysis of ethyl methanesulfonate-induced mutations at the hprt gene in the ethyl methanesulfonate-sensitive Chinese hamster cell line EM-C11 and its parental line CHO9. Cancer Res. 1994 Jun 1;54(11):3001–3006. [PubMed] [Google Scholar]
  25. Pappin D. J., Coull J. M., Köster H. Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes. Anal Biochem. 1990 May 15;187(1):10–19. doi: 10.1016/0003-2697(90)90410-b. [DOI] [PubMed] [Google Scholar]
  26. Pappin D. J., Hojrup P., Bleasby A. J. Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol. 1993 Jun 1;3(6):327–332. doi: 10.1016/0960-9822(93)90195-t. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  29. Roberts E., Nash R. A., Robins P., Lindahl T. Different active sites of mammalian DNA ligases I and II. J Biol Chem. 1994 Feb 4;269(5):3789–3792. [PubMed] [Google Scholar]
  30. Robins P., Pappin D. J., Wood R. D., Lindahl T. Structural and functional homology between mammalian DNase IV and the 5'-nuclease domain of Escherichia coli DNA polymerase I. J Biol Chem. 1994 Nov 18;269(46):28535–28538. [PubMed] [Google Scholar]
  31. Samec S., Jones T. A., Corlet J., Scherly D., Sheer D., Wood R. D., Clarkson S. G. The human gene for xeroderma pigmentosum complementation group G (XPG) maps to 13q33 by fluorescence in situ hybridization. Genomics. 1994 May 1;21(1):283–285. doi: 10.1006/geno.1994.1261. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. 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]
  35. Thompson L. H., Brookman K. W., Jones N. J., Allen S. A., Carrano A. V. Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange. Mol Cell Biol. 1990 Dec;10(12):6160–6171. doi: 10.1128/mcb.10.12.6160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  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. Webster A. D., Barnes D. E., Arlett C. F., Lehmann A. R., Lindahl T. Growth retardation and immunodeficiency in a patient with mutations in the DNA ligase I gene. Lancet. 1992 Jun 20;339(8808):1508–1509. doi: 10.1016/0140-6736(92)91266-b. [DOI] [PubMed] [Google Scholar]
  42. de Murcia G., Ménissier de Murcia J. Poly(ADP-ribose) polymerase: a molecular nick-sensor. Trends Biochem Sci. 1994 Apr;19(4):172–176. doi: 10.1016/0968-0004(94)90280-1. [DOI] [PubMed] [Google Scholar]
  43. de Thé H., Chomienne C., Lanotte M., Degos L., Dejean A. The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha gene to a novel transcribed locus. Nature. 1990 Oct 11;347(6293):558–561. doi: 10.1038/347558a0. [DOI] [PubMed] [Google Scholar]

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