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. 1998 Apr 1;26(7):1841–1847. doi: 10.1093/nar/26.7.1841

Interaction between the N-terminus of human topoisomerase I and SV40 large T antigen.

P Haluska Jr 1, A Saleem 1, T K Edwards 1, E H Rubin 1
PMCID: PMC147454  PMID: 9512561

Abstract

We have attempted to identify human topoisomerase I-binding proteins in order to gain information regarding the cellular roles of this protein and the cytotoxic mechanisms of the anticancer drug camptothecin, which specifically targets topoisomerase I. In the course of this work we identified an interaction between the N-terminus of human topoisomerase I and the SV40 T antigen that is detectable in vitro using both affinity chromatography and co-immunoprecipitation. Additional results indicate that this interaction does not require intermediary DNA or stoichiometric quantities of other proteins. Furthermore, the interaction is detectable in vivo using a yeast two-hybrid assay. Two binding sites for T antigen are apparent on the topoisomerase I protein: one consisting of amino acids 1-139, the other present in the 383-765 region of the protein. Interestingly, nucleolin, which binds the 166-210 region of topoisomerase I, is able to bind an N-terminal fragment of topoisomerase I concurrently with T antigen. Taken together with our prior identification of nucleolin as a topoisomerase I-binding protein, the current results suggest that helicase-binding is a major role of the N-terminus of human topoisomerase I and that the resultant helicase-topoisomerase complex may function as a eukaryotic gyrase.

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Selected References

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  1. Bharti A. K., Olson M. O., Kufe D. W., Rubin E. H. Identification of a nucleolin binding site in human topoisomerase I. J Biol Chem. 1996 Jan 26;271(4):1993–1997. doi: 10.1074/jbc.271.4.1993. [DOI] [PubMed] [Google Scholar]
  2. Breeden L., Nasmyth K. Regulation of the yeast HO gene. Cold Spring Harb Symp Quant Biol. 1985;50:643–650. doi: 10.1101/sqb.1985.050.01.078. [DOI] [PubMed] [Google Scholar]
  3. Brill S. J., DiNardo S., Voelkel-Meiman K., Sternglanz R. Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA. 1987 Mar 26-Apr 1Nature. 326(6111):414–416. doi: 10.1038/326414a0. [DOI] [PubMed] [Google Scholar]
  4. Christman M. F., Dietrich F. S., Fink G. R. Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerases I and II. Cell. 1988 Nov 4;55(3):413–425. doi: 10.1016/0092-8674(88)90027-x. [DOI] [PubMed] [Google Scholar]
  5. Duguet M. When helicase and topoisomerase meet! J Cell Sci. 1997 Jun;110(Pt 12):1345–1350. doi: 10.1242/jcs.110.12.1345. [DOI] [PubMed] [Google Scholar]
  6. Egyházi E., Durban E. Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation. Mol Cell Biol. 1987 Dec;7(12):4308–4316. doi: 10.1128/mcb.7.12.4308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ellis N. A., Groden J., Ye T. Z., Straughen J., Lennon D. J., Ciocci S., Proytcheva M., German J. The Bloom's syndrome gene product is homologous to RecQ helicases. Cell. 1995 Nov 17;83(4):655–666. doi: 10.1016/0092-8674(95)90105-1. [DOI] [PubMed] [Google Scholar]
  8. Fang S. H., Yeh N. H. The self-cleaving activity of nucleolin determines its molecular dynamics in relation to cell proliferation. Exp Cell Res. 1993 Sep;208(1):48–53. doi: 10.1006/excr.1993.1221. [DOI] [PubMed] [Google Scholar]
  9. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  10. Flick J. S., Johnston M. Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Sep;10(9):4757–4769. doi: 10.1128/mcb.10.9.4757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gangloff S., McDonald J. P., Bendixen C., Arthur L., Rothstein R. The yeast type I topoisomerase Top3 interacts with Sgs1, a DNA helicase homolog: a potential eukaryotic reverse gyrase. Mol Cell Biol. 1994 Dec;14(12):8391–8398. doi: 10.1128/mcb.14.12.8391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gobert C., Bracco L., Rossi F., Olivier M., Tazi J., Lavelle F., Larsen A. K., Riou J. F. Modulation of DNA topoisomerase I activity by p53. Biochemistry. 1996 May 7;35(18):5778–5786. doi: 10.1021/bi952327w. [DOI] [PubMed] [Google Scholar]
  13. Gupta M., Fujimori A., Pommier Y. Eukaryotic DNA topoisomerases I. Biochim Biophys Acta. 1995 May 17;1262(1):1–14. doi: 10.1016/0167-4781(95)00029-g. [DOI] [PubMed] [Google Scholar]
  14. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  15. Holm C., Covey J. M., Kerrigan D., Pommier Y. Differential requirement of DNA replication for the cytotoxicity of DNA topoisomerase I and II inhibitors in Chinese hamster DC3F cells. Cancer Res. 1989 Nov 15;49(22):6365–6368. [PubMed] [Google Scholar]
  16. Hsiang Y. H., Hertzberg R., Hecht S., Liu L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed] [Google Scholar]
  17. Hsiang Y. H., Lihou M. G., Liu L. F. Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by camptothecin. Cancer Res. 1989 Sep 15;49(18):5077–5082. [PubMed] [Google Scholar]
  18. Hsiang Y. H., Liu L. F. Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res. 1988 Apr 1;48(7):1722–1726. [PubMed] [Google Scholar]
  19. Hsiang Y. H., Liu L. F., Wall M. E., Wani M. C., Nicholas A. W., Manikumar G., Kirschenbaum S., Silber R., Potmesil M. DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues. Cancer Res. 1989 Aug 15;49(16):4385–4389. [PubMed] [Google Scholar]
  20. Kim R. A., Wang J. C. Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase. J Biol Chem. 1992 Aug 25;267(24):17178–17185. [PubMed] [Google Scholar]
  21. Kretzschmar M., Meisterernst M., Roeder R. G. Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11508–11512. doi: 10.1073/pnas.90.24.11508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lai J. S., Herr W. Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6958–6962. doi: 10.1073/pnas.89.15.6958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lane D. P., Crawford L. V. T antigen is bound to a host protein in SV40-transformed cells. Nature. 1979 Mar 15;278(5701):261–263. doi: 10.1038/278261a0. [DOI] [PubMed] [Google Scholar]
  24. Lanford R. E. Expression of simian virus 40 T antigen in insect cells using a baculovirus expression vector. Virology. 1988 Nov;167(1):72–81. doi: 10.1016/0042-6822(88)90055-4. [DOI] [PubMed] [Google Scholar]
  25. Liu L. F., Miller K. G. Eukaryotic DNA topoisomerases: two forms of type I DNA topoisomerases from HeLa cell nuclei. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3487–3491. doi: 10.1073/pnas.78.6.3487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liu L. F., Wang J. C. Supercoiling of the DNA template during transcription. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7024–7027. doi: 10.1073/pnas.84.20.7024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lu J., Mullen J. R., Brill S. J., Kleff S., Romeo A. M., Sternglanz R. Human homologues of yeast helicase. Nature. 1996 Oct 24;383(6602):678–679. doi: 10.1038/383678a0. [DOI] [PubMed] [Google Scholar]
  28. Merino A., Madden K. R., Lane W. S., Champoux J. J., Reinberg D. DNA topoisomerase I is involved in both repression and activation of transcription. Nature. 1993 Sep 16;365(6443):227–232. doi: 10.1038/365227a0. [DOI] [PubMed] [Google Scholar]
  29. Mirabella A., Gartenberg M. R. Yeast telomeric sequences function as chromosomal anchorage points in vivo. EMBO J. 1997 Feb 3;16(3):523–533. doi: 10.1093/emboj/16.3.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mitchell D. A., Marshall T. K., Deschenes R. J. Vectors for the inducible overexpression of glutathione S-transferase fusion proteins in yeast. Yeast. 1993 Jul;9(7):715–722. doi: 10.1002/yea.320090705. [DOI] [PubMed] [Google Scholar]
  31. Morham S. G., Kluckman K. D., Voulomanos N., Smithies O. Targeted disruption of the mouse topoisomerase I gene by camptothecin selection. Mol Cell Biol. 1996 Dec;16(12):6804–6809. doi: 10.1128/mcb.16.12.6804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Morris E. J., Geller H. M. Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity. J Cell Biol. 1996 Aug;134(3):757–770. doi: 10.1083/jcb.134.3.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rossi F., Labourier E., Forné T., Divita G., Derancourt J., Riou J. F., Antoine E., Cathala G., Brunel C., Tazi J. Specific phosphorylation of SR proteins by mammalian DNA topoisomerase I. Nature. 1996 May 2;381(6577):80–82. doi: 10.1038/381080a0. [DOI] [PubMed] [Google Scholar]
  34. Rubin E., Pantazis P., Bharti A., Toppmeyer D., Giovanella B., Kufe D. Identification of a mutant human topoisomerase I with intact catalytic activity and resistance to 9-nitro-camptothecin. J Biol Chem. 1994 Jan 28;269(4):2433–2439. [PubMed] [Google Scholar]
  35. Saleem A., Kharbanda S., Yuan Z. M., Kufe D. Monocyte colony-stimulating factor stimulates binding of phosphatidylinositol 3-kinase to Grb2.Sos complexes in human monocytes. J Biol Chem. 1995 May 5;270(18):10380–10383. doi: 10.1074/jbc.270.18.10380. [DOI] [PubMed] [Google Scholar]
  36. Scheffner M., Knippers R., Stahl H. RNA unwinding activity of SV40 large T antigen. Cell. 1989 Jun 16;57(6):955–963. doi: 10.1016/0092-8674(89)90334-6. [DOI] [PubMed] [Google Scholar]
  37. Simanis V., Lane D. P. An immunoaffinity purification procedure for SV40 large T antigen. Virology. 1985 Jul 15;144(1):88–100. doi: 10.1016/0042-6822(85)90308-3. [DOI] [PubMed] [Google Scholar]
  38. Simmons D. T., Melendy T., Usher D., Stillman B. Simian virus 40 large T antigen binds to topoisomerase I. Virology. 1996 Aug 15;222(2):365–374. doi: 10.1006/viro.1996.0433. [DOI] [PubMed] [Google Scholar]
  39. Stahl H., Dröge P., Knippers R. DNA helicase activity of SV40 large tumor antigen. EMBO J. 1986 Aug;5(8):1939–1944. doi: 10.1002/j.1460-2075.1986.tb04447.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stewart L., Ireton G. C., Parker L. H., Madden K. R., Champoux J. J. Biochemical and biophysical analyses of recombinant forms of human topoisomerase I. J Biol Chem. 1996 Mar 29;271(13):7593–7601. doi: 10.1074/jbc.271.13.7593. [DOI] [PubMed] [Google Scholar]
  41. Stillman B. W., Gluzman Y. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol. 1985 Aug;5(8):2051–2060. doi: 10.1128/mcb.5.8.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thrash C., Bankier A. T., Barrell B. G., Sternglanz R. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4374–4378. doi: 10.1073/pnas.82.13.4374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Thrash C., Voelkel K., DiNardo S., Sternglanz R. Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity. J Biol Chem. 1984 Feb 10;259(3):1375–1377. [PubMed] [Google Scholar]
  44. Tsao Y. P., Russo A., Nyamuswa G., Silber R., Liu L. F. Interaction between replication forks and topoisomerase I-DNA cleavable complexes: studies in a cell-free SV40 DNA replication system. Cancer Res. 1993 Dec 15;53(24):5908–5914. [PubMed] [Google Scholar]
  45. Tuteja N., Huang N. W., Skopac D., Tuteja R., Hrvatic S., Zhang J., Pongor S., Joseph G., Faucher C., Amalric F. Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation. Gene. 1995 Jul 28;160(2):143–148. doi: 10.1016/0378-1119(95)00207-m. [DOI] [PubMed] [Google Scholar]
  46. Uemura T., Morikawa K., Yanagida M. The nucleotide sequence of the fission yeast DNA topoisomerase II gene: structural and functional relationships to other DNA topoisomerases. EMBO J. 1986 Sep;5(9):2355–2361. doi: 10.1002/j.1460-2075.1986.tb04504.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Uemura T., Yanagida M. Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 1984 Aug;3(8):1737–1744. doi: 10.1002/j.1460-2075.1984.tb02040.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wall M. E., Wani M. C. Camptothecin and taxol: discovery to clinic--thirteenth Bruce F. Cain Memorial Award Lecture. Cancer Res. 1995 Feb 15;55(4):753–760. [PubMed] [Google Scholar]
  49. Wallis J. W., Chrebet G., Brodsky G., Rolfe M., Rothstein R. A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell. 1989 Jul 28;58(2):409–419. doi: 10.1016/0092-8674(89)90855-6. [DOI] [PubMed] [Google Scholar]
  50. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1996;65:635–692. doi: 10.1146/annurev.bi.65.070196.003223. [DOI] [PubMed] [Google Scholar]
  51. Watt P. M., Louis E. J., Borts R. H., Hickson I. D. Sgs1: a eukaryotic homolog of E. coli RecQ that interacts with topoisomerase II in vivo and is required for faithful chromosome segregation. Cell. 1995 Apr 21;81(2):253–260. doi: 10.1016/0092-8674(95)90335-6. [DOI] [PubMed] [Google Scholar]
  52. Wun-Kim K., Upson R., Young W., Melendy T., Stillman B., Simmons D. T. The DNA-binding domain of simian virus 40 tumor antigen has multiple functions. J Virol. 1993 Dec;67(12):7608–7611. doi: 10.1128/jvi.67.12.7608-7611.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yang L., Wold M. S., Li J. J., Kelly T. J., Liu L. F. Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A. 1987 Feb;84(4):950–954. doi: 10.1073/pnas.84.4.950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yocum R. R., Hanley S., West R., Jr, Ptashne M. Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Oct;4(10):1985–1998. doi: 10.1128/mcb.4.10.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yu C. E., Oshima J., Fu Y. H., Wijsman E. M., Hisama F., Alisch R., Matthews S., Nakura J., Miki T., Ouais S. Positional cloning of the Werner's syndrome gene. Science. 1996 Apr 12;272(5259):258–262. doi: 10.1126/science.272.5259.258. [DOI] [PubMed] [Google Scholar]

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