PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of narLink to Publisher's site
 
Nucleic Acids Res. 1991 January 11; 19(1): 69–75.
PMCID: PMC333535

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites.

Abstract

Camptothecin (CPT), a plant alkaloid with antitumor activity, is a specific inhibitor of eukaryotic DNA topoisomerase I. We have previously isolated and characterized a CPT-resistant topoisomerase I isolated from a CPT-resistant human leukemia cell line, CPT-K5. cDNA clones of topoisomerase I were isolated from the CPT-resistant and the parental CPT-sensitive cell lines, respectively. Sequencing of the clones identified two mutations in the cDNA isolated from the resistant cells, which cause amino acid changes from aspartic acid to glycine at residues 533 and 583 of the parental topoisomerase I. When the CPT-K5 topoisomerase I was expressed in E. coli as a fusion protein with Staphylococcal Protein A fragment, the activity was resistant to CPT at a dose level up to 125 microM, whereas the parental fusion protein was sensitive to CPT as low as 1 microM. The resistance index (greater than 125) of the CPT-K5 fusion topoisomerase I is similar to that of the native CPT-K5 topoisomerase I. These results indicate that either or both of the two amino acid changes identified in the mutant enzyme is responsible for the resistance to CPT.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Wang JC. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. [PubMed]
  • Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem. 1989;58:351–375. [PubMed]
  • Hsiang YH, Hertzberg R, Hecht S, Liu LF. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed]
  • Abelson HT, Penman S. Selective interruption of high molecular weight RNA synthesis in HeLa cells by camptothecin. Nat New Biol. 1972 May 31;237(74):144–146. [PubMed]
  • Kessel D. Effects of camptothecin on RNA synthesis in leukemia L1210 cells. Biochim Biophys Acta. 1971 Aug 26;246(2):225–232. [PubMed]
  • Kessel D, Bosmann HB, Lohr K. Camptothecin effects on DNA synthesis in murine leukemia cells. Biochim Biophys Acta. 1972 May 10;269(2):210–216. [PubMed]
  • Hsiang YH, Liu LF. 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]
  • Andoh T, Ishii K, Suzuki Y, Ikegami Y, Kusunoki Y, Takemoto Y, Okada K. Characterization of a mammalian mutant with a camptothecin-resistant DNA topoisomerase I. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5565–5569. [PubMed]
  • Kjeldsen E, Bonven BJ, Andoh T, Ishii K, Okada K, Bolund L, Westergaard O. Characterization of a camptothecin-resistant human DNA topoisomerase I. J Biol Chem. 1988 Mar 15;263(8):3912–3916. [PubMed]
  • Kunze N, Yang GC, Jiang ZY, Hameister H, Adolph S, Wiedorn KH, Richter A, Knippers R. Localization of the active type I DNA topoisomerase gene on human chromosome 20q11.2-13.1, and two pseudogenes on chromosomes 1q23-24 and 22q11.2-13.1. Hum Genet. 1989 Dec;84(1):6–10. [PubMed]
  • Lynn RM, Bjornsti MA, Caron PR, Wang JC. Peptide sequencing and site-directed mutagenesis identify tyrosine-727 as the active site tyrosine of Saccharomyces cerevisiae DNA topoisomerase I. Proc Natl Acad Sci U S A. 1989 May;86(10):3559–3563. [PubMed]
  • Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed]
  • Shero JH, Bordwell B, Rothfield NF, Earnshaw WC. High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients. Science. 1986 Feb 14;231(4739):737–740. [PubMed]
  • Young RA, Davis RW. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. [PubMed]
  • Thrash C, Bankier AT, Barrell BG, 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. [PubMed]
  • Ishii K, Hasegawa T, Fujisawa K, Andoh T. Rapid purification and characterization of DNA topoisomerase I from cultured mouse mammary carcinoma FM3A cells. J Biol Chem. 1983 Oct 25;258(20):12728–12732. [PubMed]
  • Bjornsti MA, Wang JC. Expression of yeast DNA topoisomerase I can complement a conditional-lethal DNA topoisomerase I mutation in Escherichia coli. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8971–8975. [PubMed]
  • Nishikawa K, Ooi T. Radial locations of amino acid residues in a globular protein: correlation with the sequence. J Biochem. 1986 Oct;100(4):1043–1047. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Towbin H, Gordon J. Immunoblotting and dot immunobinding--current status and outlook. J Immunol Methods. 1984 Sep 4;72(2):313–340. [PubMed]
  • D'Arpa P, Machlin PS, Ratrie H, 3rd, Rothfield NF, Cleveland DW, Earnshaw WC. cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2543–2547. [PubMed]
  • Uemura T, Morino K, Uzawa S, Shiozaki K, Yanagida M. Cloning and sequencing of Schizosaccharomyces pombe DNA topoisomerase I gene, and effect of gene disruption. Nucleic Acids Res. 1987 Dec 10;15(23):9727–9739. [PMC free article] [PubMed]
  • Shuman S, Moss B. Identification of a vaccinia virus gene encoding a type I DNA topoisomerase. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7478–7482. [PubMed]
  • Nilsson B, Holmgren E, Josephson S, Gatenbeck S, Philipson L, Uhlen M. Efficient secretion and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci. Nucleic Acids Res. 1985 Feb 25;13(4):1151–1162. [PMC free article] [PubMed]
  • Richards FM. Areas, volumes, packing and protein structure. Annu Rev Biophys Bioeng. 1977;6:151–176. [PubMed]
  • Rose GD, Gierasch LM, Smith JA. Turns in peptides and proteins. Adv Protein Chem. 1985;37:1–109. [PubMed]
  • Liu LF, Miller KG. 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. [PubMed]
  • Gupta RS, Gupta R, Eng B, Lock RB, Ross WE, Hertzberg RP, Caranfa MJ, Johnson RK. Camptothecin-resistant mutants of Chinese hamster ovary cells containing a resistant form of topoisomerase I. Cancer Res. 1988 Nov 15;48(22):6404–6410. [PubMed]
  • Huff AC, Leatherwood JK, Kreuzer KN. Bacteriophage T4 DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in T4-infected Escherichia coli. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1307–1311. [PubMed]
  • Shuman S, Golder M, Moss B. Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. J Biol Chem. 1988 Nov 5;263(31):16401–16407. [PubMed]

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