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

Hiroomi Tamura, Chie Kohchi, Ryutaro Yamada, Tomotake Ikeda, Osamu Koiwai, Eric Patterson, Jack D. Keene, Kosuke Okada, Eigil Kjeldsen, Ken Nishikawa, Toshiwo Andoh

Research output: Contribution to journalArticle

127 Citations (Scopus)

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 μM, whereas the parental fusion protein was sensitive to CPT as low as 1 μM. The resistance index (>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.

Original languageEnglish
Pages (from-to)69-75
Number of pages7
JournalNucleic Acids Research
Volume19
Issue number1
Publication statusPublished - 1991 Jan 11
Externally publishedYes

Fingerprint

Camptothecin
Cloning
Molecular Cloning
CDNA
Amino acids
Fusion
Mutation
DNA
Fusion reactions
Complementary DNA
Type I DNA Topoisomerase
Proteins
Protein
Clone
Amino Acids
Cell
Cells
Alkaloids
Line
Leukemia

ASJC Scopus subject areas

  • Genetics
  • Statistics, Probability and Uncertainty
  • Applied Mathematics
  • Health, Toxicology and Mutagenesis
  • Toxicology
  • Genetics(clinical)

Cite this

Tamura, H., Kohchi, C., Yamada, R., Ikeda, T., Koiwai, O., Patterson, E., ... Andoh, T. (1991). Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites. Nucleic Acids Research, 19(1), 69-75.

Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites. / Tamura, Hiroomi; Kohchi, Chie; Yamada, Ryutaro; Ikeda, Tomotake; Koiwai, Osamu; Patterson, Eric; Keene, Jack D.; Okada, Kosuke; Kjeldsen, Eigil; Nishikawa, Ken; Andoh, Toshiwo.

In: Nucleic Acids Research, Vol. 19, No. 1, 11.01.1991, p. 69-75.

Research output: Contribution to journalArticle

Tamura, H, Kohchi, C, Yamada, R, Ikeda, T, Koiwai, O, Patterson, E, Keene, JD, Okada, K, Kjeldsen, E, Nishikawa, K & Andoh, T 1991, 'Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites', Nucleic Acids Research, vol. 19, no. 1, pp. 69-75.
Tamura, Hiroomi ; Kohchi, Chie ; Yamada, Ryutaro ; Ikeda, Tomotake ; Koiwai, Osamu ; Patterson, Eric ; Keene, Jack D. ; Okada, Kosuke ; Kjeldsen, Eigil ; Nishikawa, Ken ; Andoh, Toshiwo. / Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites. In: Nucleic Acids Research. 1991 ; Vol. 19, No. 1. pp. 69-75.
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