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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Tamura H,Kohchi C,Yamada R,Ikeda T,Koiwai O,Patterson E,Keene JD,Okada K,Kjeldsen E,Nishikawa K

doi

10.1093/nar/19.1.69

subject

Has Abstract,Author List Incomplete

pub_date

1991-01-11 00:00:00

pages

69-75

issue

1

eissn

0305-1048

issn

1362-4962

journal_volume

19

pub_type

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