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      Identification of a mutant human topoisomerase I with intact catalytic activity and resistance to 9-nitro-camptothecin.

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          Abstract

          Human U-937 myeloid leukemia cells were selected for resistance to increasing concentrations of the camptothecin derivative, 9-nitro-20(S)camptothecin (9-NC). The isolated single cell clone, designated U-937/CR, was approximately 20-fold resistant to 9-NC. Analysis of topoisomerase I (topo I) gene expression in U-937/CR cells demonstrated similar mRNA levels as compared with U-937 cells. Immunoblotting with an anti-topo I serum revealed reactive proteins at 100, 75, and 67 kDa which were expressed at the same level in the parental and 9-NC-resistant clones. These cell lines also demonstrated similar levels of topo I catalytic activity as determined by assaying nuclear extracts for relaxation of supercoiled plasmid DNA. In contrast, catalytic assays performed in the presence of 9-NC demonstrated that topo I activity from U-937/CR cells was approximately 10-fold more resistant than that from U-937 cells. Nucleotide sequencing of topo I cDNAs revealed the substitution of phenylalanine (TTC) at residue 361 in U-937 cells with serine (TCC) in the 9-NC-resistant clone. Expression and partial purification of the mutant topo I protein in Escherichia coli demonstrated resistance of this enzyme to 9-NC in catalytic assays. Taken together, these findings identify a novel mutation in topo I which confers resistance to 9-NC and support the involvement of this region in the interaction between topo I and 9-NC.

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          Author and article information

          Journal
          J. Biol. Chem.
          The Journal of biological chemistry
          0021-9258
          0021-9258
          Jan 28 1994
          : 269
          : 4
          Affiliations
          [1 ] Division of Cancer Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
          Article
          8300570
          a43116a1-a5d9-45ca-848f-f16035bf4b91
          History

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