CLONING OF CHINESE-HAMSTER DNA TOPOISOMERASE-I CDNA AND IDENTIFICATION OF A SINGLE-POINT MUTATION RESPONSIBLE FOR CAMPTOTHECIN RESISTANCE

A camptothecin-resistant (DC3F/C-10) Chinese hamster cell line that co ntains a catalytically altered and camptothecin (CPT)-resistant DNA to poisomerase I (top 1) (Tanizawa, A., and Pommier, Y. (1992) Cancer Res . 52, 1848-1854) and the parent cell line (DC3F) were used to compare top 1 mRNAs and cDNAs. Northern blot analysis showed a single 4.1-kilo base band without quantitative reduction between the two cell lines. W e have cloned and sequenced top 1 cDNAs. DC3F and DC3F/C-10 top 1 c-DN A are 3591 and 3626 base pair long, respectively, and encode 767 amino acids. The homology of deduced amino acid sequences between Chinese h amster and mouse or human top 1 are 98.1 and 96.7, respectively. cDNAs from DC3F/C-10 and DC3F cells differ by a single base point mutation (G to A) which results in an amino acid change from Gly505 to Ser (Gly 505 --> Ser). G505 corresponds to Gly503 of human top 1 cDNA and is lo cated 220 amino acids away from the presumed catalytic Tyr725. The poi nt mutation in the Chinese hamster top 1 is located in a region that i s highly conserved among all cloned top 1 cDNAs (plant ATH, vaccinia v irus, Shope fibroma virus, Drosophila, Saccharomyces cerevisiae, Schiz osaccharomyces pombe, mouse, and Human). A mutation of Asp533 to Gly i n this same region has been shown to confer CPT resistance for human t op 1. Chinese hamster top 1 protein with a Gly505 --> Ser mutation tha t was expressed in bacteria was resistant to CPT, indicating that this single base mutation is involved in CPT resistance. Our results sugge st that the highly conserved region around Gly505 plays an important r ole in the interactions among top 1, DNA, and CPT.