Camptothecin Resistance from a Single Mutation Changing Glycine 363 of Human DNA Topoisomerase I to Cysteine

A full-length human DNA topoisomerase I complementary DNA clone was mutagenized in vitro and the mutagenized DNA was used to replace wild-type human TOPI complementary DNA in YCpGALl-hTOPl, a plasmid constructed for the expression of the human enzyme in yeast A yeast strain devoid of yeast DNA topoisomerase I and permeable to the anticancer drug camptothecin was transformed with the plasmid pool. Assays of DNA topoisomerase I in lysates of camptothecin-resistant trans-formants identified one with nearly the same level of the enzyme as transformants of unmutagenized YCpGAL1-hTOP1, and a single mutation changing Gly363 to a cysteine was found in this mutant. The G363C mutant enzyme was overexpressed in yeast and partially purified. It differed significantly from wild-type human DNA topoisomerase I similarly expressed and purified: camptothecin-stimulated cleavage of DNA was observed with the wild-type but not the G363C enzyme, and the DNA relaxation activity of the mutant enzyme, unlike that of the wild-type enzyme, was not significantly stimulated by Mg(II). The positions of the G363C and other previously reported camptothecin resistance mutations in eukaryotic DNA topoisomerase I were discussed in terms of a model in which the active site is in an interdomainal cleft. © 1993, American Association for Cancer Research. All rights reserved.