Camptothecin resistance from a single mutation changing glycine 363 of human DNA topoisomerase I to cysteine.

Abstract

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 TOP1 complementary DNA in YCpGAL1-hTOP1, 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 transformants 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 an interdomainal cleft.