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 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.
Benedetti
P
P
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Fiorani
P
P
Capuani
L
L
Wang
J C
JC
D000595:Amino Acid Sequence; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D004351:Drug Resistance; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D004247:DNA; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin