Tethering a type IB topoisomerase to a DNA site by enzyme fusion to a heterologous site-selective DNA-binding protein domain

Topoisomerase IB (Top1) has essential functions in higher eukaryotes, but e ffective anticancer agents can transform it into a lethal DNA-cleaving toxi n. Fusion of the yeast Gal4 DNA-binding protein domain (amino acids 1-105; Gal4DBD) to the NH, terminus of full-length human Top1 results in a GalTop chimera that maintains basic properties of the two parent proteins. DNA cle avage and binding activities of GalTop were then compared to Top1 to establ ish whether the fusion protein had altered site specificity. Under conditio ns of reduced binding of Top1 to DNA, Gal4DBD was able to selectively ancho r the chimera on a template containing a Gal4 consensus moth, thus bringing Top1 to cleave 20-40-bp sequences close to the Gal4 motif with high specif icity. Footprinting analyses showed that the chimera protected a DNA region that was wider than that protected by a Gal4DBD protein fragment, consiste nt with the cleavage results. The data demonstrate that a Top1 can be targe ted to a specific DNA site by protein fusion to a heterologous DNA-binding domain. Such hybrid topoisomerase-derived enzymes may be useful for directi ng Top1 activity to specific genomic loci in living cells.