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Tethering a Type IB Topoisomerase to a DNA Site by Enzyme Fusion to a Heterologous Site-selective DNA-binding Protein Domain¹

Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, 20133 Milan [G. L. B., M. B., E. Z., L. C., G. C.]; and Department of Biochemistry "Moruzzi," University of Bologna, 40126 Bologna [G. C.], Italy

Topoisomerase IB (Top1) has essential functions in higher eukaryotes, but effective anticancer agents can transform it into a lethal DNA-cleaving toxin. Fusion of the yeast Gal4 DNA-binding protein domain (amino acids 1–105; Gal4DBD) to the NH₂ terminus of full-length human Top1 results in a GalTop chimera that maintains basic properties of the two parent proteins. DNA cleavage and binding activities of GalTop were then compared to Top1 to establish whether the fusion protein had altered site specificity. Under conditions of reduced binding of Top1 to DNA, Gal4DBD was able to selectively anchor the chimera on a template containing a Gal4 consensus motif, thus bringing Top1 to cleave 20–40-bp sequences close to the Gal4 motif with high specificity. Footprinting analyses showed that the chimera protected a DNA region that was wider than that protected by a Gal4DBD protein fragment, consistent with the cleavage results. The data demonstrate that a Top1 can be targeted to a specific DNA site by protein fusion to a heterologous DNA-binding domain. Such hybrid topoisomerase-derived enzymes may be useful for directing Top1 activity to specific genomic loci in living cells.

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