A functional linker in human topoisomerase I is required for maximum sensitivity to camptothecin in a DNA relaxation assay

Human topoisomerase I is composed of four major domains: the highly charged NH2-terminal region, the conserved core domain, the positively charged lin ker domain, and the highly conserved COOH-terminal domain. Near complete en zyme activity can be reconstituted by combining recombinant polypeptides th at approximate the core and COOH-terminal domains, although DNA binding is reduced somewhat for the reconstituted enzyme (Stewart, L, Ireton, G. C., a nd Champoux, J. J. (1997) J. Mol Biol. 269, 355-372), A reconstituted enzym e comprising the core domain plus a COOH-terminal fragment containing the c omplete linker region exhibits the same biochemical properties as a reconst ituted enzyme lacking the linker altogether, and thus detachment of the lin ker from the core domain renders the linker non-functional. The rate of rel igation by the reconstituted enzyme is increased relative to the forms of t he enzyme containing the linker indicating that in the intact enzyme the li nker slows religation. Relaxation of plasmid DNA by full-length human topoi somerase I or a 70-kDa form of the enzyme that is missing only the non-esse ntial NH2-terminal domain (topo70) is inhibited similar to 16-fold by the a nticancer compound, camptothecin, whereas the reconstituted enzyme is nearl y resistant to the inhibitory effects of the drug despite similar affinitie s for the drug by the two forms of the enzyme. Eased on these results and i n light of the crystal structure of human topoisomerase I, we propose that the linker plays a role in hindering supercoil relaxation during the normal relaxation reaction and that camptothecin inhibition of DNA relaxation dep ends on a direct effect of the drug on DNA rotation that is also dependent on the linker.