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 NH(2)-terminal region, the conserved core domain, the positively charged linker domain, and the highly conserved COOH-terminal domain. Near complete enzyme activity can be reconstituted by combining recombinant polypeptides that approximate the core and COOH-terminal domains, although DNA binding is reduced somewhat for the reconstituted enzyme (Stewart, L., Ireton, G. C., and Champoux, J. J. (1997) J. Mol. Biol. 269, 355-372). A reconstituted enzyme comprising the core domain plus a COOH-terminal fragment containing the complete linker region exhibits the same biochemical properties as a reconstituted enzyme lacking the linker altogether, and thus detachment of the linker from the core domain renders the linker non-functional. The rate of religation by the reconstituted enzyme is increased relative to the forms of the enzyme containing the linker indicating that in the intact enzyme the linker slows religation. Relaxation of plasmid DNA by full-length human topoisomerase I or a 70-kDa form of the enzyme that is missing only the non-essential NH(2)-terminal domain (topo70) is inhibited approximately 16-fold by the anticancer compound, camptothecin, whereas the reconstituted enzyme is nearly resistant to the inhibitory effects of the drug despite similar affinities for the drug by the two forms of the enzyme. Based on these results and in 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 depends on a direct effect of the drug on DNA rotation that is also dependent on the linker.
Stewart
L
L
Department of Microbiology, School of Medicine, University of Washington, Seattle, Washington 98195-7242, USA.
Ireton
G C
GC
Champoux
J J
JJ
D000970:Antineoplastic Agents / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics; D004278:DNA, Superhelical / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D020860:Subtilisin / Q000378:metabolism
D000970:Antineoplastic Agents; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D020860:Subtilisin; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin