Expression of human topoisomerase I with a partial deletion of the linker region yields monomeric and dimeric enzymes that respond differently to camptothecin.
Human topoisomerase I is a 765-residue protein composed of four major domains as follows: the unconserved and highly charged NH(2)-terminal domain, a conserved core domain, the positively charged linker region, and the highly conserved COOH-terminal domain containing the active site tyrosine. Previous studies of the domain structure revealed that near full topoisomerase I activity can be reconstituted in vitro by fragment complementation between recombinant polypeptides approximating the core and COOH-terminal domains. Here we demonstrate that deletion of linker residues Asp(660) to Lys(688) yields an active enzyme (topo70DeltaL) that purifies as both a monomer and a dimer. The dimer is shown to result from domain swapping involving the COOH-terminal and core domains of the two subunits. The monomeric form is insensitive to the anti-tumor agent camptothecin and distributive during in vitro plasmid relaxation assays, whereas the dimeric form is camptothecin-sensitive and processive. However, the addition of camptothecin to enzyme/DNA mixtures causes enhancement of SDS-induced breakage by both monomeric and dimeric forms of the mutant enzyme. The similarity of the dimeric form to the wild type enzyme suggests that some structural feature of the dimer is providing a surrogate linker. Yeast cells expressing topo70DeltaL were found to be insensitive to camptothecin.
Ireton
G C
GC
Department of Microbiology, University of Washington School of Medicine, Seattle 98195-7242, USA.
Stewart
L
L
Parker
L H
LH
Champoux
J J
JJ
D016367:Baculoviridae / Q000378:metabolism; D002166:Camptothecin / Q000494:pharmacology*; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014170:Transformation, Genetic; D015003:Yeasts / Q000378:metabolism
D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D004264:DNA Topoisomerases, Type I; D005990:Glycerol; D002166:Camptothecin