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 domai ns as follows: the unconserved and highly charged NH2-terminal domain, a co nserved core domain, the positively charged linker region, and the highly c onserved COOH-terminal domain containing the active site tyrosine, Previous studies of the domain structure revealed that near full topoisomerase I ac tivity can be reconstituted in vitro by fragment complementation between re combinant polypeptides approximating the core and COOH-terminal domains. He re we demonstrate that deletion of linker residues Asp(660) to Lys(688) yie lds an active enzyme (topo70 Delta L) that purifies as both a monomer and a dimer, The dimer is shown to result from domain swapping involving the COO H-terminal and core domains of the two subunits, The monomeric form is inse nsitive to the anti-tumor agent camptothecin and distributive during in vit ro plasmid relaxation assays, whereas the dimeric form is camptothecin-sens itive and processive. However, the addition of camptothecin to enzyme/DNA m ixtures causes enhancement of SDS-induced breakage by both monomeric and di meric 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 pro viding a surrogate linker. Yeast cells expressing topo70 Delta L were found to be insensitive to camptothecin.