title: Reconstitution of enzymatic activity by the association of the cap and catalytic domains of human topoisomerase I.
abstract: When human topoisomerase I binds DNA, two opposing lobes in the enzyme, the cap region (amino acid, residues 175-433) and the catalytic domain (Deltacap, residues 433 to the COOH terminus) clamp tightly around the DNA helix to form the precleavage complex. Although Deltacap contains all of the residues known to be important for catalysis and binds DNA with an affinity similar to that of the intact enzyme, this fragment lacks catalytic activity. However, a mixture of Deltacap and topo31 (residues 175-433) reconstitutes enzymatic activity as measured by plasmid DNA relaxation and suicide cleavage assays. Although the formation of an active complex between topo31 and Deltacap is too unstable to be detected by pull-down experiments even in the presence of DNA, the association of topo31 with Deltacap persists and is detectable after the complex catalyzes the covalent attachment of the DNA to Deltacap by suicide cleavage. Removal of topo31 from Deltacap-DNA after suicide cleavage reveals that, unlike the cleavage reaction, religation does not require the cap region of the protein. These results suggest that activation of the catalytic domain of the enzyme for cleavage requires both DNA binding and the presence of the cap region of the protein.
authors.affiliation: Department of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195-7242, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Yang
authors.forename: James J
authors.initials: JJ
authors.lastname: Champoux
mesh_terms: D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D010446:Peptide Fragments / Q000302:isolation & purification; D011189:Potassium Chloride / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D011189:Potassium Chloride; D004247:DNA; D004264:DNA Topoisomerases, Type I