title: Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases.
abstract: Type IA DNA topoisomerases work with a unique mechanism of strand passage through an enzyme-bridged, ssDNA gate, thus enabling them to carry out diverse reactions in processing structures important for replication, recombination, and repair. Here we report a unique reaction mediated by an archaeal type IA topoisomerase, the synthesis and dissolution of hemicatenanes. We cloned, purified, and characterized an unusual type IA enzyme from a hyperthermophilic archaeum, Nanoarchaeum equitans, which is split into two pieces. The recombinant heterodimeric enzyme has the expected activities in its preference of relaxing negatively supercoiled DNA. Its amino acid sequence and cleavage site sequence analysis suggest that it is topoisomerase III, and therefore we named it "NeqTop3." At high enzyme concentrations, NeqTop3 can generate high-molecular-weight DNA networks. Biochemical and electron microscopic data indicate that the DNA networks are connected through hemicatenane linkages. The hemicatenane formation likely is mediated by the single-strand passage through denatured bubbles in the substrate DNA under high temperature. NeqTop3 at lower concentrations can reverse hemicatenanes. A complex of human topoisomerase 3α, Bloom helicase, and RecQ-mediated genome instability protein 1 and 2 can partially disentangle the hemicatenane network. Both the formation and dissolution of hemicatenanes by type IA topoisomerases demonstrate that these enzymes have an important role in regulating intermediates from replication, recombination, and repair.
authors.affiliation: Institute of Cellular and Organismic Biology and Molecular Cell Biology, Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan 115.
authors.forename: Shun-Hsiao
authors.initials: SH
authors.lastname: Lee
authors.forename: Grace Ee-Lu
authors.initials: GE
authors.lastname: Siaw
authors.forename: Smaranda
authors.initials: S
authors.lastname: Willcox
authors.forename: Jack D
authors.initials: JD
authors.lastname: Griffith
authors.forename: Tao-Shih
authors.initials: TS
authors.lastname: Hsieh
mesh_terms: D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D043863:Catenanes / Q000378:metabolism*; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism*; D046588:Nanoarchaeota / Q000201:enzymology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D002352:Carrier Proteins; D043863:Catenanes; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; C501527:RMI1 protein, human; C533196:RMI2 protein, human; C097151:Bloom syndrome protein; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I
keywords: DNA repair; DNA topology; double Holliday junction; genetic recombination