title: Distinct regions of the Escherichia coli ParC C-terminal domain are required for substrate discrimination by topoisomerase IV.
abstract: Type IIA DNA topoisomerases are essential enzymes that use ATP to maintain chromosome supercoiling and remove links between sister chromosomes. In Escherichia coli, the type IIA topoisomerase topo IV rapidly removes positive supercoils and catenanes from DNA but is significantly slower when confronted with negatively supercoiled substrates. The ability of topo IV to discriminate between positively and negatively supercoiled DNA requires the C-terminal domain (CTD) of one of its two subunits, ParC. To determine how the ParC CTD might assist with substrate discrimination, we identified potential DNA interacting residues on the surface of the CTD, mutated these residues, and tested their effect on both topo IV enzymatic activity and DNA binding by the isolated domain. Surprisingly, different regions of the ParC CTD do not bind DNA equivalently, nor contribute equally to the action of topo IV on different types of DNA substrates. Moreover, we find that the CTD contains an autorepressive element that inhibits activity on negatively supercoiled and catenated substrates, as well as a distinct region that aids in bending the DNA duplex that tracks through the enzyme's nucleolytic center. Our data demonstrate that the CTD is essential for proper engagement of both gate and transfer segment DNAs, reconciling different models to explain how topo IV discriminates between distinct DNAs topologies.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
authors.forename: Seychelle M
authors.initials: SM
authors.lastname: Vos
authors.forename: Imsang
authors.initials: I
authors.lastname: Lee
authors.forename: James M
authors.initials: JM
authors.lastname: Berger
mesh_terms: D027101:DNA Topoisomerase IV / Q000235:genetics* / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D027101:DNA Topoisomerase IV
keywords: 56-FAM; BME; BSA; C-terminal domain; CTD; DNA bending; DNA binding; DNA topology; EDTA; FRET; G-segment; His(6); MBP; N-terminal domain; NTD; T-segment; TEV; Topo IV; WT; bovine serum albumin; carboxyfluorescein; ethylenediaminetetraacetic acid; fluorescence resonance energy transfer; gate segment; hexahistidine; kDNA; kinetoplast DNA; maltose binding protein; substrate discrimination; tobacco etch virus; topoisomerase IV; transfer segment; type IIA topoisomerase; wild type; β-mercaptoethanol