title: E. coli Gyrase Fails to Negatively Supercoil Diaminopurine-Substituted DNA.
abstract: Type II topoisomerases modify DNA supercoiling, and crystal structures suggest that they sharply bend DNA in the process. Bacterial gyrases are a class of type II topoisomerases that can introduce negative supercoiling by creating a wrap of DNA before strand passage. Isoforms of these essential enzymes were compared to reveal whether they can bend or wrap artificially stiffened DNA. Escherichia coli gyrase and human topoisomerase IIα were challenged with normal DNA or stiffer DNA produced by polymerase chain reaction reactions in which diaminopurine (DAP) replaced adenine deoxyribonucleotide triphosphates. On single DNA molecules twisted with magnetic tweezers to create plectonemes, the rates or pauses during relaxation of positive supercoils in DAP-substituted versus normal DNA were distinct for both enzymes. Gyrase struggled to bend or perhaps open a gap in DAP-substituted DNA, and segments of wider DAP DNA may have fit poorly into the N-gate of the human topoisomerase IIα. Pauses during processive activity on both types of DNA exhibited ATP dependence consistent with two pathways leading to the strand-passage-competent state with a bent gate segment and a transfer segment trapped by an ATP-loaded and latched N-gate. However, E. coli DNA gyrase essentially failed to negatively supercoil 35% stiffer DAP DNA.
authors.affiliation: Departments of Physics and Cell Biology, Emory University, Atlanta, GA 30322, USA.
authors.forename: Mónica
authors.initials: M
authors.lastname: Fernández-Sierra
authors.affiliation: Departments of Physics and Cell Biology, Emory University, Atlanta, GA 30322, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Shao
authors.affiliation: Departments of Physics and Cell Biology, Emory University, Atlanta, GA 30322, USA.
authors.forename: Chandler
authors.initials: C
authors.lastname: Fountain
authors.affiliation: Departments of Physics and Cell Biology, Emory University, Atlanta, GA 30322, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Finzi
authors.affiliation: Departments of Physics and Cell Biology, Emory University, Atlanta, GA 30322, USA. Electronic address: ddunlap@emory.edu.
authors.forename: David
authors.initials: D
authors.lastname: Dunlap
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism; D027081:DNA Gyrase / Q000737:chemistry* / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000378:metabolism; D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012475:Salmonella / Q000201:enzymology
chemical_list: D000951:Antigens, Neoplasm; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015075:2-Aminopurine; D000255:Adenosine Triphosphate; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II
keywords: DNA wrapping; diaminopurine; gyrase; negative supercoiling; topoisomerase