E. coli Gyrase Fails to Negatively Supercoil Diaminopurine-Substituted DNA. Issue 13 (3rd July 2015)

• Record Type: Journal Article
• Authors: Fernández-Sierra, Mónica; Shao, Qing; Fountain, Chandler; Finzi, Laura; Dunlap, David
• Is Part Of: Journal of molecular biology. Volume 427:Issue 13(2015:Jul. 01)
• Page Start: 2305
• Page End: 2318
• Publication Date: 2015-07-03
• Subjects: AFM atomic force microscopy -- CTD C-terminal domain -- DAP diaminopurine -- EMSA electrophoretic mobility shift assay -- IHF integration host factor -- NEB New England Biolabs -- PBS phosphate-buffered saline; topoisomerase -- gyrase -- diaminopurine -- negative supercoiling -- DNA wrapping; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.04.006
• Languages: English
• ISSNs: 0022-2836

Abstract

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. Graphical abstract: Highlights: Stiffened DNA might interfere with type II topoisomerases. DAP-substituted DNA is 35% stiffer than normal DNA. E . coli gyrase slows and human topoisomerase IIα pauses when relaxing (+) supercoils in DAP DNA. Salmonella gyrase negatively supercoils DNA slightly faster and further than E . coli gyrase. E . coli gyrase fails to negatively supercoil DAP DNA.