RESUMO
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.
Assuntos
DNA Girase/química , DNA Girase/metabolismo , DNA Super-Helicoidal/química , Proteínas de Escherichia coli/química , 2-Aminopurina/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , DNA Super-Helicoidal/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Conformação Proteica , Salmonella/enzimologiaRESUMO
Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect.
Assuntos
Benzoxazóis/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Desoxirribonucleases/metabolismo , Corantes Fluorescentes/química , Compostos de Quinolínio/química , Sítios de Ligação , Cátions , DNA/análise , DNA/química , DNA Polimerase beta/antagonistas & inibidores , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Relação Dose-Resposta a Droga , Substâncias Intercalantes/química , Modelos Químicos , Ligação Proteica , Estrutura Secundária de Proteína , TemperaturaRESUMO
We present a single-molecule method for measuring the torque exerted by braided DNA molecules undergoing spontaneous unbraiding while attached to a paramagnetic dumbbell in the absence of external manipulation. A magnetic tweezers setup is employed to braid pairs of lambda DNA molecules covalently bound to a surface. Upon removing the magnetic field, the braided DNA molecules undergo spontaneous unbraiding, efficiently transforming the stored elastic energy into enough mechanical energy to rotate the tethered dumbbells for periods as long as 30 minutes. Using hydrodynamic equations we estimate the torque exerted on the dumbbells by the DNA braids, yielding values ranging from 47 to 166 pN nm.
RESUMO
Here we describe the use of magnetic tweezers and or microfluidics to manipulate single DNA molecules. We describe experiment which employ magnetic tweezers coupled to an inverted microscope as well as the use of a magnetic tweezers setup with an upright microscope. Using a chamber prepared via soft lithography, we also describe a microfluidic device for the manipulation of individual DNA molecules. Finally, we present some past successful examples of using these approaches to elucidate unique information about protein-nucleic acid interactions.