α32p atp

Thin layer chromatography (TLC)-based ATPase assays were performed using [α-³²P]ATP (Hartmann Analytic)^{36 (link),37 (link)}. To quantify DNA-stimulated ATPase activity, 0.5 µM protein or protein complex were combined with 1 mM of a 43-nucleotide ssDNA (5’-GGCCGCGAGCCGGAAATTTAATTATAAACCAGACCGTCTCCTC-3’). 0.5 µM protein or protein complex or equivalent protein–DNA mixtures were incubated with 1 mM [α-³²P]ATP in 50 mM HEPES-NaOH, pH 7.5, 80 mM NaCl, 5 mM MgCl₂, 2 mM DTT at 30 °C for up to 60 min. 5 µl of sample were withdrawn at selected time points and reactions were quenched with 5 µl of 100 mM EDTA. 0.8 µl of the samples were spotted on a PEI-cellulose TLC plate and chromatographed with 1 M acetic acid, 0.5 M LiCl, 20 % (v/v) ethanol. The corresponding ADP and ATP spots were visualized using a Storm 860 phosphorimager (GMI, USA) and quantified using ImageQuant software (version 5.2; Cytiva). Data were plotted and analyzed using Prism (version 9.0; GraphPad), the ATPase activity was calculated as the number of hydrolyzed ATP molecules per protein molecule per minute, by fitting quantified data to the equation V = (A_fast * V_fast² + A_slow * V_slow²)/(A_fast * V_fast + A_slow * V_slow); A_fast/slow, amplitudes of the fast/slow hydrolysis phases; V_fast/slow, rates of the fast/slow hydrolysis phases [min^-1]; V, ATP hydrolyzed as a function of time [min⁻¹].

Reactions (10 μL) were set up with 0.5 μM condensin holocomplex, with or without 24 nM relaxed circular 6.4-kb plasmid DNA in ATPase buffer (40 mM TRIS–HCl pH 7.5, 125 mM NaCl, 10% (v/v) glycerol, 5 mM MgCl₂, 5 mM ATP, 1 mM DTT and 33 nM [α³²P]-ATP; Hartmann Analytic). Relaxed circular DNA was prepared by treating the negatively supercoiled plasmid DNA with E. coli topoisomerase I (NEB). After treatment with topo I, relaxed DNA was purified by phenol-chloroform extraction and ethanol precipitation. ATP hydrolysis reactions were incubated at RT (∼25°C) and were initiated by addition of ATP. A volume of 0.8 μL of the reaction mix was spotted onto PEI cellulose F TLC plates (Merck) every 3 min for a total duration of 15 min. The reaction products were resolved on TLC plates using 0.5 M LiCl and 1 M formic acid solution and detected by exposing the TLC plates to a phosphorimager screen and analysis on a Typhoon FLA 9,500 scanner (GE Healthcare). ATP hydrolysis rates were calculated from the ADP/ATP ratios from time points in the linear range of the reaction.

ATPase activity of the Cas2/3 was assayed at 37 °C in an ATPase (A) buffer (10 mM Tris–HCl (pH 8.0 at 25 °C), 75 mM NaCl, 7% (v/v) glycerol, 0.1 mg/ml BSA, 2 mM MgCl₂, 2 mM ATP and 1 nM of [α³²P] ATP (Hartmann Analytic)) in the presence of 5 nM double-stranded pSP-CC plasmid or single-stranded M13mp18 or in the absence of DNA. Reactions were initiated by adding Cas2/3 to the reaction mixture and terminated at different time points (0–64 min) by the addition of EDTA to 25 mM final concentration. Reaction products were fractionated on thin-layer chromatography (TLC) PEI Cellulose F plates (Merck) in 0.325 M sodium phosphate buffer (pH 3.5 at 23 °C) and visualised using FLA-5100 phosphorimager (Fujifilm). The ATP depletion was measured as a function of time and fitted according to a first-order kinetical model [ATP]_t = [ATP]_oe^−kt. The reaction rate constants were estimated from at least three independent measurements (see Additional file 14 for individual data values).
Influence of different divalent metal ions for Cas2/3 ATPase activity was examined at 37 °C for 20 min in the A buffer containing 5 nM ssM13mp18 and 5 mM of divalent metal ions: Mg²⁺ (MgCl₂), Ca²⁺ (CaCl₂), Mn²⁺ (MnCl₂), Co²⁺ (CoCl₂), Ni²⁺ (NiCl₂), or Zn²⁺ (ZnCl₂).

Radioactively labeled yeast tRNA^Phe lacking the CCA terminus (tRNA) or ending with two C residues (tRNA-CC) was prepared by in vitro transcription in the presence of α³²P-ATP (3000 Ci/mmol; Hartmann Analytic, Braunschweig, Germany) and purified as described [60 (link),61 ].

Reactions (10 μL) were set up with 5 μM SMC head proteins, as indicated, in ATPase buffer (50 mM TRIS-HCl pH 7.5, 215 mM NaCl, 2% (v/v) glycerol, 10 mM MgCl₂, 5 mM ATP, 1.3 mM DTT and 33 nM [α³²P]-ATP; Hartmann Analytic). ATP hydrolysis reactions were initiated by addition of ATP and incubated at 30°C. A volume of 1.0 μL of the reaction mix was spotted onto PEI cellulose F TLC plates (Merck) every 3 min for a total of 15 min. The reaction products were resolved on TLC plates using 0.5 M LiCl and 1 M formic acid solution and detected by exposing the TLC plates to a phosphorimager screen and analysis on a Typhoon FLA 9,500 scanner (GE Healthcare). ATP hydrolysis rates were calculated from the ADP/ATP ratios from time points in the linear range of the reaction.
ATPase assays with condensin holocomplexes were carried out as described previously (Kschonsak et al., 2017 (link)).

Detergent-solubilized TmrAB (2 µM) were incubated with ATP (1 mM) traced with [α³²P]-ATP (Hartmann Analytic), MgCl₂ (5 mM), CP^Fs (4 µM), or orthovanadate (1 mM) for 5 min at 45°C. Cold ATP (10 mM) was added, and unbound nucleotides were removed by rapid gel filtration (Bio-Spin columns P-30, Bio-Rad). ATP (10 mM) was added, and samples were spotted onto polyethyleneimine cellulose plates (Merck Millipore). Thin-layer chromatography was performed using 0.75 M KH₂PO₄ pH 3.4. Plates were developed overnight on Exposure Cassette-K (Bio-Rad) and evaluated on Personal Molecular Imager System (Bio-Rad). Representative radiograms of three experiments are displayed.