Fig 1.
Cleavage of ssDNA plasmid depends on ion concentration.
Products of the cleavage of ssDNA M13mp18 plasmid by PA-Nter in the presence of increasing concentrations of divalent metal ions separated by agarose gel electrophoresis. All samples were incubated at 37°C for 2 h and inactivated at 80°C for 20 min (reaction of 500 ng M13mp18 with 1.25 μM PA-Nter).
Fig 2.
Cleavage of short nucleic acids fragments.
Activity of PA-Nter on the various nucleic acids substrates in the presence of increasing concentrations of divalent metal ions. All samples were incubated with purified 2.55 μM PA-Nter at 25°C for 1 h and inactivated at 70°C for 5 min. (A) 6.8 μM 20-nt RNA, (B) 8.5 μM 19-nt DNA, (C) 2.4 μM 66-nt DNA. Possible secondary structures of these oligonucleotides are shown in S3 Fig.
Fig 3.
Single molecule fluorescence techniques applied for PA-Nter activity measurements.
Histograms of single molecule FRET efficiency of 30-nt fluorescent hairpin DNA (A) before the reaction, (B) after the reaction with PA-Nter; E = 0 indicates no FRET, E = 1 indicates maximal FRET, S denotes photon stoichiometry. Example histogram shown from a series of three independent experiments. The correlation functions of hairpin DNA measured with FCCS, (C) maximum cross-correlation before the reaction, (D) minimum cross-correlation after the reaction with PA-Nter. Blue line indicates cross-correlation, green and red denote auto-correlation functions. (E) Cross-correlation functions in 0, 5, 10, 15, 20, 25 and 30 minute of the reaction of 15 nM hairpin DNA with 100 nM PA-Nter. Insert picture shows the concentration of the hairpin DNA (nM) throughout the reaction time. (F) Comparison with electrophoretic assay. Products of the cleavage of 5.4 μM hairpin DNA with 3.3 μM PA-Nter in the presence of 1 mM ions and 2 mM DTT. Samples were incubated at 25°C for 1 h and inactivated at 70°C for 5 min.
Fig 4.
Example data set showing the decrease of substrate concentration in time in for a range of PA-Nter concentrations (numbers in [nM]) in the presence of A) Mg2+, B) Mn2+. (C, D) Reaction rate of PA-Nter in the presence of either 1 mM Mg2+ or 1 mM Mn2+ in the function of enzyme concentration. The kinetics was monitored in the regime of enzyme excess and constant concentration of doubly labelled hairpin DNA (~15 nM) using fluorescence cross-correlation spectroscopy (FCCS) for 30 min. Errors are depicted as SEM from triplicate measurements. The line shows fitted hyperbolic dependence: (vmax • x)/(KM + x). The fitting parameters for asymptotic maximum reaction speed vmax were 0.81± 0.38 and 1.38± 0.34 nM/min for 1 mM Mg2+ and 1 mM Mn2+, respectively. Black points indicate the measurement in the reaction buffer containing 50 mM Hepes, 150 mM KCl pH 7.8 and 1 mM ions, white points (not included in fitting) indicate the measurement in the reaction buffer containing 50 mM Hepes, 150 mM KCl pH 7.8, 2 mM DTT and 1 mM ions.
Fig 5.
PA-Nter saturation with metal ions accordingly to binding constants.
The enzymatic catalysis of PA-Nter is dependent on the number of ions occupying the active site of the enzyme.
Fig 6.
PA-Nter ion occupancy in vitro.
The curves are plotted according to Eq 4, assuming 1 μM total concentration of Mn2+ and 1mM Mg2+.