Fig 4. MjAgo-mediated target DNA cleavage under single turnover conditions.
(A) Schematic representation of the experimental setup. MjAgo was premixed with guide DNA. Cleavage was started by adding a radioactively labelled DNA target strand. Cleavage experiments were conducted using 1 μM MjAgo, 100 nM guide strand (D-as2b) and 2.5 nM target strand (32P-D-s2b) at 85°C. Samples were taken at time points 0’, 5’, 10’, 15’, 20’, 25’, 30’, 60’ and 120’, separated on 20% denaturing polyacrylamide gel and visualized by autoradiography. Relative cleavage amplitudes were plotted versus time. Data could be fitted best using a single exponential equation yielding a rate constant kcleavage: 0.0012 (± 0.00007) s-1. The average of three independent measurements yielded a rate constant of 0.0014 (± 0.0003) s-1.