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. 2013 Mar 4;41(8):4525–4534. doi: 10.1093/nar/gkt138

Figure 1.

Figure 1.

The Csm2 and Psy3 heterodimer preferentially bind forked and 3′-overhang DNA substrates. (A) Csm2–Psy3 was purified using the schematic presented using the conditions outlined in the ‘Materials and Methods’ section. Coomasie stained Csm2–Psy3 heterodimer (2.4 μg) is shown. Csm2 is 25 kDa and Psy3 is 28 kDa. (B) The Csm2–Psy3 heterodimer was assayed for DNA binding. Increasing concentrations of Csm2–Psy3 were added to a reaction mixture containing 25 nM fluorescein-labelled DNA fork in a fluorescence spectrophotometer. The binding isotherm was performed on three separate days, and each data set is shown. These data were fit using a global non-linear regression to obtain the Kd and a Hill coefficient. (C) Complexes of Csm2–Psy3 bound to a fluorescein-labelled fork substrate were pre-assembled as described. Fluorescence anisotropy was then measured with increasing concentrations of the indicated unlabelled competitors. Competition curves were fit using non-linear regression to calculate the apparent Ki. The experiment was done in triplicate, and standard deviations were plotted.