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. Author manuscript; available in PMC: 2017 Jul 31.
Published in final edited form as: J Mol Biol. 2016 Jul 14;428(15):2997–3012. doi: 10.1016/j.jmb.2016.07.002

Figure 6. RecBCD unwinding of RP DNA requires its secondary translocase activity.

Figure 6

A. Substrates used to test re-initiation of unwinding by RecBCD.

B. Stopped-flow experiments were performed by mixing pre-incubated RecBCD and DNA Substrate XVII (18.75 and 25 nM, respectively) in Buffer M (250 mM NaCl) in a 1:10 volumetric ratio with ATP (1 mM) and heparin (8 mg/mL) in buffer M (8 mM NaCl) yielding the final concentrations listed above and a final NaCl concentration of 30 mM at 25°C. Cy3 fluorescence was excited at 505 nm, and Cy3 fluorescence (green) was monitored at 570 nm and Cy5 fluorescence (red) was monitored at > 665 nm.

C. Cy3 fluorescence traces for experiments as performed in panel B, at 1 mM ATP (dark blue), 200 μM ATP (pink), 40 μM ATP (light blue), and no ATP (gray).

D. Cy3 fluorescence traces for experiments as performed in panel B at 1 mM ATP using Substrate XVI (red), XVII (blue), or XVIII (orange).