Fig. 5.

The influence of PCNA loading and RFC targeting on nuclease activity of the Mus81 complex. a Effect of RFC-mediated PCNA loading on stimulation of the Mus81 complex nuclease activity. Mus81-Mms4 (40 nM) was incubated with circular 3′ flap DNA substrate (6 nM) in the presence or absence of RFC (180 nM), PCNA (70, 140, 270 nM) and ATP (1 mM) as indicated. The reactions were incubated at 37 °C for 30 min and then analyzed on denaturing gel. b RFC targets the Mus81 complex in the nicked substrate. Mus81-Mms4 (0.4 nM) was incubated with the 3′ flap substrate (4 nM) in the presence or absence of ΦX174 virion circular ssDNA (0.25 nM), and increasing concentrations of RFC (5, 12.5, 25, and 50 nM), as indicated. Reactions were incubated at 37 °C for 30 min and analyzed. c Quantification of data in (b) from three independent experiments. *Raw data provided in Additional file 2