Fig 2. Temperature-dependent dynamic deactivation of pol V Mut wt.

Activity and dynamic deactivation of pol V Mut wt (200 nM) were measured as shown in Fig 1A at (A) 37°C and (B) 30°C on 12 nt oh HP (1 μM) in the presence of saturating concentration of ATPγS (500 μM) and dNTP’s (mix of dTTP, dCTP, dGTP 500 μM each). (C) Prior to deactivation, pol V Mut wt performs one round of DNA synthesis at 37°C (black circles in the graph) and 3 rounds at 30°C (white triangles in the graph). Deactivated pol V Mut is reactivated by adding RecA* wt (200 nM). (D) Activity of pol V Mut wt measured in the presence of ATP (500 μM) and dNTP’s (dTTP, dCTP, dGTP 500 μM each). Pol V Mut wt is not active with ATP but it can synthesize DNA when trans-activated by RecA* wt (trans-activation reaction was performed for 30 min). Representative DNA synthesis gels for pol V Mut wt are presented in (A, B, and D). The experiments were repeated 3 times and the average % PE (percent p/t DNA extended) with the SD (standard deviation) for each reaction time point is graphed in panel C.