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. 2019 Feb 4;15(2):e1007956. doi: 10.1371/journal.pgen.1007956

Fig 4. Static Deactivation of pol V Mut E38K/ΔC17 at 37°C and 30°C.

Fig 4

(A) Sketch showing static deactivation of pol V Mut E38K/ΔC17 (200 nM) in the absence of DNA synthesis at 37°C and 30°C. Static deactivation was determined by measuring the extent of DNA synthesis as a function of incubation time on 12 nt oh HP p/t DNA at 37°C in the presence of ATPγS and dNTP’s (dTTP, dCTP, dGTP 500 μM each). Pol V Mut E38K/ΔC17 was incubated either alone (I.) or with ATPγS (II.), or with ATPγS and 12nt oh HP DNA (III.). (B) and (D) show representative gels for each deactivation condition. Pol V Mut E38K/ΔC17 deactivates more slowly at 30°C (D-E) compared to 37°C (B-C). At each temperature, pol V Mut is stabilized when incubated in the presence of ATPγS. Deactivated pol V Mut E38K/ΔC17 is not dead since it can be reactivated by incubation with RecA* (+RecA*). Static deactivation is expressed as the relative polymerase activity measured at each incubation time point divided by the polymerase activity measured at t = 0. Each experiment was repeated 2–3 times and average relative activity along with SD for each deactivation time point are presented in (C) and (D). (I. and black circles in the graphs) represent static deactivation of pol V Mut alone, (II. and white circles in the graphs) represent deactivation of pol V Mut +ATPγS and (III. and black triangles in the graphs) represent deactivation of pol V Mut + ATPγS + 12 nt oh HP.