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. 2015 Mar 26;43(8):4179–4190. doi: 10.1093/nar/gkv260

Figure 4.

Figure 4.

RPA relieves the inhibitory effect of long 5′flaps. (a) Effect of RPA on the DNA primer extension and strand displacement activity of Pol δDV in Buffer TM (20 mM NaCl, 30°C) as a function of the length (n) of the 5′-flap in the strand to be displaced using a T7 gap substrate. (b) Effect of RPA on a T1 gap substrate that does not contain a 5′-flap. (c) Scheme depicting the strand displacement assay using the signal from a Cy3–Cy5 couple placed at the end of the duplex region to be displaced. (d) Time courses of the Cy3 fluorescence intensity using a T1 gap substrate that does not contain a 5′-flap in the strand to be displaced. In black is the time course after addition of dNTP to a pre-formed Pol δDV–DNA complex. In this experiment RPA was added (spiked RPA) 50 min after the reaction had been started. In the gray time course RPA was added from the beginning, before adding dNTP. A control experiment with only RPA added is shown as well. (e) Same experiments as in d) but using a T1 gap substrate containing a T25 5′-flap in the strand to be displaced. The RPA-only experiment is shown offset for clarity.