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. 2007 Oct 16;35(20):6973–6983. doi: 10.1093/nar/gkm695

Figure 2.

Figure 2.

Pyrophosphorolysis of TMP-terminated primer. (A) Exonuclease-deficient holoenzyme (100 nM) was preincubated with 90 nM 25/45-mer DNA (radiolabeled primer) and mixed with 400 nM α-32P-TTP in the presence of 2.5 mM Mg2+. The reaction was aged for 5 s to permit radiolabeled TTP incorporation. A second mixing event then combined this with varying concentrations of PPi [15 (filled circle), 30 (open circle), 75 (filled square), 200 (open square), 400 (filled triangle) and 1000 (open triangle) μM] in solution with 2.5 mM Mg2+ and 20 μM unlabeled TTP. The unlabeled TTP was included to prevent the undesired incorporation of radiolabeled TTP following the pyrophosphorolysis reaction. Shown is the concentration of the 26-mer plotted as a function of time and the data were analyzed by non-linear regression using a single exponential equation to obtain the observed rate constants at the various PPi concentrations. (B) The observed reaction rates were plotted versus the concentration of PPi used and fitted using Equation (5) to yield a maximum rate of pyrophosphorolysis of 1.4 ± 0.3 s−1 and a Kd of 240 ± 80 μM.