Influenza Polymerase Fidelity Figure 3. Effect of temperature on polymerase activity and misincorporation efficiency of IAV Pol complex. (A) IAV polymerization at different temperatures. LC: loading control (B) Fully extended products for the polymerase activity of IAV Pol at the four different temperatures were compared after being normalized with the value obtained at 30uC (100%). (C) The misincorporation efficiency of IAV Pol at four temperatures were calculated as described in Figure 2F, and compared with the value obtained at 30uC (100%). C: no polymerase control. doi:10.1371/journal.pone.0010372.g003 [22,23,24]. Under this complete nucleotide bias condition, the primer extension pauses at one nucleotide before the template sequence that the deleted nucleotide is supposed to be incorpo- rated, called a ‘‘stop site’’. However, low fidelity enzymes tend to efficiently incorporate incorrect nucleotides at the pause sites, and display the efficient primer extension products beyond the stop sites, whereas high fidelity enzymes exhibit little primer extension beyond the stop sites. The efficiency of the primer extension beyond the first stop site in the total primer extension has been used to compare the enzyme fidelity between polymerases [5,22,25]. For this test, we performed the 30-nt template-based and ApG- initiated polymerization reactions in which a mixture of all four NTPs (or three in case of misincorporation reactions) are provided. The ratio of radioactive NTP to nonradioactive NTP is kept constant for all the NTPs and is approximately 1/1600, allowing us to use a low concentration of radioactive NTPs. The 30-nt template also encodes the 39 end 30-nt sequence of the viral PA gene template (Figure 2A). As shown in Figure 2B, in the reaction with all four NTPs (500 mM), H3N2 IAV Pol complex extended the ApG primer and generated the 30-nt long fully extended products, and the extents of the fully extended products were PLoS ONE | www.plosone.org 6 April 2010 | Volume 5 | Issue 4 | e10372