Influenza Polymerase Fidelity Figure 2. Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, HIV-1 RT and MuLV RT with biased nucleotide substrate pools. (A) Template sequences used for the misincorporation assay with IAV Pol, T7 RNA polymerase and RTs of HIV-1 and MuLV. The IAV Pol template used is a 30-nt sequence from the 39 end of the viral PA sequence with a ApG primer binding site (P:primer, T:template,). The first UTP or TTP incorporation site of each template was marked with ‘‘X’’, and the first stop site in the (2) UTP or TTP reaction was marked with ‘‘1*’’ under each template sequence (the second and third stop sites were marked ‘‘2*’’ and ‘‘3*’’ for the IAV template). The RNA synthesis initiation sites were marked in blue. (B) ApG-initiated RNA-dependent RNA polymerization by IAV Pol: ApG primer was extended with a 30-nt RNA template and three different amounts of H3N2 IAV Pol protein (1x, 2x and 3x) with 500 mM four NTPs (‘‘4’’) and 0.16 mM a-32P-GTP, and the same reactions were repeated except using two biased NTP pools, minus UTP (‘‘- U’’) or minus ATP (‘‘- A’’). The sequence of the incorporated nucleotides near the three UTP stop sites (red) is shown at the side. The extended products in the (2) UTP reaction, which was used for calculating the misincorporation efficiency, was marked as ‘‘]’’.Dotted lines refer to RNAs. (C) DNA dependent RNA polymerization of bacterial phage T7 RNA polymerase: A 47 bp ds DNA (box) encoding T7 promoter (grey box) and 29 bp sequence (white box) was used for RNA synthesis with T7 RNA polymerase at 37uC for 60 mins. ‘‘]’’: Fully extended misincorporated product. (D) and (E) RNA dependent DNA polymerization reaction by HIV-1 RT (D) and MuLV RT (E). A 48 mer RNA template annealed to a 18-mer single stranded DNA primer was used for the DNA polymerization by HIV-1 and MuLV RTs at 37uC for 60 mins. 500 mM dNTPs mixed with a-32P-dNTPs (0.16 mM), which is the same nucleotide concentration and ratio as used in the reactions with IAV Pol and T7 RNA polymerase, was used for DNA synthesis. ‘‘]’’: Fully extended misincorporated product. Dotted lines refer to RNA and solid lines refer to DNA. (F) and (G) Comparison of the misincorporation efficiency of the four polymerases. For calculation of the misincorporation efficiency, the fully extended misincorporated product in (2) U/(2) T reactions in all four polymerases was normalized to the total extended product in the lanes with all four NTPs (dNTPs). The fold differences of the calculated misincorporation percentages between three activities of IAV Pol (F) and between IAV Pol and three other polymerases (G) were determined. At least five repeats of the assay were conducted in this analysis. doi:10.1371/journal.pone.0010372.g002 Misincorporation efficiency of IAV Pol complex It has been well-established that a biased nucleotide pool is a major factor to promote polymerase-induced mutation synthesis [21]. Basically, polymerases pause at the template sequences where the limited nucleotides are supposed to be incorporated, and the temporal pausing of the polymerase triggers the incorporation of an incorrect nucleotide, called misincorporation, which eventually generates mutations. Therefore, another important indication of the data presented in Figures 1 would be that the full extended products observed in the significantly biased NTP pools could include the products of misincorporation to some extent, particularly at low total GTP concentrations (Fig. 1, lanes 1). In fact, one commonly used assay to investigate the enzyme fidelity of polymerases is a primer-extension based misincorpora- tion assay employing completely biased nucleotide pools that consist of only three of nucleotides instead of all four nucleotides PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e10372