proportional to the amounts of the IAV Pol complex under the reaction conditions (1x, 2x and 3x: Fig. 2B, 3X = 200 ng), confirming that the NTP incorporation kinetics was linear under the employed polymerase reaction. Upon the deletion of UTP or ATP from the four NTPs [(2) U or (2) A in Fig. 2B], however, the amount of fully extended products were significantly reduced, indicating that the biased NTP pool generated major kinetic blockages in the IAV Pol polymerization reaction. As predicted, the first ‘‘stop site’’ was observed at one nucleotide before the sites where the deleted UTP (‘‘X’’ in Fig. 2A) should be incorporated (Fig. 2A and 2B: see ‘‘1*’’ in the (2) U reactions of and the sequences of the expected products). Excitingly, primer extension was observed beyond the first stop site, generating the second (‘‘2*’’) and third (‘‘3*’’) stops sites (Figure 2A and 2B). This indicates that mutation synthesis must have occurred at not only the first stop site but also the later stop sites. Unlike the UTP stop sites, it is difficult to observe the first stop sites induced by the deletion of other three NTPs (A, G and C) because the first 5 nucleotides in the 39 end conserved sequence of the IAV genomes contain A, G and C. Thus, their first stop sites generate only 3–5 nucleotide long products which are technically difficult to separate even with 8 M urea high percentage denaturing PAGE. However, as shown in the reactions with (2) ATP pool (Fig. 2B), the full length extension was also significantly decreased which supports that the biased NTP pool generates kinetic blockage. Interestingly, the sixth ‘‘A’’ incorporation site is present at the 18th nucleotide from the 39 end of the 30-nt template sequence and this could be observed in the (2) ATP reaction (‘‘6*’’) in Figure 2B. The product seen at the sixth A incorporation site must have been generated after IAV Pol made mutations at the five different stop sites prior to the fifth sixth stop site seen in the gel. Importantly, we observed that the reactions with (2) CTP or (2) GTP biased pools also induced the reduction of the fully extended product (data not shown). Indeed, this is the first biochemical observation reported for mutation synthesis by IAV Pol. Misincorporation efficiency of bacterial T7 RNA polymerase, HIV-1 RT and MuLV RT with templates encoding identical sequences Next, we compared the enzyme fidelity of IAV Pol complex with three other polymerases, bacterial phage T7 DNA-dependent RNA polymerase as well as two retroviral RNA-dependent DNA polymerases which are well-characterized for their fidelity [26]: 1) murine leukemia virus reverse transcriptase (MuLV RT) with high fidelity and 2) human immunodeficiency virus Type 1 reverse transcriptase (HIV-1 RT) with low fidelity [18,24,26,27] Impor- tantly, these polymerases initiate the nucleic acid synthesis by using their own unique template and primer complexes (i.e. double stranded DNA with promoter sequence for T7 RNA polymerase, and a DNA primer annealed to a RNA template for RTs) as illustrated in Figure 2A. First, we conducted a RNA synthesis reaction with T7 RNA polymerase under the same NTP concentration and ratio between radioactive and nonradioactive GTP (1/1600) as the IAV reaction except for the template sequence. As illustrated in Figure 2A, the T7 Pol template used is a 47-bp double stranded DNA (Fig. 2C) containing 18-bp T7 promoter and 29-bp of the sequence which is the same as the IAV RNA Pol template. The first nucleotide of the RNA product starts from G instead of A because T7 Pol prefers G as the initial nucleotide in RNA synthesis [28]. We also used the T7 Pol activity which displayed a similar level of the fully extended product as the IAV Pol reaction (Fig. 2B). As shown in Figure 2C, the first (2) UTP stop site was observed in the T7 Pol reaction (‘‘1*’’ in Influenza Polymerase Fidelity Fig. 2C). However, a significantly high level of the fully extended product was observed (see ‘‘]’’ in Fig. 2C), compared to the IAV Pol reaction with (2) UTP (see ‘‘]’’ in Fig. 2B). Next, we conducted the (2) dTTP and (2) dATP misincorpora- tion assay with low fidelity HIV-1 and high fidelity MuLV RT proteins. The RT reactions employed a 48-nt RNA template encoding the 39 end 30-nt sequence of the PA gene and 18-nt non- viral sequence annealed to an 18-nt DNA primer (Fig. 2A). Thus, the single stranded 30-nt PA sequence of the 48-mer RNA template where DNA synthesis is catalyzed by RTs encodes the exactly same 30-nt sequence as the influenza Pol 30-nt template used in Figure 2B. The RT reactions were conducted using the same nucleotide substrate (dNTP) concentration and the same ratio between nonradioactive and radioactive dNTP (1/1600) as the IAV Pol and T7 RNA Pol reactions. First, we normalized the polymerase activity of HIV-1 and MuLV RTs showing a similar band intensity of the fully extended product with all four NTPs as the full length bands in the 1x activity of the IAV Pol reactions (Fig. 2B). Importantly, note that the specific activities of the a-32P- NTP and a-32P-dNTP, which were used in the IAV Pol and RT reactions, respectively, were identical (3000 Ci/mmole), and thus the same band intensity of the fully extended products in the reactions with different polymerases indicates the same level of nucleotide incorporation and nucleic acid synthesis. Next, we repeated the same RT reactions with the equal activity of low fidelity HIV-1 and high fidelity MuLV RTs but with the (2) TTP or (2) dATP biased pools. Indeed, HIV-1 RT displayed much more extension beyond the first stop site (‘‘1*’’: see the sites marked with ‘‘U’’ in Figure 2D) and full length products (see ‘‘]’’ in Fig. 2D) than MuLV RT (Fig. 2E). This observation not only confirms that HIV-1 RT is more error-prone than MuLV RT, but also validates the fidelity assay condition established in this study. We also confirmed the first stop sites in these RT reactions using the same primer extension reactions but with 59 end c-32P-labeled DNA primer and four different biased dNTP pools (data not shown). Now with the misincorporation assays completed with the three other polymerases, we were able to compare their enzyme fidelity by comparing the extent of the primer extension beyond the first stop site (see ‘‘*’’ in Fig. 2). First, as seen in Figure 2B (1x) for IAV Pol with (2) UTP and Figure 2D for HIV-1 RT with (2) TTP, IAV Pol generated much shorter and fewer RNA products beyond the first stop site than HIV-1 RT even if there was a 2-fold more total polymerase activity of the 1x IAV Pol than HIV-1 RT, which was estimated from the reaction with all four nucleotides present. This suggests that IAV Pol complex has a lower misincorporation capability than HIV-1 RT. Also, HIV-1 RT showed much higher extension beyond the first stop site (Fig. 2D) than MuLV RT (Fig. 2E), suggesting that HIV-1 RT has lower fidelity than MuLV RT, which has been demonstrated previously (25). Interestingly, IAV Pol complex generated similar or slightly less primer extension beyond the first stop site than the high fidelity MuLV RT even if a lower polymerase activity of MuLV RT, compared to IAV Pol, was used, supporting that IAV Pol complex has at least similar fidelity with MuLV RT. Overall, the data shown in Figure 2 qualitatively suggests that IAV Pol complex is a high fidelity enzyme. It is important to address that IAV Pol may fall off from the template during the initial synthesis [14], which can induce the pausing of the primer extension reaction, and this is also true for several RNA polymerases using the promoter such as T7 RNA polymerase used in this study [29]. Since our study observes the pausing events at 11–13 nt away from the first nucleotide incorporation, the intrinsic pausing nature of these polymerases PLoS ONE | www.plosone.org 7 April 2010 | Volume 5 | Issue 4 | e10372