5264 Nucleic Acids Research, 2008, Vol. 36, No. 16 Figure 2. Determination of A. aeolicus primase initiation speciﬁcity. (A) Selected sequences of the 23-mer templates containing the trinu- cleotide of interest that was tested in (B). (B) Denaturing HPLC detec- tion and assessment of primer products obtained using selected trinucleotide-speciﬁc templates. The trinucleotide of interest in the tem- plate as originally designed is denoted in bold and the trinucleotide recognized by A. aeolicus primase is in bold. All reactions were performed in triplicate, but only representative chromatograms are shown. The elution times for the 15-mer and 17-mer primers are shown and were based on the elution proﬁles obtained for the RNA standards. The minor peak adjacent to the predominant full-length 17-mer is due to misincorporation and is discussed in the results. The asterisk indicates the elution time for a 16-mer primer synthesized by primases from either S. aureus or G. stearothermophilus on the d(CTA) template. products obtained were longer than the predicted 16-mer when compared with the RNA standards (Figure 2B). The size of these full-length RNA polymers generated by A. aeolicus primase on the 23-mer templates were also compared with the more hydrophobic full-length 16-mer primers produced by the well-characterized primases from S. aureus and G. stearothermophilus on the d(CTA) 23-mer template (25,35). The 16-mer primers synthesized by S. aureus and G. stearothermophilus on their preferred tri- nucleotide contained the most hydrophobic ribonucleotide base, adenine, instead of a guanine. Together these com- parisons indicated that the full-length RNA products pro- duced by A. aeolicus primase on templates 1 through 4 were 17 nt in length. These ﬁndings prompted a review of the initiation sequence and it was noted that in each of the three templates, an additional nucleotide 30 to the originally designed unique trinucleotide was potentially responsible for the longer product (Figure 2A). This notion was consistent with the model in which A. aeolicus primase initiates primer synthesis on the middle nucleotide in the ﬁrst recognition trinucleotide nearest to the 30 end of the ssDNA template. As shown in Figure 2B, template 1 yielded the largest number of primers (17.4 0.5 pmol) in the presence of 2 mM primase (200 pmol) and 2 mM template (200 pmol). Template 2 generated the second highest amount of pri- mers (7.6 0.4 pmol), whereas templates 3 and 4 produced fewer primers (5.4 0.4 and 2.9 0.5 pmol, respectively). The minor peak adjacent to the predominant full-length 17-mer was due to misincorporation, as is discussed later. The predominant and full-length RNA polymers derived from template 1 eluted at 8.71 min, later than the full- length primers synthesized on templates 2 (8.57 min), 3 (8.62 min) and 4 (8.62 min). These primer elution proﬁles were consistent with the slightly more hydrophobic 17-mer primer derived from the overlined d(CCC) in template 1 (Figure 2A) eluting shortly after the full-length 17-mer RNAs derived from the initiation trinucleotides d(GCC) and d(CGC) in templates 3 and 4, respectively. This primer elution proﬁle was also consistent with A. aeolicus primase initiating on d(CCC) in template 2 and the result- ing 17-mer primer eluting slightly earlier than the 17-mer RNA polymer derived from d(CCC) in template 1, due to the presence of a hydrophilic cytosine instead of a more hydrophobic guanine. However, although A. aeolicus pri- mase initiated primer synthesis on d(CCC) in both tem- plates 1 and 2, the diﬀerence in the amount of RNA polymers produced on these ssDNA templates suggested that context, such as a single base adjacent to the initiation trinucleotide, inﬂuences enzyme activity. The speciﬁcity of A. aeolicus primase activity was further evaluated by testing templates that contained the currently known recognition trinucleotides for the pri- mases from E. coli, S. aureus and G. stearothermophilus, speciﬁcally d(CTG), d(CTA) and d(TTA) (Figure 2A). No observable primer products were synthesized by A. aeoli- cus primase on these templates (Figure 2B), conﬁrming that this enzyme preferentially initiates de novo primer synthesis on trinucleotides that diﬀer from those used by other bacterial primases. Collectively, these results indicated that the trinucleo- tides d(CCC), d(GCC) and d(CGC) in templates 1 through 4, respectively, were generating a signiﬁcant amount of primers (Figure 2). In addition, these results indicated that the majority of RNA polymers generated with TEOS 2 and eluting at 10.6 min were initiating from d(CGC) within that template, correlating with a RNA polymer that is also 17 nt in length (Figure 1). The broader peak that eluted at 13.1 min with the reactions containing TEOS 2 was consistent with primers initiating from the preferred trinucleotide d(CCC), as well as from d(GCC). The less than optimum positioning of these two later tri- nucleotides and the apparent limitations of A. aeolicus primase to produce primers that extend to the end of the TEOS 2 template most likely contributed to a reduction in the amount of full-length, template-derived, 25-mer and 26-mer RNA polymers. Moreover, the 9- to 13-mer pri- mers observed at approximately 8.3 min in Figure 1B most likely initiated from d(CCC) or d(GCC) in TEOS 2 with