5266 Nucleic Acids Research, 2008, Vol. 36, No. 16 Figure 4. Ribonucleoside triphosphate dependence of A. aeolicus primase activity on the preferred d(CCC) template. (A) Chromatograms of primers synthesized in which the concentration of all four NTPs were varied simultaneously as indicated and detected by denaturing HPLC. (B) The picomole of primers produced were quantiﬁed and plotted versus NTP concentration. The error bars represent the SD of triplicate experiments. E. coli, indicating that all primases share a common elongation mechanism. This elongation kinetic aﬃnity appears to be orders of magnitude weaker than the overall NTP kinetic aﬃnity (Figure 4), demonstrating the vast diﬀerence between the initiation and elongation phases of primer synthesis for the A. aeolicus primase. However, caution is warranted in these analyses since the kinetics of the incorporation of a single nucleotide during elongation is compared with overall primer synth- esis in the presence of all four NTPs. Aquifex aeolicus primase activity dependence for ribonucleotides The concentration of the NTP mixture was varied to opti- mize A. aeolicus primase activity using the preferred d(CCC) template. The striking results showed that primer synthesis activity rose almost linearly to a maxi- mum with 1.2 mM of each NTP and then fell even more rapidly until there was virtually no activity with 2.8 mM of each NTP (Figure 4). At the optimum NTP concentra- tion, the enzyme synthesized 15.7 0.3 pmol primers even though the reaction contained 200 pmol of primase and 200 pmol of template. These data indicated that only 8% of the templates were primed at the optimum temperature and NTP concentration for maximum activity. Under similar conditions with the primases from S. aureus and E. coli and their recognition trinucleotides, 49 and 25% of the templates were primed, respectively (28,34). Aquifex aeolicus primase appeared to have a very high KM for the NTP mixture, but was even more strongly inhibited by it. Since the NTP inhibitory eﬀect was certainly exert- ing itself as the activity neared its peak, 400 mM of each NTP was used in the primer synthesis reactions to mini- mize inhibition. Temperature optimum for A. aeolicus primase activity To delineate the optimum temperature for maximum activ- ity of A. aeolicus primase, the primer synthesis assay was performed at separate temperatures from 258C to 858C using the d(GCC) template. Based on the amount of pri- mers synthesized, A. aeolicus primase activity was observed to be the highest at 558C (3.4 0.4 pmol primers) and 658C (3.2 0.4 pmol primers) with about half the activity at 758C (2.4 0.2 pmol primers) (Figure 5). Little or no activ- ity was observed at 428C and below (Figure 5). At 558C, the predominant primer synthesized was a full-length 17-mer, whereas at 65 and 758C the abundance of the 17-mer decreased. The minor peak that eluted just prior to the full-length RNA polymer for the reactions incubated at 65 and 758C indicated an increase in the amount of misinsertion, especially at the higher temperature. At 858C, A. aeolicus primase was only slightly active, although this is the optimal temperature for culturing this microbe. A small amount of template hydrolysis was also seen at this elevated temperature. Collectively, these results sug- gested that auxiliary proteins and/or factors are most likely required to stabilize the interaction between the genomic template and primase at the higher physiological temperatures for this extreme thermophile. DISCUSSION Although neither the initiation speciﬁcity nor the opti- mum functional temperature of a hyperthermophilic bac- terial primase had previously been determined, the two-step empirical method used in this study showed that A. aeolicus primase was active and that the preferred initiation trinucleotide for A. aeolicus primase was d(CCC) and to a lesser degree d(GCC) and d(CGC). These recognition sequences are not used by any other