Nucleic Acids Research, 2008, Vol. 36, No. 16 5267 Figure 5. Temperature dependence of A. aeolicus primase activity using the d(GCC) template. (A) Chromatograms of primers synthesized by A. aeolicus primase and detected by denaturing HPLC, after incubation at the temperatures speciﬁed. (B) Plot of percentage activity versus tempe- rature in which the total area under the RNA primer peaks was ﬁrst normalized to the areas under the template peaks and was then normalized to the highest activity. The error bars represent the standard deviation of triplicate experiments. bacterial or phage primase examined to date. Previous studies have demonstrated that the RNA primers gener- ated by primase from both S. aureus and E. coli initiate complementary to the middle T in their respective initia- tion trinucleotide sequence (25,31). This study demon- strated that A. aeolicus primase also initiates de novo primer synthesis complementary to the middle nucleotide in the recognition trinucleotide, indicating that this is a conserved property for all microbial primases examined to date. Knowledge of the preferred recognition sequence d(CCC) and temperature optimum for A. aeolicus primase has provided important information for further biochem- ical characterization of this enzyme. Although the A. aeolicus primase used in these studies did not have the C-terminal domain which had been deleted to obtain a soluble recombinant protein (26), the N-terminal zinc binding domain and RNA polymerase domain were suﬃ- cient for de novo primer synthesis. This is similar to ﬁnd- ings in the E. coli and bacteriophage T7 systems in which only the domains containing the Cys4 zinc ﬁnger and active site for RNA polymerization were necessary to retain initiation speciﬁcity and catalyze template-directed oligoribonucleotide synthesis (26,37). In general, enzymes from thermophiles tend to be more stable, less ﬂexible and less active than related proteins from mesophilic organisms (38). Although the relationship of molecular ﬂexibility and activity is poorly deﬁned, inter- molecular and intracellular molecular associations most likely play a vital role in thermostability. For instance, crystal structure analyses indicated that the linker between the zinc binding domain and the RNA polymerase domain in A. aeolicus primase is shorter than the correspond- ing region in E. coli primase (26). Although the shorter linker in A. aeolicus primase is less ﬂexible than the corresponding linker in E. coli primase, the proposed two dominant transition states of this enzyme do not appear to have been compromised in terms of productive and nonproductive conformations (26). In the extended productive form, the zinc-binding domain is uncoupled from the associated RNA polymerase domain and pre- sumably interacts with the template DNA that is bound to another primase’s active site in the RNA polymerase domain in trans, resulting in primer synthesis. Therefore, the low activity of A. aeolicus primase may be due in part to reduced ﬂexibility and the intermolecular protein–pro- tein interactions required for primer synthesis, compared with the intramolecular interactions observed between the zinc binding and RNA polymerase domains of a primase from a mesophilic bacterium (34). Moreover, the C-term- inal domain of A. aeolicus primase most likely plays a critical role in stabilizing intermolecular interactions in vivo. Hyperthermophiles do not appear to replicate or repair their DNA by error-prone mechanisms (3). Despite the misincorporation propensities observed for A. aeolicus pri- mase in this study and for other bacterial primases (31,34), other replications proteins may modulate primase activity as is implicated for archaeal primases (39). These misin- sertions are most certainly corrected by primer-removing enzymes such as DNA polymerase I. In addition, although the deleterious eﬀects of high temperatures on nucleic acids have been well documented (10,40), there is some evidence that biomolecular stability at high temperatures is achieved by elevated salt concentrations, as well as by the presence of polyamines, cationic proteins and/or supercoiling (38). Therefore, further study is required to provide additional insight on the mechanisms by which hyperthermophiles such as A. aeolicus accurately replicate their genome.