Nucleic Acids Research, 2008, Vol. 36, No. 16 5263 concentration of A. aeolicus primase to maximize the length of the primers produced. These experiments tested overall trinucleotide speciﬁcity and allowed for kinetic studies of A. aeolicus primase. species. Total primer synthesis is the sum of moles of all primer lengths. This approach also normalized the variability introduced to the system during sample pre- paration and injection into the HPLC column. RNA primer synthesis assay and thermally denaturing HPLC analysis All RNA primer synthesis reactions were performed in 100 ml nuclease-free water containing 50 mM HEPES, 100 mM potassium glutamate, pH 7.5, 10 mM DTT, 10 mM magnesium acetate and either 1 mM of TEOS 1 or TEOS 2 or 2 mM of the 23-mer trinucleotide-speciﬁc template. The concentration of each NTP was 400 mM unless otherwise noted and the primase concentration was 2 mM. The composition of these reaction mixtures were similar to those conditions previously determined to be optimal for E. coli and S. aureus primases (25,31). Since the optimum temperature for A. aeolicus activity was unknown, this primase was incubated with each of the TEOS templates sequentially for 30 min at 65, 75 and 858C in the ﬁrst set of experiments. After its tem- plate speciﬁcity was determined, the temperature for max- imum enzyme activity was determined by incubating A. aeolicus primase in separate reactions, each incubated at a diﬀerent temperature that ranged from 258C to 858C for 1 h. Since A. aeolicus primase exhibited the highest activity at 55 or 658C, all subsequent primer synthesis reactions were incubated at 558C for 1 h. After incubation, the samples were desalted in a Sephadex G-25 spin column and dried using a speed vacuum. The pellet was resus- pended in water to 1/10th of the original volume of the sample and 8 ml of that sample was analyzed by HPLC under thermally denaturing conditions at 808C as pre- viously described (28). The denaturing HPLC elution gradient was adjusted for optimal separation of primer products and ssDNA tem- plate peaks on a DNA Sep column. For the primase activ- ity reactions, the primers synthesized in the reactions containing either TEOS 1 or TEOS 2 were eluted with a ﬂow rate of 0.9 ml/min using a gradient of 0–10% aceto- nitrile over 24 min and for the reactions with the 23-mer trinucleotide-speciﬁc template, a gradient 0–8.8% acetoni- trile over 16 min was used. Both the RNA and ssDNA templates were detected by UV absorbance at 260 nm. Retention times of the single-stranded oligoribonucleotide and oligodeoxyribonucleotide standards were correlated to the retention times of the appropriate oligonucleotide standard to conﬁrm composition and length. The RNA standards utilized in this study consisted of the sequence 50-AGUGUGUGUGUG-30 (12-mer), 50-AGUGUGUG UGUGUC-30 (14-mer) and 50-AGUGUGUGUGUGUC UG-30 (16-mer). The moles of primers were quantiﬁed by using the tem- plate as an internal standard (34). First, the area under each RNA peak and the template peak were background- corrected and each peak area summed such that it had units of mV min. Next, all areas were then divided by their relative extinction coeﬃcients. Finally, the moles of template in each reaction were used in conjunction with its mole-adjusted area to determine the moles of each primer RESULTS Functional assessment of TEOS 1 and TEOS 2 oligonucleotide templates To determine whether oligonucleotides that together com- prise all 64 trinucleotides would support primase activity, TEOS 1 and TEOS 2 (Figure 1A) were tested in the primer synthesis assay using the primases from E. coli and S. aureus. Primers were produced by E. coli primase using TEOS 1 and by S. aureus primase with TEOS 2 (data not shown). This was expected since TEOS 1 con- tained the E. coli initiation trinucleotide d(CTG) and since d(CTA), the preferred recognition sequence for S. aureus, was present in TEOS 2. These results demonstrated that both TEOS 1 and TEOS 2 could be useful in evaluating the activity of an uncharacterized bacterial primase. Analysis of A. aeolicus primase activity using TEOS 1 and TEOS 2 Since the optimum temperature for A. aeolicus primase activity was unknown, the samples were initially incubated at three sequential reaction temperatures, speciﬁcally 65, 75 and 858C. TEOS 1 did not generate any primers and was not further utilized, whereas TEOS 2 generated two species of primers that eluted at 10.6 and 13.1 min (Figure 1B). These results demonstrated that the truncated A. aeolicus primase was active and contained the necessary components for de novo primer synthesis. The predominant peak at 10.6 min using TEOS 2 com- prised the most homogeneous and abundant population (peak a in Figure 1B), presumably due to A. aeolicus pri- mase initiating primer synthesis from a single position in TEOS 2 and terminating elongation at the 50 nucleotide of this template. A comparison of the elution proﬁle of RNA polymer standards with the primer peak at 10.6 min indi- cated that this RNA polymer was 17 nt in length. Similarly, a comparison of the peak that eluted at 13.1 min with the RNA oligonucleotide standards indi- cated that these primer products were about 26 nt in length. Examination of the TEOS 2 sequence suggested that initiation of primer synthesis by A. aeolicus primase for the 10.6 min peak corresponded to d(CGC), d(GCG), d(CGG) or d(GGT), whereas the 13.1 min peak correlated with d(GGG), d(GGC), d(GCC) or d(CCC). These pre- dictions were then tested using trinucleotide-speciﬁc templates. Preferred recognition trinucleotide sequence for A. aeolicus primase is d(CCC) To identify the initiation trinucleotides recognized by A. aeolicus primase, a series of trinucleotide-speciﬁc tem- plates were synthesized for testing as described in Materials and methods section. When templates 1 through 4 (Figure 2A) were analyzed, the full-length primer