Figure 6: Both PriSX and PriSLX differ from PriSL in primer synthesis.

(a–c) Effects of temperature and substrates on the primase activity of PriSL, PriSX and PriSLX. PriSL, PriSX or PriSLX was incubated with M13 ssDNA for 30 min in the presence of indicated substrates and at indicated temperatures. Bands indicated by an arrow (a) are short products that PriSL was able to synthesize in the absence of a template. Recently, the two spots were identified as dAMP-glycerol (the lower band) and dAMP-Tris (the higher band) ³⁶. The exposure time for the gel in a was about three times as long as those for the gels in b,c. (d–f) Incorporation of dNMPs or rNMPs by PriSL, PriSX and PriSLX in the presence of rNTPs or dNTPs, respectively. Primase was incubated with M13 ssDNA at 55 °C (PriSL), 65 °C (PriSX) or 75 °C (PriSLX) in the presence of a fixed amount (10 μM) of radiolabelled dNTPs and an increasing amount (0, 1, 10 and 100 μM) of unlabelled rNTPs or a fixed amount (10 μM) of radiolabelled rNTPs and an increasing amount (0, 1, 10 and 100 μM) of unlabelled dNTPs. For PriSX, incorporation of dNMPs was also tested in the presence of rNTPs at increased concentrations (0, 0.25, 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 μM) (panel to the right of Fig. 2e).