Eucaryotic DNA primase does not prefer to synthesize primers at pyrimidine rich DNA sequences when nucleoside triphosphates are present at concentrations found in whole cells.
The critical role of NTP concentration in determining where calf thymus DNA primase synthesizes a primer on a DNA template was examined. Varying the concentration of NTPs dramatically altered the template sequences at which primase synthesized primers. At the low NTP concentrations typically used for in vitro experiments (100 microM), primase greatly preferred to synthesize primers at pyrimidine rich DNA sequences. However, when the concentrations of NTPs were increased to levels typically found in whole cells, primers were now synthesized in all regions of the template. Importantly, synthesis of primers in all regions of the DNA template, not just the pyrimidine rich sequences, is the pattern of primer synthesis observed during DNA replication in whole cells. With low concentrations of NTPs (i.e., Vmax/K(M) conditions), primers are only synthesized at the most preferred synthesis sites, namely, those that are pyrimidine rich. In contrast, under conditions of high NTP concentrations, primer synthesis will occur at the first potential synthesis site to which primase binds. Now, the primase x DNA complex will be immediately converted to a primase x DNA x NTP x NTP complex that is poised for primer synthesis.