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^†

Abstract

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.

In the Simian Virus 40 In Vitro Replication System, Start Site Selection by the Polymerase α-Primase Complex Is Not Significantly Altered by Changes in the Concentration of Ribonucleotides

“…It follows that an increase in the concentration of a particular ribonucleotide might stabilize the Pol ␣-primase complex at different template sequences and thereby alter the initiation profile. Indeed, it has been reported that at elevated ribonucleotide concentrations, the Pol ␣-primase complex purified from calf thymus initiates synthesis in a sequence-independent manner (43). Therefore, we next addressed whether elevated ribonucleotide concentrations had similar effects on start site selection in the SV40 replication system.…”

“…In order for primase to initiate DNA synthesis, a stable complex must form between primase and the pyrimidine-rich initiation sites. Recent experiments indicate that stable binding of two NTPs to primase is a prerequisite for stable complex formation between primase and single-stranded DNA templates (43,64). Related studies demonstrated that fluctuations in ribonucleotide pools can influence exactly which template sequences are selected as initiation sites (43).…”

“…The concentration of ribonucleotides used in one set of pulse reactions were those found in the standard SV40 in vitro replication reactions (200 M each for CTP, UTP, and GTP and 4 mM ATP) (48,70,86). The second set of pulse reactions contained the high ribonucleotide levels suggested to reflect the in vivo concentrations (1 mM CTP, 1 mM UTP, 2 mM GTP, and 4 mM ATP) (43). The elevated ATP concentration (4 mM) is required to support T-ag assembly and subsequent unwinding events (9,21,24).…”

“…However, whether these sequences will serve as recognition sites for primase under conditions other than those present in the standard in vitro SV40 replication assays has yet to be examined. For example, the ribonucleotide concentrations used in standard SV40 replication assays are 200 M GTP, UTP, and CTP and 4 mM ATP (48,70,86); however, it has been proposed that the actual ribonucleotide concentrations present in cells are higher than the standard ribonucleotide concentrations (43). Moreover, using the purified Pol ␣-primase complex isolated from calf thymus and the elevated nucleoside triphosphate (NTP) levels suggested to reflect in vivo concentrations (e.g., 1 mM CTP, 1 mM UTP, 2 mM GTP, and 4 mM ATP), it was reported that primer synthesis occurs at the first potential site to which primase binds (43).…”

“…For example, the ribonucleotide concentrations used in standard SV40 replication assays are 200 M GTP, UTP, and CTP and 4 mM ATP (48,70,86); however, it has been proposed that the actual ribonucleotide concentrations present in cells are higher than the standard ribonucleotide concentrations (43). Moreover, using the purified Pol ␣-primase complex isolated from calf thymus and the elevated nucleoside triphosphate (NTP) levels suggested to reflect in vivo concentrations (e.g., 1 mM CTP, 1 mM UTP, 2 mM GTP, and 4 mM ATP), it was reported that primer synthesis occurs at the first potential site to which primase binds (43). These observations raise the question of whether the previously described 3Ј-NTT-5Ј and 3Ј-NT-5Ј initiation sites will be selected in SV40 replication reactions conducted in the presence of the alleged in vivo concentrations of ribonucleotides.…”