In the simian virus 40 in vitro replication system, start site selection by the polymerase alpha-primase complex is not significantly altered by changes in the concentration of ribonucleotides

The simian virus 40 (SV40) in vitro replication system was previously used to demonstrate that the human polymerase (Pol) ol-primase complex preferent ially initiates DNA synthesis at pyrimidine-rich trinucleotide sequences. H owever, it has been reported that under certain conditions, nucleoside trip hosphate (NTP) concentrations play a critical role in determining where euk aryotic primase initiates synthesis. Therefore, we have examined whether in creased NTP concentrations alter the template locations at which SV40 repli cation is initiated. Our studies demonstrate that elevated ribonucleotide c oncentrations do not significantly alter which template sequences serve as initiation sites. Of considerable interest, the sequences that serve as ini tiation sites in the SV40 system are similar to those that serve as initiat ion sites for prokaryotic primases. It is also demonstrated that regardless of the concentration of ribonucleotides present in the reactions, DNA synt hesis initiated outside of the core origin. These studies provide additiona l evidence that the Pol alpha -primase complex can initiate DNA synthesis o nly after a considerable amount of single-stranded DNA is generated.