Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase α (pol α-primase) was compared with those synthesized by a multiprotein form of DNA polymerase α (pol α₂) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G^*pppN-RNA-DNA chains and identifying their 5'-terminal ribo-nucleotide. Pol α₂ strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol α-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vitro. Pol α₂ had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol α-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase a affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.