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 alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) 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 ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-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 vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-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 alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.
D001483:Base Sequence; D004257:DNA Polymerase II / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012313:RNA / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic
D004279:DNA, Viral; D009097:Multienzyme Complexes; D011506:Proteins; D012313:RNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II