Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex.

Abstract

Yeast DNA primase and DNA polymerase I can be purified by immunoaffinity chromatography as a multipeptide complex which can then be resolved into its functional components and further reassembled in vitro. Isolated DNA primase synthesizes oligonucleotides of a preferred length of 9-10 nucleotides and multiples thereof on a poly(dT) template. In vitro reconstitution of the DNA primase:DNA polymerase complex allows the synthesis of long DNA chains covalently linked to RNA initiators shorter than those synthesized by DNA primase alone. The SS (single-stranded) circular DNA of phage M13mp9 can also be replicated by the DNA primase:DNA polymerase complex. Priming by DNA primase occurs at multiple sites and the initiators are utilized by the DNA polymerase moiety of the complex, so that almost all the SS template is converted into duplex form. The rate of DNA synthesis catalyzed by isolated yeast DNA polymerase I on the M13mp9 template is not constant and is characterized by distinct pausing sites, which partly correlate with secondary structures on the template DNA. Thus, replication of M13mp9 SS DNA with the native primase:polymerase complex gives rise to a series of DNA chains with significantly uniform termini specified by the primase start sites and the polymerase stop sites.