DNA replication in vitro by recombinant DNA‐polymerase‐α‐primase

DNA‐polymerase‐α‐primase complex contains four subunits, p180, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA‐polymerase‐α‐primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over‐expressed the single polypeptides and co‐expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co‐expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co‐expression of all four subunits yielded large quantities of tetrameric DNA‐polymerase‐α‐primase. The p180, p58 and p48 polypeptides were also co‐expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA‐polymerase‐α‐primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA‐polymerase‐α‐primase synthesized double‐stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.