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Comparative Study
. 1994 Jun 15;222(3):781-93.
doi: 10.1111/j.1432-1033.1994.tb18925.x.

DNA replication in vitro by recombinant DNA-polymerase-alpha-primase

F Stadlbauer  1 , A Brueckner, C Rehfuess, C Eckerskorn, F Lottspeich, V Förster, B Y Tseng, H P Nasheuer
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Free article
Comparative Study

DNA replication in vitro by recombinant DNA-polymerase-alpha-primase

F Stadlbauer et al. Eur J Biochem. .
Free article

Abstract

DNA-polymerase-alpha--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-alpha--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-alpha--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-alpha--primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA-polymerase-alpha--primase synthesized double-stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.

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