F. Stadlbauer et al., DNA-REPLICATION IN-VITRO BY RECOMBINANT DNA-POLYMERASE-ALPHA-PRIMASE, European journal of biochemistry, 222(3), 1994, pp. 781-793
DNA-polymerase-alpha-primase complex contains four subunits, p180, p68
, p58, and p48, and comprises a minimum of two enzymic functions. We h
ave cloned cDNAs encoding subunits of DNA-polymerase-alpha-primase fro
m human and mouse. Sequence comparisons showed high amino acid conserv
ation among the mammalian proteins. We have over-expressed the single
polypeptides and co-expressed various subunit complexes using baculovi
rus vectors, purified the proteins and investigated their biochemical
properties. The purified mouse p48 subunit (Mp48) alone had primase ac
tivity. Purification of co-expressed Mp48 and Mp58 subunits yielded st
able DNA primase of high specific activity. Co-expression of all four
subunits yielded large quantities of tetrameric DNA-polymerase-alpha-p
rimase. The p180, p58 and p48 polypeptides were also co-expressed and
immunoaffinity purified as a trimeric enzyme complex. The tetrameric a
nd trimeric DNA-polymerase-alpha-primase complexes showed both DNA pri
mase and DNA polymerase activities. The tetrameric recombinant DNA-pol
ymerase-alpha-primase synthesized double-stranded M13 DNA and replicat
ed polyoma viral DNA in vitro efficiently.